Abstracts - Microbiology Society

Transcription

Microbiology
society
for
general
Spring Conference
25–28 March 2013
SGM Spring Conference | Manchester | 25–28 March 2013
Manchester Central Convention Complex
Abstracts
Contents
Prize Lectures 2
Sessions
MA01 Viruses and human cancer: causes to cures
3
MA02 Metabolic interactions at the host–pathogen interface
MA03 Co-infections and co-colonisation
15
MA04 Antimicrobial resistance: are scientists getting the message right?
20
MA05 New approaches to exploit Streptomyces21
9
MA06 Next-generation antimicrobials
27
MA07 Bacterial–fungal interactions
31
MA08 RNA – so much more than just a genome
36
MA09 Virology workshop: Vaccines and antivirals
38
MA10 Virology workshop: The virome and viromics
40
MA11 Virology workshop: Assembly and structure
43
MA12 Virology workshop: Innate immunity
45
MA13 Virology workshop: RNA – so much more than just a genome
48
MA14 Virology workshop: Pathogenesis
50
MA15 Virology workshop: Gene expression and replication
53
MA16 Virology workshop: Clinical virology
55
MA01 Viruses and human cancer: causes to cures
59
MA02 Metabolic interactions at the host–pathogen interface
60
64
Posters
MA03 Co-infections and co-colonisation
MA05 New approaches to exploit Streptomyces66
MA06 Next-generation antimicrobials
73
MA09 Virology workshop: Vaccines and antivirals
83
MA10 Virology workshop: The virome and viromics
87
MA11 Virology workshop: Assembly and structure
88
MA12 Virology workshop: Innate immunity
89
MA13 Virology workshop: RNA – so much more than just a genome
90
MA14 Virology workshop: Pathogenesis
90
MA15 Virology workshop: Gene expression and replication
96
MA16 Virology workshop: Clinical virology
99
CMM Clinical and medical microbiology
102
ENV Environment
107
FB Fermentation and bioprocessing (industry)
110
GM General microbiology
110
SC Systems and cells
118
Prize Lecture abstracts
Prize Lectures
COLWORTH PRIZE LECTURE – MON 1210
Vaccines R&D: challenges for the 21st century
Jeffrey W. Almond
Vice President of Research, Sanofi Pasteur, Campus Mérieux, 1541 avenue
Marcel Mérieux, F-69280 Marcy-L’Etoile, France
In terms of impact on human health, vaccines are one of medical sciences
most spectacular successes. However, there are still a good number of
infectious diseases against which we do not yet have effective vaccines
and where existing vaccines are less than perfect.
This presentation will review the process of vaccine development,
identify priorities for the next decade from an industry perspective and
illustrate research & development approaches using examples of the
more difficult pathogens that have devised strategies for immune evasion
or modulation..
Peter Wildy Prize for Microbiology
Education LECTURE – MON 1720
Beautiful and a little bit scary … viruses and science communication
David Bhella
Programme Leader, MRC Centre for Virus Research, University of Glasgow,
Glasgow, UK
My enthusiasm to tell the world about viruses began over twenty years
ago while working as a diagnostic electron microscopist in a London
hospital. Captivated by the sinister beauty of these tiny pathogens I would
stay after work in the darkroom printing micrographs before rushing
home to show them to my friends. In the intervening years a fascination
with virus structure, interest in digital media and my close relationship
with the science-learning manager at Glasgow Science Centre (to
whom I am married) have led me to become deeply involved in public
engagement. Working as a group leader in the MRC Centre for Virus
Research I have developed a thriving programme of outreach activities
including an extraordinarily successful molecular biology workshop for
high-school students and an exhibition of images and movies from virus
research. Activities such as these have the potential to educate and
delight audiences, influence opinions and occasionally change lives. In this
lecture I will try to share what I have learned through the development
of these and other projects including thoughts on working with museums
and science centres, connecting with schools and public audiences and
how different modes of engagement can balance audience size and
depth of interaction.
SGM Prize Medal Lecture – Tue 1210
Infections causing human cancers: why do some ubiquitous infections
mainly cause regional cancers?
Harald zur Hausen
Deutsches Krebsforschungszentrum (DKFZ), Im Neuenheimer Feld 280,
69120 Heidelberg, Germany
Slightly more than 20% of the global incidence of human cancers
can be linked to infectious events. Commonly, long latency periods
between primary infection and cancer occurrence are characteristic
for these types of tumours.They vary between 3 -15 years for some
cancers and may last up to 60 years for others. During the past years it
became evident that several infections are necessary for the induction
of specific cancers (e.g. HPV in cervical cancer), but are not sufficient.
Development of malignant disease presumably requires in each case
specific modifications in host cell genes.The length of the latency period
seems to reflect the number of cellular gene modifications necessary
to permit unimpaired expression of viral oncogenes in cells still capable
to proliferate. In some instances, the persisting infectious agent may
cause such modifications, either by inducing mutational events or by
epigenetically silencing genes.
Regional clustering of cancers caused by ubiquitous infections regularly
seems to be caused by local synergistic functions of mutagenic factors
or by epigenetic events acting on the previously infected cell. A number
of examples will be presented (e.g. Burkitt’s lymphoma, nasopharyngeal
cancer, both linked to Epstein–Barr virus infections, chronic hepatitis
B and C infections linked to liver cancer, cancers of the cervix and
oropharyngeal cancers caused by high risk human papillomavirus
infections and some others), where epidemiology clearly points to an
important role of co-factor activity. For primary prevention knowledge
of these co-factors could be of great importance. Regional clustering
of tumours is not only found in cancer-linked infections. Smoking habits,
dietary customs, sun exposure and other occupational factors may
contribute to regional clustering of specific cancers.Yet, even in some of
these latter malignancies, a search for synergistically interacting infections
appears to be worthwhile.
HOT TOPIC LECTURE – TUE 1735
The human microbiome: overdue recognition for our fellow travellers
Paul W. O’Toole
Department of Microbiology and Alimentary Pharmabiotic Centre, University
College Cork, Ireland (Email: pwotoole@ucc.ie)
The microbiota associated with the human body is now intensively
studied as an environmental risk factor for disease, and a modulator of
health.The coding capacity of the microbiome significantly extends that
of the human genome. Exposed body surfaces are colonized by generally
stable microbial consortia that range from the relatively simple, to some
of the most complex microbial ecosystems known.The composition and
function of these consortia, in healthy adult subjects, is generally neither
haphazard nor randomly variable over time, suggesting co-evolution
and stabilizing interactions between the microbiome and host.The
development of culture-independent methods for microbiota analysis has
allowed identification of alterations in the microbiota associated with the
extremes of life, with functional gastrointestinal diseases, with endocrine
disease, with antibiotic therapy, and with habitual diet. However, there
are few examples of microbiome configurations or missing taxa that
are unequivocally established as causative of disease in humans.The
complexity of the microbiota and its multifaceted interaction with
host physiology, especially in the gut, mean that emulating a Koch’s
postulates approach for microbiota-related pathophysiological changes
will be challenging.This presentation will review the explosion of human
microbiome research, summarize the achievements, and will discuss the
major challenges.
FLEMING PRIZE LECTURE – WED 1210
Cyclic di-GMP signalling and the regulation of bacterial virulence
Robert Ryan1,2
1
Department of Microbiology, Biosciences Institute, University College Cork,
Cork, Ireland; 2Division of Molecular Microbiology, College of Life Sciences,
University of Dundee, Dundee, UK
Signal transduction pathways involving the second messenger cyclic
di-GMP occur widely in bacteria where they act to link perception of
environmental or intracellular cues and signals to specific alterations
in cellular function. Such alterations can contribute to bacterial lifestyle transitions including biofilm formation and virulence.The cellular
levels of the nucleotide are controlled through the opposing activities
of diguanylate cyclases (DGCs) and phosphodiesterases (PDEs).The
GGDEF domain of DGCs catalyses the synthesis of cyclic di-GMP
from GTP whereas EAL or HD-GYP domains in different classes of
PDE catalyse cyclic di-GMP degradation to pGpG and GMP. We are
now beginning to understand how alterations in cyclic di-GMP exert a
regulatory action through binding to diverse receptors or effectors that
include a small ‘adaptor’ protein domain called PilZ, transcription factors
and riboswitches.The regulatory action of enzymatically active cyclic diGMP signalling proteins is however not restricted to an influence on the
level of nucleotide. Here I will discuss our recent findings that highlight
the role that protein-protein interactions involving these signalling
proteins have in regulating functions that contribute to bacterial virulence.
Please note: Abstracts are published as received from the authors and are not subject to editing.
2
Sessions
Viruses and human cancer:
causes to cures
MA01
MA01 – Mon 0900
Overview of viruses and cancer
Robin A Weiss
Division of Infection & Immunity, University College London, London, UK
Oncogenic viruses are important in understanding cancer in two major
ways. First, oncogenic viruses in animals and humans have provided
us with great insight into molecular mechanisms of cancer generally,
irrespective of whether they are caused by infectious agents. Both
oncogenes and tumour suppressor genes first came to light through
the study of oncogenic viruses such as avian Rous sarcoma virus and
simian polyoma virus. Second, at least 15% of the global human cancer
burden is attributable to virus infection, and a further 5% to bacteria
and helminths. Although the virus is only one component in a complex
multifactorial pattern of carcinogenesis, prevention of virus infection
removes a necessary cause; hence the importance of viral vaccines and
screening methods that reduce the prevalence of infection. Viruses may
also be exploited as vectors of gene therapy or as oncolytic agents for
the specific destruction of cancer cells. Malignant transformation of cells
by viruses may be viewed as a relatively rare ‘side effect’ of infection with
delayed but disastrous consequences for the host.
MA01 – Mon 0930
Infections and cancer – the global perspective
Denise Whitby
Frederick National Laboratory for Cancer Research, PO Box B, Frederick,
MD 21701, USA
It has been estimated that 18% of all cancer are attributable to infectious
agents, notably, viruses. However, the proportion of cancer caused
by infections varies geographically. In developing countries, over 26%
of cancer is caused by infections, whereas in wealthier countries, less
than 8% of cancer is attributed to infection. The major reason why
infection-related cancers disproportionately affect low income countries
is variation in the prevalence of the microorganisms concerned Kaposi’s
Sarcoma (KS) is infrequent in the US,but in certain area of Africa, it is
the commonest cancer. The geographic disparities in the incidence of
KS are almost entirely explained by variations in the prevalence of the
aetiologic agent, Kaposi’s Sarcoma-Associated Herpesvirus (KSHV).
What remains unexplained is the wide geographical variability in KSHV
prevalence. Recent studies in Africa have provided some insights and will
be presented.
MA01 – Mon 1000
Virus and host genome variation and cancer
Paul Kellam
Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton,
Cambridge, UK
A virus infection can result in different disease courses, ranging from
mild to moderate disease for many to severe life threating disease for
an unfortunate few. In viral oncology such severe disease is cancer,
although not all individuals infected by viruses with oncogenic potential
will develop such a disease. Recent estimates indicate that the average
proportion of malignancies worldwide that could be avoided in the
absence of an infectious agent is 18%, with this figure being higher at
26% in developing countries. Genetic changes in the host genome
underpin malignancy and whole genome sequencing of cancer genomes
is revealing the extent and variety of cancer driver mutations and
the evolving nature of cancer. Similarly, it is likely that virus genome
variation will influence cancer development and large scale virus genome
Session abstracts
sequencing will reveal such variation. Here I will describe insights
gained in sequencing host and virus genomes in cancer and show how
functional consequences may be gleaned from such information.
MA01 – Mon 1100
Mechanisms of transformation by tumour viruses
Ron Javier, Kathleen Kong, Midori Taruishi, Manish Kumar
Department of Molecular Virology and Microbiology, Baylor College of
Medicine, Houston, Texas, USA
Viral infections contribute to an estimated 20% of all human cancers,
and represent the major factors for liver and cervical cancers. Currently,
eight viruses – Epstein–Barr virus, Kaposi’s sarcoma herpes virus,
high-risk papillomaviruses, Merkel cell polyomavirus, human T-cell
lymphotrophic virus type 1, hepatitis B virus, hepatitis C virus, and
human immunodeficiency virus – have strong associations with human
cancer. The mechanisms whereby these viruses promote cancers
are slow, inefficient multi-step processes that may include activation
of protooncogenes, inactivation of tumour suppressors, inhibition of
DNA damage response pathways, accumulation of somatic mutations,
increased cellular proliferation, immune evasion, induction of host
inflammatory responses, and immunosuppression. Also relevant to this
discussion are results stemming from work on a group of viruses – avian
and murine retroviruses, simian and mouse polyomaviruses, and human
adenoviruses – that produce tumors only in experimentally infected
animals. These findings led to the concept of the oncogene, identification
of many proto-oncogenes and the p53 tumour suppressor, and functions
of the retinoblastoma tumour suppressor – landmark discoveries that
apply not only to virus-associated human cancers but also to human
cancers in general. This talk explores the mechanisms of transformation
by tumour viruses with an emphasis on common themes that have
emerged.
MA01 – Mon 1130
Offered paper Different patterns of Epstein–Barr virus latency in
endemic Burkitt lymphoma (BL) lead to distinct variants within the
BL-associated gene expression signature
Gemma Kelly1,5, Julianna Stylianou1, Jane Rasaiyaah2, Wenbin Wei1,
Wendy Thomas1, Debbie Croom-Carter1, Christian Kohler3,
Rainer Spang3, Ciaran Woodman1, Paul Kellam2,4, Alan Rickinson1,
Andrew Bell1
1
The University of Birmingham, Birmingham, UK, 2University College London,
London, UK, 3University of Regensburg, Regensburg, Germany, 4Wellcome
Trust Sanger Institute, Cambridge, UK, 5Walter and Eliza Hall Institute,
Melbourne, Australia
Endemic Burkitt lymphoma (BL) is a B cell malignancy whose
pathogenesis involves complementation between a chromosomal
translocation leading to c-myc deregulation, and infection with Epstein–
Barr virus (EBV). However the virus’ role in BL development remains to
be fully elucidated, in part because two alternative forms of EBV latency
have been described in BL tumour cells. Thus classical BLs express a
single viral protein EBNA1 (a form of infection termed Latency I), while
atypical tumours (which we term Wp-restricted) carry an EBNA2deleted virus genome and express a broader range of antigens including
EBNA3A, 3B, 3C and the viral bcl2-homologue BHRF1. Here we show
that the cellular gene expression profiles of Latency I and Wp-restricted
BL cells both fall within the recently described ‘molecular BL’ signature.
However Wp-restricted BLs can be distinguished by a detectable
down-regulation of the germinal centre (GC)-associated marker Bcl6
and up-regulation of certain plasmacytoid differentiation genes, notably
IRF4 and BLIMP1. Moreover, these same changes could be induced by
in vitro infection experiments using an EBNA2-knockout virus. These
findings suggest Latency I and Wp-restricted BLs evolve from a common
progenitor cell but that different patterns of virus antigen expression can
impose a subtly different BL phenotype.
3
Session abstracts
MA01 – Mon 1145
Offered paper The Merkel cell polyomavirus small T antigen promotes
cell motility, migration and invasion
Laura M Knight, David A Griffiths, Adrian Whitehouse
University of Leeds, Leeds, UK
Merkel cell carcinoma (MCC) is a highly aggressive non-melanoma skin
cancer arising from epidermal mechanoreceptor Merkel cells. In 2008,
a novel human polyomavirus, Merkel cell polyomavirus (MCV), was
identified and is strongly implicated in MCC pathogenesis. Currently,
little is known regarding the virus-host cell interactions which support
virus replication and viral-induced mechanisms in cellular transformation
and metastasis. This study focuses on characterising the functions of
the MCV small tumour antigen (ST) with regards to its possible roles in
tumourigensis. SILAC-based quantitative proteomics demonstrated that
MCV-ST expression results in upregulation of proteins which regulate
the cellular cytoskeleton. Stathmin is a phosphoprotein which regulates
microtubule stability and its overexpression has been associated with
increased migration and invasion in cancer cells. Western blot analysis
confirmed the proteomic data, demonstrating stathmin was upregulated
upon MCV-ST expression and in a primary MCC cell line positive for
the integrated MCV genome. MCV-ST expression leads to the perinuclear redistribution of β-tubulin, typical of microtubule destabilisation.
Scratch assays and matrigel/fibronection transwell assays demonstrated
that MCV-ST mediated microtubule destabilisation promotes motility,
migration and invasion. Moreover stathmin depletion confirmed that the
ability of MCV-ST to promote migration and invasion is a result of MCVST mediated microtubule destabilisation.
MA01 – Mon 1400
EBV-induced aberrant cell signalling as a potential anti-cancer target
Paul Murray
The University of Birmingham, St Vincents Drive, Edgbaston, Birmingham, UK
The Epstein–Barr virus uses the host B cell differentiation programme
to persist in memory B cells. In doing so it is able to survive, usually
asymptomatically, for the lifetime of the host. In rare circumstances EBV
can contribute to the development of cancer, such events are presumed
to be ‘accidents’ of the viral strategy for persistence. Many studies have
elucidated the signalling pathways aberrantly activated by EBV and its
latent virus genes in B cells and have shown that the same pathways are
activated in EBV-associated lymphomas, such as Hodgkin’s lymphoma.
These observations have been taken as evidence that these pathways
are alone sufficient for EBV-induced lymphoma development. Recently,
we have sought to distinguish the impact of EBV induced signalling events
relevant on the pathogenesis of lymphomas from those associated with
normal virus persistence. In doing so we have provided fresh clues to the
contribution of EBV to lymphomagenesis and identified novel targets for
therapeutic intervention.
MA01 – Mon 1430
Offered paper Levels of viral oncogenes expressed from integrated
HPV16 genomes are related to the epigenetic state of the long control
region
Ian J Groves, Mark R Pett, Cinzia G Scarpini, Dawn M Ward,
Nicholas Coleman
Department of Pathology, University of Cambridge, Cambridge, UK
High-risk human papillomavirus (HR-HPV) is usually integrated into the
host genome in cervical cancer cells. However, the factors that provide
a competitive advantage to particular integrants in a mixed population of
HPV-infected cells remain poorly understood. Using a single culture of
polyclonal episome-containing W12 cervical keratinocytes, we generated
a panel of 24 isogenic clones that differed only by the site of HPV16
integration into host chromosomes. The observed sites were typical
of those seen in cervical carcinomas in vivo, suggesting that integration
occurs at host genomic sites that are relatively accessible for insertion
of foreign DNA. Across clones suitable for analysis, E7 protein levels
per cell varied by over 10-fold, with only a third of clones showing
significantly greater E7 abundance than the parental episome-containing
cells. In addition, levels of viral E7/E6 transcripts per template varied by
over 40-fold. Detailed epigenetic characterisation of the viral long control
region (LCR) showed that transcript levels per template correlated
with chromatin modifications and RNA polymerase-II recruitment and
activation. Together, these data indicate that different levels of integrantderived oncogene expression are associated with epigenetic differences
at the LCR and that elevated levels of viral oncogenes are not an
inevitable consequence of HPV16 integration.
MA01 – Mon 1445
Offered paper The HCV NS5A protein causes EGFR accumulation by
inhibiting ubiquitination of the receptor and interfering with normal
endocytic trafficking
Zsofia Igloi, Jamel Mankouri, Mark Harris
University of Leeds, Leeds, West Yorkshire, UK
Hepatitis C virus (HCV) can establish a persistent infection, leading to
cirrhosis and hepatocellular carcinoma. In this context the HCV NS5A
protein has been intensely studied as it contains a C-terminal polyproline
motif (PxxPxR) that binds to the Src homology 3 (SH3) domains of a
number of cellular proteins involved in signal transduction. Previously,
we observed elevated levels of Epidermal Growth Factor Receptor
(EGFR) in cells harbouring HCV subgenomic replicons (SGR). Here we
demonstrate that this elevation is dependent on the NS5A PxxPxR motif
as it was abolished by alanine substitution of these prolines (termed the
PA mutant). Furthermore, ubiquitination of the receptor was impaired
in cells harbouring wild type, but not a PA mutant SGR. This could
explain the increase in the amount of the receptor since ubiquitination
is an important signal for trafficking from endosomes to the lysosome.
To understand the mechanism of this impairment, we have used
coimmunoprecipitation analysis to investigate the effects of NS5A on the
extensive net of protein-protein interactions contributing to the trafficking
of EGFR. As elevated EGFR levels have been described to contribute to
cancer formation, our studies could contribute to a deeper understanding
of how HCV infection potentially leads to hepatocellular carcinoma.
MA01 – Mon 1530
Human papillomaviruses and the DNA damage response
Laimonis A. Laimins
Dept. of Microbiology-Immunology, Northwestern University, Chicago, IL
60611, USA
The infectious HPV life cycle is closely linked to the differentiation state
of the host epithelia, with viral genome amplification and late gene
expression restricted to suprabasal cells. The E6 and E7 proteins provide
an environment conducive to DNA synthesis upon differentiation, but
little is known concerning the mechanisms that regulate productive
viral genome amplification and late gene activation. Using keratinocytes
that stably maintain HPV-31 episomes, and chemical inhibitors, we
demonstrate that viral proteins activate the ATM DNA damage
response in differentiating cells, as indicated by phosphorylation of
CHK2, BRCA1 and NBS1. This activation is necessary for viral genome
amplification, as well as for formation of viral replication foci. In contrast,
inhibition of ATM kinase activity in undifferentiated keratinocytes had no
effect on the stable maintenance of viral genomes. Using Fluorescence
In Situ Hybridisation for HPV DNA coupled with immunofluoroscence
for DNA damage proteins, we have found that DNA repair factors
colocalise with viral replication centers. We have also determined that
the SCM1 protein, which is part of the cohesin complex as well as the
ATM DNA damage pathway, is activated in HPV positive cells and
localised to HPV replication foci. In these HPV positive cells, SCM1 is
found in complexes with ATM, CHK2 and g-H2AX and ist activation
is necessary for genome amplification. Our studies identify important
mechanisms that regulate the productive phase of the viral life cycle in
differentiating cells.
4
Session abstracts
MA01 – Mon 1600
Offered paper The Epstein–Barr virus lytic cycle is regulated by human
Notch ligand-expressing stromal cells
Claire Shannon-Lowe, Martin Rowe
The University of Birmingham, Birmingham, UK
Epstein–Barr virus (EBV) is a gamma-herpesvirus that remains latent in
resting memory B cells for the lifetime of the host, with a very restricted
gene expression profile. However, EBV infection of B cells in vitro initiates
a growth transformation programme, generating lymphoblastoid cell
lines, in which spontaneous lytic replication is frequently observed. In
vivo, the growth transformation programme is only observed during
infectious mononucleosis and lytic replication is observed very rarely. We
suggest both the virus lytic replication and the growth transformation
programme may be regulated by the interaction between infected B
cells and stromal cells. Using a model B cell line, expressing only the virus
maintenance protein (EBNA1), grown on mouse stromal cells expressing
human notch ligand Delta-like-1, we have shown notch ligation strongly
inhibits EBV lytic cycle. Upon removal of notch ligation, (removal from
stromal cells or gamma-secretase inhibitor), EBV lytic cycle spontaneously
occurs in up to 40% of cells. We observed an immediate reduction in
the presence of the transcriptionally-active intracellular Notch (Notch
IC) followed by a reduction in Zeb2, a negative regulator of BZLF1. We
have also demonstrated a significant change in the growth transformation
programme of resting B cells when cultured on stroma.
MA01 – Mon 1615
Offered paper Investigating mechanisms of influenza polymerase host
adaptation
Anna V Cauldwell, Olivier Moncorgé, Hongbo Zhou,
Wendy Barclay
Imperial College London, London, UK
Typical avian influenza A viruses do not replicate efficiently in mammals.
Many host adaptive mutations have been mapped to the polymerase
complex (PB1, PB2 and PA) as well as the nucleoprotein (NP) which are
required for transcription and replication of influenza viral RNA, however
the mechanism/s of host adaption are unknown. Polymerase activity is
routinely measured using a cell-based activity assay. Using this approach
we suggest that PB2 humanising mutations don’t enhance polymerase
activity by a universal mechanism and furthermore are not all hostspecific as some can enhance activity in avian as well as mammalian cells.
We wish to address why certain mutations which enhance influenza
polymerase activity in a cell-based polymerase assay aren’t selected for in
nature by the virus and to explore whether the polymerase assay truly
reflects viral fitness, as well as influenza virus transcription and replication.
To investigate this we have created a series of viral variants altered in
polymerase genes using a reverse genetics approach and have carried
out virological assays whose results can be compared with data obtained
during cell based polymerase assays. Through this approach we hope to
further elucidate the various selective pressures that drive host adaptation.
MA01 – Mon 1630
Offered paper Modulation of enhancer looping and differential gene
targeting by Epstein–Barr virus transcription factors directs epigenetic
reprogramming
Michael J McClellan1, C David Wood1, Opeoluwa Ojeniyi1,
Aditi S Kanhere2,4, Richard G Jenner2, Aaron Arvey3, Tim Cooper1,
Richard D Palermo1,5, Michelle J West1
1
University of Sussex, Brighton, UK, 2University College London, London,
UK, 3Howard Hughes Medical Institute, New York, USA, 4University of
Birmingham, Birmingham, UK, 5London Research Institute, London, UK
Epstein–Barr virus (EBV) epigenetically reprogrammes host
B-lymphocytes creating immortal cells to facilitate viral persistence.
Host-cell transcription is deregulated principally through the cooperative
actions of EBV EBNA 2, 3A, 3B and 3C, with cellular genes deregulated
by unique EBNA 3 subsets through largely unknown mechanisms.
Importantly we have elucidated the mechanism of gene targeting by
specific EBNA 3 family members demonstrating that this is driven by
differential binding of EBNA 3A, 3B or 3C to regulatory elements in both
a gene and cell-type specific manner. Strikingly, we also detect extensive
targeting of common sites by EBNA 2 and 3 proteins, predominantly
in long-range enhancers. Investigating shared sites at the novel targets
WEE1 and CTBP2 we have demonstrated that EBNA 3 proteins
repress transcription by modulating enhancer-promoter loop formation
establishing repressive chromatin hubs or preventing active hub assembly.
Re-ChIP analysis revealed that EBNA 3 proteins do not bind shared sites
simultaneously with EBNA 2 but compete for binding thus modulating
enhancer-promoter interactions. At a unique intergenic EBNA 3A and
3C binding site at the ADAM28/ADAMDEC1 locus we found that
enhancer-promoter looping directed epigenetic repression of both
genes. Our findings provide a paradigm for host-cell reprogramming
through modulation of enhancer-promoter interactions.
MA01 – Mon 1645
Offered paper Mechanism of superior B cell immortalisation activity of
type 1 Epstein–Barr virus
Stelios Tzellos1, Laila Cancian1, Michael J McClellan2,
Michelle J West2, Paul J Farrell1
1
Imperial College London, London, UK, 2University of Sussex, Brighton, UK
EBV isolates worldwide are grouped into type 1 or type 2 based on
the EBNA-2 gene sequence. Type 1 EBV is more efficient at B cell
transformation, a property previously mapped to the EBNA-2 locus.
We showed that the superior ability of type 1 EBNA-2 to sustain
proliferation of a lymphoblastoid cell line (LCL) is mostly determined by
its C-terminal region. We have now shown that D442 of type 1 EBNA-2
plays a key role. Converting this single residue of type 2 EBNA-2 from
serine to the aspartate found in type 1 EBNA-2 confers the strong
growth-promoting phenotype in the EREB2.5 LCL assay. The mechanism
of the greater transformation efficiency of type 1 EBV involves differential
regulation of EBNA-2 target genes. One such cellular target, CXCR7,
is more strongly induced by type 1 EBNA-2 and is required for EBVLCL proliferation. 5’ RACE has now been used to characterise the
transcription start site for the CXCR7 promoter transcribed in response
to EBNA-2. ChIP-seq has identified novel sequences bound by EBNA2 100kb upstream of the CXCR7 transcription start. We are now
using ChIP assays to analyse the mechanism of differential regulation of
CXCR7 by type 1/type 2 EBNA-2.
MA01 – Mon 1700
Offered paper HIV-1 cell–cell spread: virus-induced T-cell remodeling
and organelle polarisation for efficient and persistent transmission
Elisabetta Groppelli, Shimona Starling, Clare Jolly
University College London, London, UK
HIV-1 dissemination to target CD4+-T cells is mediated by two
mechanisms: virion release followed by fluid-phase diffusion and the
considerably more efficient mode of direct cell-cell spread. Cell-cell
spread contributes to enhancing viral diversity and permits virus to
evade neutralising antibodies and anti-retroviral therapy, therefore
greatly contributing to persistence of infection. Cell-cell spread occurs
at supramolecular structures called Virological Synapses that form at
intercellular contacts where HIV-1 preferentially assembles and buds
towards a susceptible target cell. Polarisation of viral egress at the VS is
associated with striking recruitment of the cellular secretory apparatus,
the microtubule organising centre and mitochondria within the infected
T-cell that was not seen in the target cell, nor in the absence of cell-cell
contact. This suggests that viral egress at the VS is regulated. Using a
combination of approaches including immunofluorescence microscopy
of live cells infected with replication competent HIV-1, RNAi and
pharmacological inhibitors, we show that HIV-1 cell-cell spread is
significantly inhibited when organelle trafficking to the VS is impaired and
5
Session abstracts
also when organelle functions are disrupted. This work contributes to
the understanding of the mechanisms used by HIV-1 to hijack the cellular
machinery and specifically direct its most efficient and immune-evasive
mode of transmission.
MA01 – Tue 0900
Oligoclonality and leukaemia in HTLV-1 infection
Charles R M Bangham
Department of Medicine, Imperial College, London, UK (Email: c.bangham@
imperial.ac.uk)
HTLV-1 persists in vivo by driving oligoclonal proliferation of infected
T cells. In 5% of HTLV-1-infected individuals this process culminates in
malignant transformation of one or more T-cell clones, resulting in the
aggressive and usually fatal disease adult T-cell leukaemia/lymphoma
(ATLL). However, it remains unknown what determines the selective
expansion of certain HTLV-1-infected T cells in both non-malignant and
malignant infection.
We hypothesise that the genomic integration site (IS) of the HTLV-1
provirus determines the pattern and intensity of spontaneous proviral
expression; the proviral expression in turn determines the abundance
of the clone and the rate of CTL-mediated killing. We aim to identify
the factors that determine the integration site targeting, expression and
abundance of the HTLV-1 provirus in natural infection. I shall present
data that lead to the following conclusions:
• Each infected individual carries about 100,000 distinct clones of
HTLV-1-infected T cells – about 1000 times more than previously
believed
• HTLV-1 integration in the host DNA is not random: the virus
preferentially integrates into host DNA within 100 to 1000 bases of
specific transcription factor binding sites, notably P53 and STAT1.
• The frequency of latency or spontaneous HTLV-1 expression in an
infected T-cell clone is determined by the genomic integration site
of HTLV-1 in that clone.
The results suggest that transcriptional interference and chromatin
remodelling are critical determinants of proviral latency in natural HTLV1 infection. These results open the way to mechanistic tests of the
molecular mechanisms of targeting, expression and clonal proliferation
in vivo.
MA01 – Tue 0930
The role of miR-122 in the hepatitis C virus lifecycle
Stanley M Lemon
Departments of Medicine and Microbiology & Immunology, Lineberger
Comprehensive Cancer Center, University of North Carolina at Chapel Hill,
Chapel Hill, NC 27517 USA
Hepatitis C virus (HCV) is a positive-strand RNA virus that is strongly
associated with hepatocellular carcinoma. We are interested in
understanding why its replication is dependent upon miR-122, a liverspecific microRNA that binds to two sites near the 5’ end of the
(+)-strand RNA genome. We have shown that miR-122 binds HCV
RNA in association with Argonaute 2, stabilising the viral genome and
slowing its decay both in cell-free reactions and in infected cells. More
recent work has demonstrated that miR-122 protects the viral RNA
from degradation by the cellular 5’ exonuclease, Xrn1, and that Xrn1 and
the viral RNA replicase compete to determine the abundance of HCV
RNA in infected cells. However, while Xrn1 knockdown and miR-122
supplementation have equal, redundant, and non-additive effects on the
rate of viral RNA decay, Xrn1 knockdown does not rescue replication
of a viral mutant defective in miR-122 binding. Thus, while miR-122
enhances HCV replication by protecting the viral RNA against Xrn1, it has
additional, essential function(s) in the viral lifecycle. Since supplementing
infected cells with miR-122 neither alters polysome profiles of HCV RNA,
nor enhances viral protein expression in Xrn1-depleted cells, we suspect
miR-122 functions directly during viral RNA synthesis.
MA01 – Tue 1000
Lymphatic reprogramming in cancer – cues provided by virus–host
interactions
Pirita Pekkonen1, Elisa Kaivanto1, Raquel Diaz Martinez1, Olga Tatti3,
Kari Alitalo4, Kaisa Lehti3, Päivi M Ojala1,2
1
Institute of Biotechnology, University of Helsinki, Finland; 2Foundation for the
Finnish Cancer Insititute, Helsinki, Finland; 3Research Programs Unit, Genome
Scale Biology, University of Helsinki, Finland; 4Research Programs Unit,
Translational Cancer Biology, University of Helsinki, Finland
Kaposi sarcoma herpesvirus (KSHV) is a human tumour virus and the
causative agent of KS. However, the definitive cellular origin of the KS
tumour cells and mechanisms of endothelial cell transformation remain
unresolved. We recently showed that Kaposi sarcoma herpesvirus
reprograms lymphatic endothelial cells into a mesenchymal and invasive
cell type via endothelial-to-mesenchymal transition. This revealed a novel
viral oncogenesis mechanism where viral proteins, vFLIP and vGPCR,
co-opt the Notch pathway to modify the cellular microenvironment
more permissive for viral gene expression. Reprogramming involved
activation of PDGF receptors and was dependent on MT1-MMP activity.
Transcriptome analyses of reprogrammed cells showed induction of a
number of transcription factors and genes involved in invasion. We are
currently investigating the KSHV-induced lymphatic reprogramming at the
level of chromatin and testing its contribution to initiation and progression
of Kaposi sarcoma. This intriguing finding of Notch-mediated alterations of
lymphatic plasticity has prompted us to address if reprogramming would
occur also in other, non-viral human cancers where lymphangiogenesis
is important. Our work aims to contribute to deeper molecular
understanding of endothelial plasticity and mechanisms of reprogramming.
MA01 – Tue 1100
Infection and cancer: clues from people with HIV and other immune
deficiencies
Andrew E Grulich
Head, HIV Epidemiology and Prevention Program, The Kirby Institute,
University of New South Wales, Sydney, Australia
Immune deficiency offers a window through which to examine the
infection-cancer relationship. Simply stated, if a cancer is caused by the
consequences of infection, then it is likely to occur at increased rates in
people with immune deficiency. Over the past decade, epidemiological
research has shown that a wide variety of mainly infection-related cancers
occurs at increased rates in people with immune deficiency. These
include cancers related to Epstein–Barr virus (Non Hodgkin Lymphoma,
Hodgkin’s Disease), human herpesvirus-8 (Kaposi’s sarcoma), human
papillomavirus (anogenital and oropharyngeal cancers), hepatitis B and
hepatitis C virus (liver cancer) and helicobacter pylori (stomach). A range of
other cancers appear to occur at slightly increased rates including cancers
of the lung and lip, non-melanoma skin cancer, melanoma, myeloma and
leukaemia. Most of the common epithelial cancers, including cancers of
the colorectum, breast, prostate and ovary, do not occur at increased
rates in people with HIV or in organ transplant recipients, suggesting a
lack of association with infection. In some cases, the increased cancer
risk associated with immune deficiency is reversible. In people with HIV,
effective anti-retroviral therapy reduces the risk of many types of infectionrelated cancer, but the pattern of risk reduction varies by type of cancer
and by infective cause. Similarly, in kidney transplant recipients, cessation
of immune suppression reduces risk of some but not all infection-related
cancer. ecently completed epidemiological studies suggest a broader than
previously appreciated role of infection and the immune response system
in the causation of cancer.
MA01 – Tue 1130
Offered paper KSHV-encoded vIRF3 modulates MHC-II antigen
presentation through CIITA-dependent and -independent mechanisms:
implications for oncogenesis
Jianmin Zuo
School of Cancer Sciences, Birmingham, UK
6
Session abstracts
circulation showed that reovirus could be detected in plasma and blood
mononuclear, granulocyte, and platelet cell compartments after infusion.
Despite the presence of neutralising antibodies before viral infusion in all
patients, replication-competent reovirus that retained cytotoxicity was
recovered from blood cells but not plasma, suggesting that transport
by cells could protect virus for potential delivery to tumors. Analysis of
surgical specimens demonstrated greater expression of reovirus protein
in malignant cells compared to surrounding normal liver tissue. Hence,
reovirus could be protected from neutralising antibodies after systemic
administration by immune cell carriage, which delivered reovirus to
tumour. We believe the insights provided by such translational studies
will be critical in realising the full immunotherapeutic and cytotoxic
potential of OV treatment for cancer. Further translational clinical trials
with other OV and in other tumours are currently in development.
Evasion of immune T cell responses is crucial for persistent viruses
to establish a normal carrier state. KSHV, which is carried as lifelong
infections and can cause various tumours, the immune evasion strategies
can be relevant in the context of tumourigenesis. In this study, we
report that the vIRF3 latent viral gene expressed in KSHV-related
tumours functions as a potent immunevasin. Expression of vIRF3 downregulates surface MHC-II DR expression with a slow kinetics but, more
importantly can substantially inhibit the recognition by KSHV-specific
CD4 T cells. This property of vIRF3 is only partly due to its ability to
inhibit the transcription of CIITA, and thus MHC-II expression; an
as yet unidentified CIITA-independent mechanism is also involved
in qualitatively modulating the availability of specific peptide/MHC-II
complexes at the cell surface. Consistent with these observations, the
vIRF3 expressing KSHV-associated PEL lines are generally resistant to
recognition by KSHV-specific CD4 T cells. Interestingly, some PEL lines
exhibit small subpopulations with lower vIRF3 expression that can be
recognised. These data implicate vIRF3 as a critical determinant of the
MHC-II antigen presentation function in KSHV-associated PEL that is
likely to be important in the pathogenesis of these tumours.
MA01 – Tue 1145
Offered paper The identification and characterisation of novel
phosphorylation sites of the HCV protein NS5A
Douglas Ross-Thriepland, Yutaka Amako, Mark Harris
The non-structural 5A (NS5A) protein of HCV acts at multiple stages of
the HCV lifecycle including RNA replication, assembly and virus release. It
is extensively phosphorylated and this is proposed to regulate its functions.
To identify phosphorylation sites we used tryptic digestion and mass
spectrometry on NS5A purified from Huh7 cells stably harbouring an
HCV sub genomic replicon. Four residues of NS5A were unambiguously
identified as phosphor-acceptors -S146, S222, S225 and T348 . Our data
also showed that S222 and S225 were contained within a short serinerich region of NS5A that was multiply phosphorylated, as the tryptic
digest revealed a mixture of species containing up to 7 phosphorylated
residues. Disruption of phosphorylation sites by mutation to either alanine
(phosphoablatant) or aspartate (phosphomimic) produced a variety of
phenotypes, in particular mutation of serines 222/225 to alanine resulted in
a significant impairment of replication. Utilising live cell confocal microscopy
we investigated whether phosphorylation sites affect the trafficking
of NS5A in infected cells and identified pS225 as a key determinate
in the distribution of NS5A. Lastly we generated a phospho-specific
antibody against pS222 and used this to further investigate the role of this
phosphorylation event in the virus lifecycle
MA01 – Tue 1400
Oncolytic viruses for cancer therapy
Alan Melcher
Leeds Institute of Molecular Medicine, Section of Oncology & Clinical
Research, University of Leeds, Wellcome Trust Brenner Building, Level 5, St
James’s University Hospital, Beckett Street, Leeds, UK
Oncolytic viruses (OV), which have now reached Phase III clinical trial
testing, were initially designed to kill tumour cells directly. However,
OV can also trigger an anti-tumour immune response, even in the
absence of viral replication and direct cell lysis. Our laboratory has mainly
focused on reovirus, a naturally occurring non-pathogenic dsRNA virus,
which is thought to specifically target ras-activated tumour cells. In both
mouse and human systems, reovirus infection of melanoma, colorectal
and other cancers is cytotoxic and supports priming of an adaptive,
specific cytotoxic T cell response, as well as innate activation of NK
cells mediated by type 1 interferons. In parallel with standard pre-clinical
and early clinical development, we have completed a trial with defined
biological endpoints, in which patients having resection of colorectal
cancer liver metastases were treated with a single cycle of intravenous
reovirus prior to their planned surgery. Tracking the viral genome in the
MA01 – Tue 1430
Offered paper Epstein–Barr virus-associated cancer: exploring factors
determining the effectiveness of immune-based therapies
Tracey Haigh, Lindsay Paling, Martin Rowe, Graham S Taylor
The University of Birmingham, Birmingham, West Midlands, UK
Background Epstein–Barr Virus (EBV) is associated with several different
types of cancers including epithelial carcinomas and lymphomas of B cells
and T/NK cells. Since all are MHC-II-positive and express the viral protein
EBNA1 there is considerable interest in the ability of EBNA1-specific
CD4+ T-cells to recognise and kill EBV-infected and transformed cells.
Methods CD4+ T cell clones specific for different EBNA1 epitopes
were tested for their ability to recognise EBV-positive B-lymphoblastoid
cell lines (LCLs) or T/NK lymphoma cell lines using a variety of
immunological assays.
Results Remarkably, an EBNA1 CD4+ T cell epitope not presented
by LCLs was strongly presented by three T/NK lymphoma lines.
Furthermore, two T cell clones specific for this epitope, both able
to efficiently kill LCLs, differed markedly in their ability to kill a T/
NK lymphoma line. This result reflected differences in the cytolytic
mechanisms utilised by the CD4+ T cell clones; further work
demonstrated some T/NK lymphoma lines are completely resistant to
being killed by the Fas/FasL pathway.
Conclusion These results show that both the antigen processing
environment within the target cell and the cytolytic mechanisms used by
effector T cells are important factors that could influence the therapeutic
effectiveness of T-cell based immunotherapies.
MA01 – Tue 1445
Offered paper Oncolytic reovirus as a novel therapy for hepatitis C
virus-associated liver cancer
Adel Jebar1,2, Fiona Errington-Mais1,4, Daniel Swinson3, Matt Coffey5,
Peter Selby2,1, Alan Melcher1,4, Stephen Griffin1,4
1
University of Leeds, Leeds, West Yorkshire, UK, 2Cancer Research UK, Leeds,
West Yorkshire, UK, 3Leeds Teaching Hospitals NHS Trust, Leeds, West
Yorkshire, UK, 4Leeds Institute of Molecular Medicine, Leeds, West Yorkshire,
UK, 5Oncolytics Biotech (Barbados) Inc., Worthing, Christ Church, Barbados
Hepatocellular carcinoma (HCC) is the 3rd leading cause of cancer
deaths worldwide with a 5-year survival rate less than 10%. Over 30% of
HCC cases are attributable to infection with Hepatitis C virus (HCV). The
pan-tyrosine kinase inhibitor, Sorafenib, comprises standard of care for
patients with advanced disease. Interestingly, Sorafenib shows enhanced
benefit for HCV-HCC patients, potentially linked to dual anti-HCV/HCC
effect. Oncolytic Reovirus is a wild-type dsRNA virus that selectively
replicates in malignant cells, stimulating both direct and immune-mediated
killing. Here, we demonstrate the potential of Reovirus to enhance
HCV-HCC therapy via three distinct mechanisms. First, Reovirus displays
superior killing of HCV +ve/-ve HCC cell lines compared with other
oncolytic viruses, and is additive with Sorafenib effects. Second, Reovirus
induces high levels of IL-28b production from HCC lines, a critical
mediator of HCV clearance. Accordingly, Reovirus-conditioned HCC cell
7
Session abstracts
line supernatants potently diminish HCV replication and this is again cooperative with Sorafenib, which also displays anti-HCV effects in culture.
Finally, Reovirus activates liver-resident Natural Killer Cells and overcomes
the immunosuppressive effects of Sorafenib, enhancing killing of HCC
lines. These results indicate that combination Reovirus and Sorafenib may
yield significant clinical benefit.
MA01 – Tue 1500
HPV vaccines – not just the cervix
Margaret Stanley
Department Pathology, University of Cambridge, Cambridge, UK
Viral infections cause at least 15% of all human cancers; one of the most
important oncogenic viruses is the human papillomavirus (HPV) a causal
agent in 5% of all malignancies. The unfolding of the HPV story started in
the 1970’s with the recognition that HPVs were a large family of viruses
that included cancer causing types. It has resulted in the development
of two prophylactic virus like particle (VLP) vaccines using sophisticated
recombinant molecular techniques and protein expression. Both vaccines
target infection by the oncogenic HPV’s 16 and 18 and one also targets
the low risk HPVs 6 and 11 that cause genital and laryngeal warts.
Population effectiveness is now being demonstrated for these vaccines in
countries with high vaccine coverage. The vaccines are well tolerated and
highly immunogenic generating serum neutralising antibody that persists
for at least 9 years and a robust recall response at 60 months post
vaccination. At present the assumption is that the protection achieved
by VLP vaccines against HPV induced ano-genital pathology is mediated
via serum neutralising IgG and this is consistent with what is known of
the mechanism of HPV infection in the genital tract. Emerging evidence
shows that very low antibody concentrations are protective and at the
present there is no immune correlate of protection.
MA01 – Tue 1600
Offered paper Induction of p16INK4a is the major barrier to
proliferation when Epstein–Barr virus (EBV) transforms primary B
cells into lymphoblastoid cell lines
Rob E White1, Lenka Skalska1, Gillian A Parker1, Alison Sinclair2,
Kostas Paschos1, Martin J Allday1
1
Imperial College London, London, UK, 2University of Sussex, Brighton, UK
Epstein–Barr virus (EBV) is implicated in around 1% of cancers worldwide.
In vitro EBV transforms primary B cells into lymphoblastoid cell lines (LCLs).
The EBV nuclear antigen 3C (EBNA3C) is essential for this transformation.
LCLs lacking functional EBNA3C undergo cell cycle arrest previously
attributed to the induction of the CDKN2A-encoded tumour suppressors
p16INK4a and/or p14ARF. To explore the role of p16INK4a as a barrier to B cell
transformation, we infected primary B cells from an individual homozygous
for a CDKN2A deletion that disrupts the function of p16INK4a but not
p14ARF. LCLs established with recombinant EBV expressing conditional
EBNA3C (EBNA3C-estrogen receptor fusion) that lacked functional
p16INK4a exhibited the same epigenetic regulation of the CDKN2A locus
as p16-competent LCLs, but failed to alter the proliferation rate when
EBNA3C was inactivated. Hence repression of INK4a is a major function of
EBNA3C in EBV-driven LCL proliferation. Microarray analysis allowed the
identification of EBNA3C-regulated genes without changes in proliferation
confounding the analysis. Infections of normal primary B cells with EBNA3Cdeficient EBV revealed that EBNA3C is necessary to overcome an EBVdriven increase in p16INK4a expression and concomitant block to proliferation
2-4 weeks post-infection. Thus EBNA3C is unnecessary for the outgrowth
of LCLs from p16INK4a-null B cells.
MA01 – Tue 1615
Offered paper Overturning the epigenetic silencing of Epstein–Barr
virus genome in cancer cells
Sharada Ramasubramanyan1, Kay Osborne1, Richard Jenner2,
Aditi Kanhere2, Kirsty Flower1, Alison Sinclair1
1
University of Sussex, Brighton, UK, 2UCL, London, UK
The ability of Epstein–Barr Virus to establish latency in cancer cells
allows it to evade the immune system. One distinguishing characteristic
is the lack of transcription of the majority of viral genes. We explored
the chromatin context at key EBV lytic cycle promoters and the origins
of lytic replication during latency and lytic replication and identified a
repressive heterochromatin-like environment (tri-methylation of histone
H3 at lysine 9 (H3K9me3) and lysine 27 (H3K27me3) encompassing the
key early lytic regulatory regions during latency. Entry into the lytic cycle
is co-ordinated by the viral transcription factor, Zta (BZLF1, ZEBRA,
EB1). We used ChIP-sequencing to identify Zta binding sites across the
EBV genome. Sequential ChIP analyses reveal that Zta interacts with
the repressive chromatin. Strikingly, a genome-wide comparison of
Zta binding pre-and post-viral genome replication revealed a dramatic
reduction in the association of Zta with ZREs that contain CpG motifs
(CpG ZREs). We show that this is linked to reduced methylation of
these sites. The repressive epigenetic state of the EBV genome aids
cancer persistence by preventing viral replication. However, once lytic
replication has been initiated and Zta is expressed, Zta plays a crucial
role overturning the epigenetic environment.
MA01 – Tue 1630
Offered paper The inflammatory cell infiltrate in primary Merkel cell
carcinoma
Rachel Wheat1, Claudia Roberts1,2, Tim Waterboer3, Lalit Pallan1,2,
Jane Steele4, Jerry Marsden2, Michael Pawlita3, Andrew Hislop1,
Neil M. Steven1,2, David J. Blackbourn1
1
School of Cancer Sciences and CR UK Centre for Cancer Research, College
of Medical and Dental Sciences, The University of Birmingham, Birmingham,
UK, 2University Hospitals Birmingham NHS Foundation Trust, Queen
Elizabeth Hospital, Birmingham, UK, 3German Cancer Research Center
(DFKZ), Heidelberg, Germany, 4Human Biomaterials Resource Centre,
College of Medical and Dental Sciences, The University of Birmingham,
Birmingham, UK
Merkel cell carcinoma (MCC) is an aggressive malignancy associated with
Merkel cell polyomavirus (MCPyV). Here, we describe the inflammatory
and immune cells within primary MCC (20 patients) and characterise
the CD8+ T lymphocytes. Immunohistochemical staining (10 patients)
revealed monotonous tumour sheets crossed by septa including CD34+
and D240+ blood and lymphatic vessels, respectively. Most tumours
stained for CD68+ macrophages, CD4+, CD3+, CD8+ cells and
CD20+ B lymphocytes. All these cells were predominantly located at the
tumour periphery and within septa. Given their reported significance as a
prognostic marker, CD8+ lymphocytes were studied (13 patients’ MCC)
by multicolour immunofluorescent staining and confocal microscopy.
Co-staining tumour for CD8+ cells with markers for tumour cells
(CK20 and MCPyV large T) and vessels (CD34 or D240) demonstrated
lymphocytes extravasate, but fail to penetrate the tumour mass.
Granzyme B+ cells, with presumed cytotoxic potential, were present,
but were not CD8+. CXCR3, which might drive lymphocytes into
tissue sites of inflammation, was absent on CD8+ cells. However, the
lymphocyte chemokine CXCL12 was abundant where the immune cells
were located. In summary, in most MCC, CD8+ cells are present but
appear functionally deficient and stalled in perivascular regions, lacking
the properties to contact and kill malignant cells.
MA01 – Tue 1645
Offered paper Entosis in the liver and hepatitis C virus infection: more
than a tumour invasion mechanism
Zania Stamataki, Rebecca Rose, Omar Qureshi, David Adams,
Jane McKeating
Hepatitis C virus (HCV) infection leads to progressive liver disease and
is associated with extrahepatic manifestations including hepatocellular
carcinoma, a cancer with increasing incidence in the UK. Understanding
lymphocyte-hepatocyte interactions is important for the design of
8
Session abstracts
Metabolic interactions at the
host–pathogen interface
effective antiviral and tumour-targeting therapies. Recent studies have
identified the process of entosis, whereby one cell invades another, as
a cell behaviour that could promote tumour progression by leading to
aneuploidy. This form of cell engulfment involving live cells also leads to
polyploidy, as internalised cells disrupt cytokinesis of their engulfing cell
hosts (Krajcovic and Overholtzer M, Cancer Res. 2012). We report the
internalisation of live lymphocytes by healthy primary hepatocytes and
hepatocellular carcinoma cell lines HepG2 and Huh-7 and explore the
consequences of this internalisation for lymphocytes and hepatocytes
in vitro. Hepatitis C virus infection perturbs entosis in infected Huh-7
cells. Our experiments provide a mechanism for this perturbation and
investigate the implications for lymphocyte and hepatocyte biology.
MA02
MA02 – Mon 0900
Listeria speedy ways to pathogenesis
J.Vazquez-Boland, M. Scortti, J. Bell, L. Han, S. Alvarez, R.J. Cai
Microbial Pathogenesis Unit, School of Biomedical Sciences, University of
Edinburgh, Edinburgh, UK
The gram-positive pathogen Listeria monocytogenes is the causative
agent of listeriosis, an invasive foodborne infection with severe clinical
manifestations including meningoencephalitis, septicemia, stillbirth
and neonatal sepsis. Listeria virulence depends on the ability of these
bacteria to survive and proliferate within macrophages and a variety
of non-professional phagocytes. In contrast to other major intracellular
pathogens such as Mycobacterium tuberculosis or Salmonella, L.
monocytogenes replicates in the cytosol and not in a membranebound vacuole. Internalisation into host cells permits Listeria to evade
extracellular host defenses while vacuolar escape is essential to prevent
lysosomal killing. After replication in the cytosol, Listeria avoids the
cytotoxic immune response directed against infected cells via a “runaway”
strategy involving actin-based cell-to-cell spread. Listeria possesses
dedicated virulence factors to ensure the efficient completion of each
of the key steps of its intracellular lifestyle.This presentation will discuss
some of the L. monocytogenes strategies that provide a competitive
edge in the race with the host immune response, with a focus on the
metabolic determinants of intracellular growth and the interference with
endocytic traffic.
MA01 – Tue 1700
Offered paper Kaposi’s sarcoma-associated herpesvirus enhanced K+
channel currents are required for virus reactivation
Mark L Dallas1, David J Hughes2, Chris Peers2, Adrian Whitehouse2,
Jamel Mankouri2
1
University of Reading, Reading, UK, 2University of Leeds, Leeds, UK
Infection with Kaposi’s sarcoma-associated herpesvirus (KSHV/
HHV8) frequently results in the development of fatal tumors in
immunocompromised patients. Whilst numerous cellular proteins are
known to be required for KSHV reactivation from latency, the role
of host cell ion channels in this process have not been investigated.
Ion channels play a key role in all aspects of cell physiology and have
recently been implicated in the mechanisms underlying persistent
viral infection. The current study investigated whether ion channel
function contributes to the KSHV lifecycle. Using a panel of ion channel
modulators we demonstrate that K+ channel blockers, but not inhibitors
of calcium, sodium or chloride channels, inhibit KSHV entry into the lytic
replication cycle. Electrophysiological approaches demonstrated that
KSHV reactivation results in an increased outward K+ current which was
selectivity inhibited by nanomolar concentrations of margatoxin (MgTX)
a specific inhibitor of Kv1.3 channels. Furthermore, MgTX drastically
reduced KSHV reactivation, confirming the function of this channel as
necessary for virus replication. This study is the first to demonstrate the
role of a host cell ion channel in the KSHV lifecycle and establishes Kv1.3
inhibition as a novel anti-KSHV target.
MA01 – Tue 1715
Offered paper Pre-clinical studies on oncolytic HSV1716 in
hepatocellular carcinoma
Lynne Braidwood, Kirsty Learmonth, Joe Conner
Virttu Biologics Ltd, Glasgow, UK
Oncolytic HSV1716, lacking the neurovirulence factor ICP34.5, has
exquisite replication competence for cancer cells and has been used in
safety trials in glioma, melanoma, H&NSCC and paediatric non-CNS
solid tumours. In total 77 patients have received HSV1716 to date
with no evidence of toxicity, no spread to surrounding normal tissue,
no shedding in patients and the selectivity of HSV1716 for replication
only in tumour cells has been demonstrated. We have assessed the
potential for therapeutic use of HSV1716 in hepatocellular carcinoma
(HCC) in non-clinical studies. HSV1716 showed high levels of replication
competence in vitro in three human HCC cell lines, HuH7, HepG2-luc
and Hep3B. Using in vitro combination studies, HSV1716 was synergistic
with standard of care HCC drug doxorubicin. HSV1716 demonstrated
strong efficacy signals in subcutaneous HuH7 and HepG2-luc xenografts
in nude mice after intratumoural or intravenous administration. In
the HuH7 model the intravenously injected virus replicated rapidly
immediately after tumour localisation and very high titres of virus (c1x108
pfu/ml) were recovered from tumour extracts at 72 hours post injection.
Our preclinical data have demonstrated excellent tumour uptake with
prolific replication and potentially synergistic interactions with approved
HCC drug and therefore strongly support clinical studies of HSV1716 in
HCC.
MA02 – Mon 0930
Metabolic determinants of infection in Salmonella and
Campylobacter
P A Barrow, M A Jones
School of Veterinary Medicine and Science, University of Nottingham,
Loughborough, Leicestershire, UK
The ability of Salmonella and Campylobacter to colonise the intestine
of chickens is the key stage leading to food poisoning. Arguments have
fluctuated between colonisation being solely or mainly a metabolic
attribute or whether a physical relationship with the host may be
involved. Rather than study mutant libraries as we have done previously
we decided to look at patterns of gene expression of S. Typhimurium
in the caeca of newly hatched chickens compared with those in nutrient
broth cultures using microarray technology. Levels of transcription,
translation, and cell division in vivo were lower than those in vitro. The
Salmonella appeared to be using carbon sources, such as propionate,
1,2-propanediol, and ethanolamine, melibiose and ascorbate. Amino acid
starvation also appeared to be a factor. Bacteria in the lumen showed
upregulation of a number of fimbrial and SPI-3, suggesting a close
physical association with the host. In bacteria harvested from the cecal
mucosa levels of transcription, translation, and cell division were higher
and glucose may also have been used as a carbon source.
The data for C. jejuni suggests simultaneous use of different electron
acceptors suggesting a different metabolic profile to Salmonella.
MA02 – Mon 1030
Insights from Plasmodium metabolomics
Manuel Llinás
Department of Molecular Biology & Lewis-Sigler Institute of Integrative
Genomics, Princeton University, USA
The genome of Plasmodium falciparum indicates that the metabolic
pathways utilised by this organism are highly unique. Recent efforts to
comprehensively examine the biology of P. falciparum have focused on
transcriptome and proteome analysis to gain insight into Plasmodiumspecific pathways. The third crucial component that remains to be
9
Session abstracts
established is the metabolome: the complement of small-molecule
metabolites and their relative levels. Our lab has begun to characterise
various aspects of parasite metabolism using high accuracy massspectrometry to simultaneously measure metabolites from complex
cellular extracts from parasite-infected cells. The approaches we are using
allow us to assay various aspects of the P. falciparum metabolome. One
approach has been to examine the interaction of Plasmodium with the
host red blood cell using targeted measurements of specific metabolites
shared with the host erythrocyte. We are also using 13C and 15N isotopic
labeling experiments to directly trace carbon flux through known
biochemical pathways. Finally, we are using metabolite measurements
to map genetic control of metabolism by assaying global metabolite
patterns in the parents and progeny of a Plasmodium falciparum genetic
cross. Results from these studies are beginning to unravel the divergence
of metabolism in P. falciparum and promise to provide unique avenues
for future drug intervention strategies.
MA02 – Mon 1100
Offered paper Metabolomics approaches to studying the intracellular
nitrogen metabolism of Listeria monocytogenes
Mohammed Shahraz, David Corbett, Warwick Dunn, Roy Goodacre,
Ian Roberts
The University of Manchester, Manchester, UK
Listeria monocytogenes is a foodborne intracellular pathogen that
following invasion of the cell it escapes the phagosome and replicates in
the cytoplasm. A key challenge to L. monocytogenes is to acquire nutrients
from the host. We have been using combination of metabolomics and
cellular microbiology to study nitrogen metabolism of L. monocytogenes
in infected epithelial cells. Using GC-MS we were able to establish that
there is a significant change in the metabolome of infected HeLa cells
10 hours post infection. By univariate analysis we were able to show
that out of 231 identified metabolites, 53 metabolites were showing
significant changes at 10 hours post infection. There was a significant
reduction in the levels of a number of nitrogenous compounds including
glutamine, lysine, alanine and valine in keeping with these amino acids
being depleted through the intracellular replication of L. monocytogenes.
In contrast a number of nitrogenous metabolites such as spermine and
amino acids tryptophan, leucine and aspartic acid were significantly
increased. We have now extended these studies by studying the ability
of mutants defective for nitrogen assimilation to grow inside HeLa cells.
This use of metabolomics offers an exciting route to study the physiology
of the infected cell.
MA02 – Mon 1115
Offered paper A new link between virulence and methionine
biosynthesis in the potato pathogen Pectobacterium atrosepticum
MArion F Cubitt1,2, Peter E Hedley2, Emma Campbell2, Ian K Toth2,
George P C Salmond1
1
University of Cambridge, Cambridge, UK, 2James Hutton Institute, Dundee,
UK
Pectobacterium atrosepticum is an agriculturally significant Gram-negative
phytopathogen which infects potato, causing soft rotting of tubers
and blackleg disease of stems. The production of virulence factors
such as exoenzymes is tightly controlled by a variety of regulatory
networks, including the quorum sensing and Rsm (regulator of
secondary metabolites) systems. In this study, a random transposon
mutagenesis was carried out on an rsmB mutant strain (in which
virulence phenotypes such as exoenzyme production are repressed) and
mutants displaying restored protease production were identified. Three
independent insertions were identified in the gene coding for MetJ, a
well-characterised DNA binding protein and repressor of methionine
biosynthesis. Disruption of this gene affected growth, increased
production of the quorum sensing signal (OHHL) and increased
virulence in planta in an rsmB mutant. Transcriptomic and proteomic
analysis showed pleiotropic effects on gene expression, including up-
regulation of virulence-associated transcripts and increased transcription
of expI, the OHHL synthase gene, and increases in the production of
several exoenzymes. The P. atrosepticum MetJ regulon was predicted
bioinformatically, and direct targets identified. To our knowledge, this is
the first report of MetJ being linked to virulence in a pathogen.
MA02 – Mon 1130
Carbon fixation and a mixed diet for the intracellular tuberculosis
bacillus
Dany J V Beste1, Katharina Nöh2, Sebastian Niedenführ2,
Tom A Mendum1, Nathaniel D Hawkins3, Jane L Ward3,
Michael H Beale3, Wolfgang Wiechert2, Johnjoe McFadden1
1
Faculty of Health and Medical Sciences, Department of Microbial and
Cellular Sciences, University of Surrey, Guildford, UK; 2Forschungszentrum
Jülich GmbH, IBG-1: Biotechnology & JARA-HPC, Jülich, Germany; 3National
Centre for Plant and Microbial Metabolomics, Rothamsted Research,
Harpenden, Herts, UK (Email: d.beste@surrey.ac.uk)
Intracellular carbon metabolism has emerged as an attractive drug target
yet the major carbon sources of intracellularly replicating pathogens
remain poorly defined. This is particularly pertinent for the tuberculosis
bacillus Mycobacterium tuberculosis which causes a long-term latent
infection in one third of the world’s population and is resistant to many
drugs. Here, we use a novel systems-based approach, 13C-flux spectral
analysis (FSA) to measure, for the first time, the metabolic interaction
between M. tuberculosis and its macrophage host cell. 13C-FSA analysis
of experimental data showed that M. tuberculosis obtains a mixture
of amino acids, C1 and C2 substrates from its host cell. The C2 source
is consistent with acetate derived from host lipid catabolism and we
experimentally confirmed that the C1 substrate was derived from
CO2. 13C labelling experiments performed on a phosphoenolpyruvate
carboxykinase (PEPCK) deletion mutant demonstrated a deficiency
in gluconeogenesis of the C2 and C1 substrates and thereby revealed
the additional uptake and metabolism of a host-derived C3 glycolytic
compound by intracellular M. tuberculosis. The finding that intracellular
M. tuberculosis has access to diverse carbon sources within a
macrophage provides new opportunities for development of novel
chemotherapeutics that target nutrient uptake and/or intracellular
metabolism of the TB bacillus.
MA02 – Mon 1400
Intestinal colonisation by commensal and pathogenic Escherichia coli
Paul S Cohen1,Tyrrell Conway2
1
University of Rhode Island, Kingston, RI 02881 USA; 2University of
Oklahoma, Norman, OK 73019 USA
This past decade we endeavored to characterise the carbon and
energy metabolism of several commensal and pathogenic E. coli in
a streptomycin-treated mouse model of intestinal colonisation. We
learned all strains use the TCA cycle and the same pathways for aerobic
and anaerobic electron flow, but differ markedly in the carbon sources
each uses to colonise. These experiments were predicated on the
nutrient-niche hypothesis that each species colonises by preferential
use of a nutrient not used by others, but more recent data suggests
this hypothesis is oversimplified. We find that interactions between
E. coli strains can be modified by mutations selected in the intestine
that decrease the growth rate on several sugars yet the mutants
co-colonise with their parent strain. On this basis we propose the
‘restaurant hypothesis’ that various E. coli physically interact with
different members of the microbial community, which make alternative
carbon sources available locally, i.e., in that restaurant. Hence, we have
recently undertaken a microbial community approach to the symbiotic
relationships between E. coli and anaerobes. This talk will cover the
animal system employed, an overview of published findings, and recent
consideration of E. coli as a foundational member of the intestinal
microbial community.
10
Session abstracts
MA02 – Mon 1430
Offered paper Unusual regulation and vulnerability of glutamate
metabolism in Mycobacterium tuberculosis
Helen M O’Hare1, Barbara Rieck1, Faisal Alzaidi1, Andrew Bottrill1,
Marcello Ventura2, Francesca Boldrin2, Riccardo Manganelli2,
Michael Zimmermann3, Uwe Sauer3, Nathalie Barilone4,
Marco Bellinzoni4, Pedro M Alzari4
1
The University of Leicester, Leicester, UK, 2University of Padua, Padua, Italy,
3
Swiss Federal Institute of Technology Zurich, Zurich, Switzerland, 4Pasteur
Institute, Paris, France
Mycobacterium tuberculosis is a metabolically versatile facultative
intracellular pathogen, able to utilise diverse carbon and nitrogen
sources and to survive long periods of starvation. Correct regulation
of metabolism is essential for M. tuberculosis to adapt to the different
environments encountered in the host. Protein kinase G, which regulates
glutamate metabolism via its substrate GarA, has also been identified as
a virulence factor. GarA binds and regulates three enzymes of glutamate
metabolism and the TCA cycle.
We have found that garA is essential in M. tuberculosis but dispensable in
the model organism M. smegmatis. Deletion of garA in M. smegmatis led
to a specific, nutrient-dependent growth defect, disruption of intracellular
metabolite pools, and an inability to utilise acetate and propionate. We
propose that GarA may be important during intracellular growth on
lipids.
In both organisms the level of phosphorylation of GarA was strongly
dependent on the growth phase and nutrient availability. This confirms
the role of GarA as a metabolic regulator integrating signals from PknG
and other pathways. The data underline the importance of glutamate
synthesis and the TCA cycle in M. tuberculosis and reveal vulnerability of
these pathways to disruption.
MA02 – Mon 1445
Offered paper Metabolic regulation of the Type III secretion machinery
in the opportunistic human pathogen, Pseudomonas aeruginosa
Jade C S Chung, Martin Welch
University of Cambridge, Cambridge, UK
Pseudomonas aeruginosa is an opportunistic human pathogen and
common cause of chronic infections in individuals with cystic fibrosis
(CF). In this work, we show that expression of the T3SS in oxygenlimited growth conditions is strongly dependent on the glyoxylate shunt
enzyme, isocitrate lyase (ICL; encoded by aceA), which was previously
shown to be highly-expressed in CF isolates. ICL-dependent regulation of
the T3SS did not alter the expression level of the master transcriptional
regulator, ExsA, and was dependent upon the ICL activity of the AceA
protein. An aceA mutant displayed enhanced biofilm formation during
anaerobic growth, which suggested that AceA-dependent modulation
of T3S might impinge upon the RetS/LadS signalling pathways. Indeed,
our data suggest that RetS is able to mediate some of its effects through
AceA, as expression of aceA in trans partially restored T3SS expression
in a retS mutant. Our findings indicate that AceA is a key player in
the metabolic regulation of T3SS expression during oxygen-limited
growth of P. aeruginosa. To the best of our knowledge, this is the first
demonstration that the T3SS can be regulated by factors that do not
affect ExsA expression levels.
MA02 – Mon 1500
Profiling metabolic activities in single cells
M M M Kuypers
MPI for Marine Microbiology, Bremen, Germany
Coupling the identity of microbes with their activity in the environment
remains an important gap in our ability to explore the role of
microorganisms in global biogeochemical cycles. The development of
techniques to quantify the metabolic activity of single microbial cells has
been especially challenging, mostly due to their small sizes. This problem
has recently been solved, however, by the development of the nanoscale
secondary ion mass spectrometry (nanoSIMS), which for the first time
makes it possible to determine the chemical, radioisotopic, or stable
isotopic composition of biomass at the sub-micron level. An even more
powerful technique emerges when nanoSIMS is combined with in situ
hybridisation using oligonucleotide
probes specific to the organisms of interest. We used 19F-labeled
oligonucleotide probes to directly identify individual microbial cells
inhabiting various environments by nanoSIMS. The hybridisation
procedure is essentially identical to that used for CARD-FISH. By
combining this probing technique with in situ incubation experiments
with isotope-labeled substrates, we assessed the metabolic activity
of microorganisms and simultaneously identified their phylogenetic
characteristics during a single nanoSIMS scan. This approach directly
linked the identity of microbial cells in a complex microbial community to
biogeochemical processes.
NanoSIMS is truly an imaging breakthrough, whose application is only just
beginning to yield information once considered inaccessible.
MA02 – Mon 1600
The regulation of symbiosis in Photorhabdus
Lea Lango, Susan A Joyce, David J Clarke
Department of Microbiology, University College Cork, Cork, Ireland
Photorhabdus is a genus of bioluminescent, Gram-negative bacterium that
is highly virulent to insect larvae whilst, at the same time, maintaining a
mutualistic relationship with nematodes of the family Heterorhabditiae.
Photorhabdus elaborates an extensive secondary metabolism during
post-exponential growth and the products of this secondary metabolism
include an antibiotic called 3-5-dihydroxy-4-isopropylstilbene (ST), an
anthraquinone pigment (AQ) and bioluminescence. We identified a
mutant in the mdh gene, encoding malate dehydrogenase (involved in
the TCA cycle), which was unable to produce ST, AQ and light. The
mdh mutant was unaffected in virulence but, importantly, this mutant
was not able to support nematode growth and development in vivo or
in vitro. Furthermore the construction of mutations in key genes in other
metabolic pathways confirmed the critical role for the TCA cycle in both
secondary metabolism and mutualism, but not in virulence. Therefore,
the TCA cycle is required for the transition of Photorhabdus from
pathogen to mutualist suggesting that a metabolic switch plays a key
role in the regulation of life-style decisions in this bacterium. Moreover,
recent array studies have revealed the full extent of the changes in gene
expression that occur during this metabolic switch and this data can be
used to describe the molecular contributions made by the bacteria to
the mutualistic association.
MA02 – Mon 1630
Lifetime imaging of Chlamydia metabolism and its crosstalk with the
host cell
Márta Szaszák1, Kensuke Shima1, Regina Orzekowsky-Schröder2,
Gereon Hüttmann2, Jan Rupp1,3
1
Institute of Medical Microbiology and Hygiene, University of Lübeck,
Germany; 2Institute of Biomedical Optics, University of Lübeck, Germany;
3
Medical Clinic III, University Hospital Schleswig-Holstein/Campus Lübeck,
Germany
Chlamydia trachomatis is an obligate intracellular bacterium that
alternates between two metabolically different developmental
forms. We performed fluorescence lifetime imaging (FLIM) of the
metabolic coenzymes NAD(P)H, by two-photon microscopy for
separate analysis of host and pathogen metabolism during intracellular
chlamydial infections. NAD(P)H autofluorescence was detected inside
the chlamydial inclusion and showed enhanced signal intensity on
the inclusion membrane. An increase of the fluorescence lifetime of
protein-bound NAD(P)H [τ2-NAD(P)H] inside the chlamydial inclusion
strongly correlated with enhanced metabolic activity of chlamydiae
during the mid-phase of infection. Inhibition of host cell metabolism
11
Session abstracts
that resulted in aberrant intracellular chlamydial inclusion morphology
completely abrogated the τ2-NAD(P)H increase inside the chlamydial
inclusion. τ2-NAD(P)H also decreased when the cells were treated
with IFNγ reflecting the reduced metabolism of persistent chlamydiae.
Furthermore, a significant increase in τ2-NAD(P)H and a decrease in the
relative amount of free NAD(P)H inside the host cell nucleus indicated
cellular starvation during intracellular chlamydial infection. FLIM analysis by
two-photon microscopy is a useful tool to visualise metabolic pathogenhost interactions with high spatial and temporal resolution in living cells.
We suggest that intracellular chlamydial metabolism is directly linked
to cellular NAD(P)H signaling pathways that are involved in host cell
survival and longevity.
MA02 – Mon 1700
Offered paper Evidence that the inactivation of iron–sulfur cluster
enzymes contributes to microaerophily in Campylobacter jejuni and the
protective role of hemerythrins
John J Kendall, David J Kelly
University of Sheffield, Sheffield, UK
The important human food-borne pathogen Campylobacter jejuni is
adapted for growth in low-oxygen environments, specifically 5 – 10%
O2. However, it is also exposed to atmospheric oxygen levels (21%)
and the oxidative stress response during infection. The molecular
bases for this microaerophily and the mechanism by which oxygen
damages Campylobacter are not well understood. We have previously
hypothesised that the major reason why Campylobacter is damaged
by excessive oxygen is due to the presence of essential oxygen-labile
Fe-S cluster-containing enzymes within its metabolic pathways. We
now provide experimental evidence to support this hypothesis. We
have shown that two crucial iron-sulfur cluster containing TCA-cycle
enzymes in Campylobacter, 2-oxoglutarate:acceptor oxidoreductase and
pyruvate:acceptor oxidoreductase, are inactivated with similar kinetics to
the decrease in cell viability after exposure of microaerobically growing
cells to atmospheric oxygen levels in batch cultures. A mutant in cj0241c,
a gene encoding a hemerythrin-like protein, has been shown to have
reduced pyruvate:acceptor oxidoreductase and 2-oxoglutarate:acceptor
oxidoreductase activity when compared to the wild-type strain grown
microaerobically and aerobically. Mutants in other C. jejuni hemerythrins
also show increased susceptibility to damage by peroxide, suggesting a
protective role for these di-iron proteins during oxidative stress.
MA02 – Tue 0900
Sugar me: the intracellular metabolism of Salmonella Typhimurium in
host cells
Arthur Thompson
Salmonella Molecular Microbiology Group, Gut Health and Food Safety,
Institute of Food Research, Norwich Research Park, Colney, Norwich, UK
Salmonella is a widespread zoonotic pathogen that can cause
gastroenteritis and fatal typhoidal disease in mammals. Within the human
gut, S. Typhimurium replicates within the microfold (M) epithelial cells
lining the small intestine and causes bloody diarrhoea. During systemic
infections in mice and immunocompromised humans, S. Typhimurium
passes through the intestinal epithelial cells and is taken up by
macrophages where it replicates within a specialised vacuole resistant to
host cell defence mechanisms. We have begun to identify the substrates
and metabolic adaptations that allow S. Typhimurium to replicate and
survive within these intracellular environments. We have shown that
sugar and glycolysis play a key role in the ability of S. Typhimurium
to replicate and survive within murine macrophages. Interestingly the
TCA cycle appears to be dispensable for S. Typhimurium replication
in murine macrophages and in fact an intact TCA cycle appears to
be a disadvantage. More recently we have become interested in the
metabolism required for S. Typhimurium to replicate within epithelial cell
lines. The metabolic requirements that allow S. Typhimurium to divide
and survive within host cells and the implications of these adaptations are
discussed in the presented talk.
MA02 – Tue 0930
Shared animal–bacterial metabolic pathways: from genomes and
metabolic models to metabolite exchange
Angela E Douglas1, Calum W Russell1, Sandy J MacDonald2,
Gavin H Thomas2
1
Cornell University, New York, USA; 2University of York, York, UK
Various bacteria obligately associated with animals have reduced
genomes (<1 Mb), with genome sequences indicative of limited
metabolic capabilities including incomplete metabolic pathways. For
bacteria in obligate symbiosis with animals, we have hypothesised that
the missing reactions are mediated by animal enzymes, such that the
bacterial metabolic capabilities are subsidised by the host. We have
tested for shared metabolic pathways in the symbiosis between the
bacterium Buchnera aphidicola and the pea aphid Acyrthosiphon pisum,
to determine the contribution of host metabolism to the synthesis of
essential amino acids, nutrients that Buchnera provides to the animal host.
Following evidence from quantitative proteomics that host enzymes are
not transferred to the bacterial cells, metabolic models were constructed
to test the feasibility of the shared metabolic pathways in the context
of the wider metabolic networks. Experimental analysis of metabolite
uptake and release from isolated Buchnera cells provided empirical tests
of model outputs. This integrated analysis revealed that essential amino
acid provisioning and nitrogen recycling by Buchnera are founded on
coevolved integration of host and bacterial metabolic networks, with
translocation of multiple metabolites between the partners.
MA02 – Tue 1030
Co-dependence of host and pathogen metabolism
Elaine Holmes
Imperial College London, UK
Abstract not received
MA02 – Tue 1100
Offered paper Biting the hand that feeds you: sialic acid utilisation at
the host interface
Chatchawal Phansopa1, John Rafferty2, David J Kelly2, C W Ian Douglas1,
Graham P Stafford1
1
School of Clinical Dentistry, The University of Sheffield, Sheffield, UK,
2
Department of Molecular Biology and Biotechnology, The University of
Sheffield, Sheffield, UK
Many human dwelling bacteria have evolved the ability to acquire
sialic acid from glycoproteins. This is especially true in the oral cavity
where several pathogens possess sialidases that liberate sialic acid from
epithelial and salivary sources at the cellular interface, often to supply
bacterial metabolism. Our work has focused on a novel integrated sialic
acid acquisition and utilisation system in the Gram-negative periodontal
pathogen Tannerella forsythia that allows sialic acid dependent growth in
oral biofilms. This system contains a novel TonB-dependent permease
with homologues in a range of gut commensal and pathogenic members
of the Bacteroidetes group. Transport of sialic acid across the outer
membrane is facilitated by new members of the SusCD family of
proteins named NanOU. Here evidence will be presented highlighting
the role of sialidases and recent work showing that NanU is a highaffinity outer membrane associated sialic acid binding protein that
enables growth on sialic acid will be presented. In addition we have
solved the structure of NanU at 1.6A revealing that it has an unusual
binding pocket which may explain the basis for its specificity for sialic
acid. These data highlight a new mode for acquisition of this important
human-derived sugar at the pathogen-host interface.
12
Session abstracts
MA02 – Tue 1115
Offered paper Hydrogen-stimulated carbon catabolism aids Salmonella
virulence
Robert Maier1, Reena Lamicchane-Khadka2
1
University of Georgia, Athens, Georgia, USA, 2Saint Mary’s College,
Notre Dame, IN, USA
Among the genes up-expressed by exposure of Salmonella enterica
serovar typhimurium to H2 (a major gaseous byproduct of intestinal
commensal metabolism) are carbon uptake and catabolism-related
genes. Glucarate, an oxidised product of glucose, is a major serum
organic acid in humans, and it is readily detected in tissues and body
fluids. Still, its role as a carbon source for a pathogen has not been
studied. High-level expression of a potential glucarate permease gudT
occurs when Salmonella is exposed to H2 gas. A gudT mutant strain of
Salmonella is deficient in glucarate dependent growth, and it exhibits
attenuated virulence (i.e. morbidity and mortality) in mice. The mean
time of death for wild type-infected mice was two days earlier than for
gudT-inoculated animals. At four days post inoculation, liver and spleen
homogenates from GudT-inoculated mice contained fewer viable
Salmonella than parental strain-inoculated animals. The parent strain
grew well H2-dependently in a minimal medium with amino acids and
glucarate as the primary C-sources, whereas the gudT strain achieved
~60% of the parent strain’s yield. Glucarate-mediated growth of a hyc
mutant (cannot produce H2) was stimulated by H2, presumably due to
the positive transcriptional response of added H2.
MA02 – Tue 1130
The metabolic interface between Pseudomonas syringae and plant cells
Gail M Preston
Department of Plant Sciences, University of Oxford, South Parks Road,
Oxford, UK
The bacterial plant pathogen Pseudomonas syringae pv. tomato (Pst)
is the causal agent of bacterial speck of tomato. It is also widely used
as a model organism for studying the molecular interactions between
pathogens and plants due to its ability to infect the model plant
Arabidopsis thaliana. P. syringae infects plants by colonising the apoplastic
compartment of plant tissues and uses a combination of toxins and
secreted proteins to disable plant defences. We have performed
metabolite profiling of tomato leaf apoplast extracts in conjunction with
nutrient utilisation assays, comparative genomics and metabolic flux
analyses to provide detailed insight into the ability of Pst to assimilate
metabolites present in plant tissues and into the molecular mechanisms
underpinning nutrient acquisition by Pst during infection. Metabolic
footprinting experiments have revealed a distinct pace and preference
to nutrient assimilation in Pst, which is consistent with adaptation of this
bacterium for growth in plants. Interestingly, in these experiments Pst
shows delayed assimilation of the most abundant amino acid in tomato
fruit and in the tomato leaf apoplast, the non-protein amino acid GABA.
Further work has shown that Pst has a complex relationship with GABA,
which can be used as a carbon and nitrogen source, but can also act to
suppress virulence gene expression. I will discuss the implications of these
experiments for our understanding of plant pathogen interactions and
the development of novel strategies for disease control.
MA02 – Tue 1400
Novel riboswitch ligand analogues as selective inhibitors of guaninerelated metabolic pathways
Jérôme Mulhbacher1, Eric Brouillette1, Marianne Allard1,
Louis-Charles Fortier2, François Malouin1, Daniel A Lafontaine1
1
Université de Sherbrooke, Département de biologie, Sherbrooke, Canada;
2
Université de Sherbrooke, Département de microbiologie et d’infectiologie,
Sherbrooke, Canada (Email: Daniel.Lafontaine@usherbrooke.ca)
Keywords: Riboswitch, antimicrobial compounds, purine biosynthesis,
GMP synthetase, Staphylococcus aureus
Riboswitches are regulatory elements modulating gene expression in
response to specific metabolite binding. It has been recently reported
that riboswitch agonists may exhibit antimicrobial properties by binding
to the riboswitch domain. Guanine riboswitches are involved in the
regulation of transport and biosynthesis of purine metabolites, which
are critical for the nucleotides cellular pool. Upon guanine binding, these
riboswitches stabilise a 5’-untranslated mRNA structure that causes
transcription attenuation of the downstream open reading frame.
In principle, any agonistic compound targeting a guanine riboswitch
could cause gene repression even when the cell is starved for guanine.
Antibiotics binding to riboswitches provide novel antimicrobial
compounds that can be rationally designed from riboswitch crystal
structures. Using this, we have identified a pyrimidine compound (PC1)
binding guanine riboswitches that shows bactericidal activity against
a subgroup of bacterial species including well-known nosocomial
pathogens. This selective bacterial killing is only achieved when guaA, a
gene coding for a GMP synthetase, is under the control of the riboswitch.
Among the bacterial strains tested, several clinical strains exhibiting
multiple drug resistance were inhibited suggesting that PC1 targets a
different metabolic pathway. As a proof of principle, we have used a
mouse model to show a direct correlation between the administration
of PC1 and the reduction of Staphylococcus aureus infection in mammary
glands. This work establishes the possibility of using existing structural
knowledge to design novel guanine riboswitch-targeting antibiotics as
powerful and selective antimicrobial compounds. Particularly, the finding
of this new guanine riboswitch target is crucial as community-acquired
bacterial infections have recently started to emerge.
MA02 – Tue 1430
Understanding folate/pterin metabolism in trypanosomatids to assess
and exploit potential drug targets
William N Hunter
Division of Biological Chemistry and Drug Discovery, College of life Sciences,
The protozoan parasites, Trypanosoma and Leishmania are auxotropic
for folate and pterin cofactors that are essential for growth and
development. In theory, the inhibition of enzymes responsible for the
salvage and manipulation of these nutrients presents opportunities
for therapeutic attack relevant to the treatment of several diseases.
We have been studying two enzymes from this class of parasite; a
broad spectrum pteridine reductase (PTR1) and the bifunctional N5,
N10 methylenetetrahydrofolate dehydrogenase/cyclohydrolase (FolD)
seeking to understand the structure-reactivity relationships that guide
function. High-resolution crystallographic data and access to small
molecule reagents are key to progress in this area and the results that
will be described allow for an assessment of how tractible the potential
drug targets might be and of some progress towards useful trypanocidal
agents.
MA02 – Tue 1500
Offered paper Manipulation of cell metabolism by vaccinia virus: a
mechanism for the stabilisation of the transcription factor HIF-1
leading to a hypoxic response
Michela Mazzon1, Nicholas E Peters2, Christoph Leonarz3, Ewelina
Krysztofinska2, Stuart W Ember1, Brian J Ferguson1,2, Geoffrey L Smith1
1
Department of Pathology, University of Cambridge, Cambridge, UK,
2
Department of Virology, Faculty of Medicine, Imperial College London,
London, UK, 3Chemistry Research Laboratory and the Oxford Centre for
Integrative Systems Biology, University of Oxford, Oxford, UK
To establish infection, viruses have evolved strategies to manipulate
cellular machinery, including regulation of stress responses and immunity.
Several viruses also regulate cellular biochemistry to create a better
environment for replication. Some viruses, for example, express
biosynthetic enzymes to expand nucleotide pool size, or growth factors
to stimulate cell division. Here we report another example of virus
13
Session abstracts
manipulation of cellular biochemistry by the induction of a hypoxic
response.
Vaccinia virus (VACV) is a poxvirus and the vaccine used to eradicate
smallpox. VACV has a large dsDNA genome and replicates in the
cytoplasm. We show that early after infection VACV induces stabilisation
of the cellular transcription factor HIF-1α. Upon nuclear translocation,
HIF-1α activates transcription of cellular genes which co-ordinated a
hypoxia response. HIF-1α stabilisation is mediated by VACV protein C16
that binds to the cellular enzyme PHD2, which under normoxia causes
hydroxylation and degradation of HIF-1α. Interestingly, the interaction
of C16 with PHD2 is mediated via the N-terminal region of C16 that
is predicted to have a PHD2-like structural fold, but to be catalytically
inactive. Modulation of the cell`s hypoxic response is reminiscent of
the biochemical consequences of solid tumours, and suggests a novel
pathway of poxvirus modulation of cellular biochemistry.
MA02 – Tue 1515
Offered paper A host of culture complexities: what tn-seq can show
us about physiology, metabolism and microbial environments
Brian A Klein1, Jodie Scott3, Margaret J Duncan3, Linden T Hu1,2
1
Tufts University Sackler School of Biomedical Sciences, Boston, MA, USA,
2
Tufts Medical Center, Boston, MA, USA, 3The Forsyth Institute, Cambridge,
MA, USA
Porphyromonas gingivalis, a Gram-negative, asaccharolytic, anaerobic,
black-pigmented bacterium, is associated with periodontal disease
and systemic comorbidities. A Mariner-based transposon system was
used to generate insertional mutant libraries of P. gingivalis that are
amenable to next-generation sequencing technologies and analysis
methods, allowing for specific phenotype screening and direct linkage of
phenotypes to genotypes. Such libraries provide a way to quantitatively
assess fitness of a particular mutant under a specific selection condition.
Survival of mutants on blood agar (BAP) was quantitatively analysed
using the Tn-seq method; 463 genes were identified as essential for
BAP growth. These libraries were then placed under various selective
nutrient conditions and re-screened by Tn-seq. Following serial passaging,
insertions into 99 genes, all of which are non-essential on solid BAP
medium, exhibited extreme growth defects in liquid (brain-heart infusion,
BHI) medium. An additional 210 genes were identified as being 100fold regulated by or dependent on hemin concentrations. Screening the
library under different environmental conditions, particularly those found
within a host niche, can provide insight into gene function, pathway
structure and conditional essentiality. Experiments using medium
containing varied concentrations of environmental niche metabolites will
allow for nutritional analyses and gene function identification/verification
as well as potential therapeutic targets.
MA02 – Tue 1600
Plasmodium falciparum lipid metabolism and the search for drug targets
Henri Vial
Dynamique des Interactions Normales et Pathologiques, CNRS UMR 5235,
Université Montpellier 2, Place Eugène Bataillon, 34095 Montpellier Cedex
05, France (E-mail: vial@univ-montp2.fr)
Lipids are of crucial importance for pathogens. They serve as a
cellular building block, signaling molecules and pathogenesis effectors.
Phospholipids are the major lipid component of malaria parasites. Our
studies are geared towards the functional characterisation of molecular
cellular and membrane events that lead to membrane biogenesis in
Plasmodium development within host cells. Tremendous advances
have been made in the understanding of the synthesis pathways for
phospholipid acquisition through a bewildering variety of even novel
metabolic pathways, in clarifying the processes that could be major and/
or essential for the differentiation and development of these parasites,
and finally in determining the original features in terms of biological
processes versus mammalian cells. Trancriptomics and metabolomics
studies show that the parasite changes dramatically as it transits through
the various stages of its life cycle.
The program provides opportunities for drug development and has
resulted in the development of a new chemotherapy strategy against
P. falciparum malaria, targeting phospholipid plasmodial metabolism.
The most advanced antimalarial drug development program
targeting the parasite metabolism is focused on inhibition of de novo
phosphatidylcholine biosynthesis. The choline analog albitiazolium, which
is currently being clinically tested against severe malaria, represents a
new type of antimalarial whose mechanism of action differs from those
currently being marketed.
MA02 – Tue 1630
Microbial interactions in the upper gut
George T Macfarlane
University of Dundee, Microbiology and Gut Biology Group, Ninewells
Hospital Medical School, Dundee, UK
The mouth and large bowel are the principal sites of permanent
bacterial colonisation of the digestive tract in healthy people, however,
significant microbial communities can also exist in the stomach and
small bowel in certain disease states. Recent work has also shown that
mucosal surfaces in the oesophagus support rich and diverse microbial
communities, that are distinct from those found in oesophageal aspirates.
The composition of these microbiotas has been shown to change in
diseases such as Barrett’s oesophagus and adenocarcinoma, where
species such as Campylobacter concisus occur in high numbers. These
organisms were not detected in healthy biopsies. Studies with biopsy
material and oesophageal cell lines have shown that this bacterium is
pro-inflammatory, but that its effects on the innate immune system can
be mitigated through the use of probiotics such as Bifidobacterium longum
and Lactobacillus acidophilus.
MA02 – Tue 1700
Trypanosome metabolism and the search for drug targets and
elucidation of the mechanism of action of anti-trypanosomal drugs
Michael P Barrett
Glasgow Polyomics & Wellcome Trust Centre for Molecular Parasitology,
College of Medical, Veterinary and Life sciences, University of Glasgow,
Glasgow, UK
Many anti-parasitic drugs have unknown modes of action. Metabolomics
allows the simultaneous identification and quantification of many
metabolites in a given cell. We have developed a metabolomics
platform, based on LC-MS, focusing on exact mass identification to
dissect metabolism in various protozoan parasites. Using eflornthine,
used in stage 2 human African trypanosomiasis, we showed the drug
specifically targets ornithine decarboxylase with no obvious off-target
effects. This was based on the observation that cellular ornithine
increased and putrescine decreased in response to treatment while
other metabolites did not change significantly in abundance until shortly
before cell death. We have also identified modes of action for several
compounds being developed as trypanocides. We have also used
metabolomics to determine metabolic requirements of trypanosomes
grown in medium and in mice. In Leishmania, we have taken a polyomic
based approach to understand drug resistance and revealed, using
metabolomics, that changes in sterol metabolism relate to the acquisition
of drug resistance. Several alterations in genes and proteins related to
the sterol biosynthetic pathways were also identified as altered in these
cells. Metabolomics has therefore proven highly effective in identifying
modes of action and mechanisms of resistance to drugs used against
trypanosomatid protozoa.
14
Session abstracts
Co-infections and co-colonisation
superimposed on a setting of airway colonisation, creating a dynamic
interaction between pathogens and the host.
MA03
MA03 – Mon 0900
Co-infections and influenza: experiences during and after the 2009
pandemic
Richard Pebody
Health Protection Agency, UK
Abstract not received
MA03 – Mon 0930
Bacterial super-infections: the other side of influenza pathogenesis
Jonathan A McCullers
Department of Infectious Diseases, St. Jude Children’s Research Hospital, 262
Danny Thomas Place, Memphis, TN 38105, USA
Secondary bacterial infections are a major cause of morbidity and
mortality following influenza. This was especially true during past
pandemics, where 50-95% of all deaths were complicated by or
attributed to bacterial pathogens including Streptococcus pneumoniae,
Staphylococcus aureus, and Streptococcus pyogenes.. The emergence
in 2009 of a new H1N1 pandemic strain and the threat of a more
sever pandemic in the near future has increased the urgency for us
to understand how bacteria work together with influenza viruses to
cause pneumonia. In 2009-2010, 25-56% of severe and fatal cases had
evidence of bacterial super-infections complicating pandemic influenza.
Several mechanisms have been postulated to explain this interaction.
Mechanical disruptions of physical barriers enhance access to sterile sites.
Increased bacterial receptor availability through cytotoxic damage or viral
neuraminidase activity enhances adherence. The lack of glycosylation
of the surface proteins of viruses emerging from the avian reservoir
contributes to both primary virulence and secondary bacterial infections
by facilitating deep lung infection and preventing viral clearance. Alteration
of lung homeostatis and interference with the immune pathways and the
immune cells that that control influenza or bacterial invaders have effects
on bacterial clearance. Enhanced inflammation due to the expression
of cytotoxins such as the influenza A virus protein PB1-F2 potentiate
disease manifestations associated with pneumonia. New insights into
the mechanistic underpinnings of viral-bacterial interactions will allow
improved means of prevention and treatment for co-infections.
MA03 – Mon 1000
Dynamics of respiratory tract colonisation in chronic obstructive
pulmonary disease
Timothy F Murphy
University at Buffalo, The State University of New York, 875 Ellicott Street,
Buffalo, USA
Chronic obstructive pulmonary disease (COPD) is a chronic debilitating
disease and a leading cause of mortality worldwide. The course of
COPD is characterised by intermittent exacerbations. Analysis of
prospectively collected respiratory tract samples for viral infection by
culture, serology and PCR, and assays for bacterial infection by culture
and immunoassays for specific antibody responses to bacteria, reveal
that exacerbations are associated with infection by virus alone, bacteria
alone and virus and bacteria simultaneously. Acquisition of new bacterial
pathogens drives exacerbations caused by bacteria.
Bacteria also play a more subtle and perhaps even more important role
in the course and pathogenesis of the disease. The presence of bacterial
pathogens in the airways in COPD, particularly nontypeable Haemophilus
influenzae (NTHI) is common. Molecular typing of prospectively
collected isolates of NTHI and analysis of sputum by PCR shows that
many patients with COPD are persistently colonised with NTHI and
that culture underestimates the frequency of colonisation. Furthermore,
recent studies of the airway microbiome suggest that the airways in
COPD may harbor a more complex flora than previously recognised.
Thus, infectious exacerbations in COPD represent acute infections
MA03 – Mon 1100
Exploring the respiratory microbiome in health and disease
William O C Cookson, Michael J Cox, Miriam F Moffatt
National Heart and Lung Institute, Imperial College London, UK
The mucosa of the airways of the lung carries a similar density of
bacteria to the mucosa of the upper intestine. Characterisation of the
airway microbiome by 16S sequencing shows that in health the airway
microbiome resembles that of the oropharynx, and is dominated
by Streptococcus spp. and anaerobes such as Veilonella and Prevotella
spp. Approximately 100 different OTUs may be detected in a typical
European airway sample. Cigarette smoking reduces the absolute
numbers of bacteria as well as their diversity. The airway microbiome
of asthmatics is characterised by expanded numbers of Proteobacteria
(most commonly Haemophilus spp.) and in more severe cases the
presence of Streptococcus and Staphylococcus spp. Cystic Fibrosis and
Bronchiectasis are both associated with chronic bacterial infections. 16S
sequencing in these patients shows marked reductions in community
diversity and domination by single organisms such as Pseudomonas
spp. Long-term high-dose antibiotic therapy is of undoubted benefit to
these patients, but the loss of diversity and richness suggests that timing
and design of current courses of antibiotics may be having undesirable
consequences. Use of 16S sequencing in patients with acute lung
infections highlights the limitations of bacterial culture, and suggests the
presence of previously unrecognised pathogens.
MA03 – Mon 1130
Offered paper Inducibe prophages of an epidemic strain of
Pseudomonas aeruginosa are highly active in the cystic fibrosis lung
Chloe E James1,2, Jo L Fothergill2, Michael A Brockhurst3,
Craig Winstanley2
1
School of Environment and Life Sciences; The University of Salford, Manchester,
UK, 2Institute of Infection and Global Health, The University of Liverpool,
Liverpool, UK, 3Department of Biology, The University of York, York, UK
Pseudomonas aeruginosa is the most common bacterial pathogen
infecting the lungs of cystic fibrosis (CF) patients. The Liverpool Epidemic
Strain (LES) is associated with increased morbidity and is widespread in
CF patients across the UK. The LES genome harbours multiple inducible
prophages that confer a competitive advantage in vivo. Evidence suggests
that induction of the lytic cycle and phage production plays a role in
niche establishment. However, the level of lytic LES phage activity in the
lungs of CF patients is unknown. This longitudinal study uses quantitative
PCR (Q-PCR) to measure the density of five inducible LES phages in the
sputum of 10 LES-infected patients over two years. Our data suggest
that free LES-phage particles consistently out-number their P. aeruginosa
host by 10-1000 fold in all infected patient sputa. Detecting specific LESphage targets by Q-PCR is, therefore, highly sensitive and may be useful
for early diagnosis of LES infection. These results provide clues to how
carriage of inducible LES-phages could contribute to the competitiveness
of their host. Active phage virions have the potential to kill other P.
aeruginosa competitors through lytic infection. Furthermore, lysogenic
infection has the capacity to mobilise genes through horizontal gene
transfer within heterologous P. aeruginosa populations.
MA03 – Mon 1145
Offered paper Nasal co-colonisation with MRSA variants
Kinga I Stanczak-Mrozek, Alex McCarthy, Patrick Houston,
Jodi A Lindsay
St George’s University of London, Division of Clinical Sciences, London, UK
Staphylococcus aureus nasal colonisation is a risk factor for subsequent
infection. The aim of this study was to determine whether methicillinresistant S. aureus strains (MRSA) from individual anterior nares of human
15
Session abstracts
patients are homogenous or vary phenotypically and genotypically.
Twenty two MRSA nasal swabs from patients admitted to St George’s
Hospital were streaked on mannitol salt agar. Twenty positive subisolates were selected from each plate and each tested for susceptibility
to 19 antibiotics and with PCR for lineage, selected bacteriophage
(φ2, φ3, φ6) and plasmid (rep10, rep20) genes. Only one patient was
colonised by sub-isolates belonging to more than one lineage. However
7/22 (31.8%) of the patient’s sub-isolates were phenotypically and/or
genotypically different, including variation in antibiotic susceptibility and
genes interacting with host. Microarray analysis revealed further genetic
differences. Free bacteriophages were found in 10 nasal specimens and
these phages were capable of successfully transferring antibiotic resistant
genes between sub-isolates. Genetic variation in nasal colonisation
population is common and may play an important role in host-pathogen
adaptation. Diagnostic antibiotic susceptibility testing should not rely on
sampling a single sub-isolate or resistance may be missed.
MA03 – Mon 1400
Microbial Facebook: probing bacterial social networks
Marvin Whiteley
The University of Texas at Austin, Section of Molecular Genetics and
Microbiology, 1 University Station, A5000 Austin, Texas, USA
The survival of pathogens in the human body has been rigorously studied
for well over a century. Bacteria are able to colonise, persist and thrive in
vivo due to an array of capabilities, including the ability to attach to host
tissues, produce extracellular virulence factors, and evade the immune
system. Most bacterial pathogenesis studies have focused on monoculture infections; however, it is clear that many bacterial infections are
not simply the result of colonisation with a single species, but rather
ensue from the actions of polymicrobial communities. Microbes within
polymicrobial infections often display synergistic interactions that result in
enhanced colonisation and persistence in the infection site, although the
molecular processes controlling these synergistic interactions are generally
not known. Here, I will discuss how interactions between biofilm bacteria
impact community development, resistance to host innate immunity, and
in vivo persistence. I will also discuss the use of novel technologies for
probing bacterial interactions in small biofilm populations.
MA03 – Mon 1430
Offered paper Respiratory viral co-infection is not associated with
increased clinical severity in a paediatric case-control study
Naomi J Gadsby1, Ivy Simm2, Alison Hardie1, Ulf Theilen2,
Kate E Templeton1
1
Specialist Virology Centre, Dept. Laboratory Medicine, Royal Infirmary of
Edinburgh, Edinburgh, UK, 2Royal Hospital for Sick Children, Edinburgh, UK
Background Clinical microbiology laboratories are increasingly using
multiplex real-time PCR to test single respiratory samples simultaneously
for a large number of respiratory viruses. This means we can diagnose
many more viral co-infections than ever before, as part of routine clinical
practise. However, whether respiratory viral co-infections are more
severe than single viral infections remains an important clinical question.
Methods We retrospectively analysed viral co-infection diagnosed by
real-time PCR and clinical severity in 175 case patients admitted to a
Paediatric Intensive Care Unit (PICU) and 178 non-PICU admitted
controls presenting to hospital, matched by age and time of year.
Results PCR detected at least 1 of 9 different respiratory viruses in
194/353 patients (55.0%), with rhinovirus, RSV and adenovirus most
common. The presence of a respiratory virus was positively associated
with a clinical diagnosis of lower respiratory tract infection (OR 4.41
(2.77-7.01), p<0.05). However, there was no association between
viral co-infection and key markers of disease severity; PICU admission,
ventilation requirement or length of hospital or PICU admission
(p=0.1 to 0.51).
Conclusions Our study shows that single respiratory viruses can cause
severe lower respiratory tract in children and the presence of additional
viruses does not appear to further increase clinical severity.
MA03 – Mon 1445
Offered paper A PCR high-resolution melt for the rapid differentiation
of non-typeable Haemophilus influenzae and Haemophilus haemolyticus
Janessa L Pickering1, Michael J Binks2, Jemima Beissbarth2,
Kim M Hare2, Heidi C Smith-Vaughan2, Lea-Ann Kirkham1
1
School of Paediatric and Child Health, University of Western Australia,
Western Australia, Australia, 2Menzies School of Health research, Charles
Darwin University, Northern Territory, Australia
Nasopharyngeal colonisation by nontypeable Haemophilus influenzae
(NTHi) can lead to a range of respiratory related infections. Accurate
identification of NTHi is paramount for disease surveillance and vaccine
efficacy studies. The co-coloniser Haemophilus haemolyticus rarely causes
disease but its similar colony phenotype impedes NTHi surveillance. We
developed an efficient protein D (hpd) based PCR high-resolution melt
(PCR-HRM) that permits identification of NTHi and H. haemolyticus
for surveillance and clinical trials. DNA sequencing was conducted on
the hpd gene from 31 H. haemolyticus and ambiguous Haemophilus
isolates. Using this sequence data we designed primers to amplify a
50bp product within hpd of NTHi and H. haemolyticus for interrogation
by high resolution melt (Rotorgene 6000). Four reference strains, 109
nasopharyngeal, 52 bronchoalveolar lavage, and 50 throat isolates
were screened to validate the assay. PCR-HRM analysis of colony boils
identified 192 isolates as NTHi or H. haemolyticus, with 96% sensitivity
and 92% specificity compared with the standard hpd#3 assay. Nineteen
isolates could not be amplified and may lack hpd entirely. The PCR-HRM
has the advantage of low cost and short preparation time, and allows
high throughput identification of both NTHi and H. haemolyticus.
MA03 – Mon 1500
HIV/HBV interactions – bench to bedside
A M Geretti
Institute of Infection & Global Health, University of Liverpool, UK
Background Globally, ~10% of HIV-positive patients have concurrent
HBV infection. HIV promotes HBV replication and markedly increases
the risk of liver-related disease and mortality. European HIV treatment
guidelines recommend routine HBV screening and maximal suppression
of both HIV and HBV replication with agents offering a high genetic
barrier to resistance, typically tenofovir plus lamivudine or emtricitabine
as part of triple antiretroviral therapy (ART).
Methods We are evaluating the virological and liver disease profile of HIVHBV co-infected cohorts accessing ART in Ghana, Malawi and Uganda. We
employ next generation sequencing (NGS) to characterise the evolution of
HBV drug-resistance and Elastometry to assess liver fibrosis.
Results HBV co-infection rates ranged from 7% to 17%. Yet, HBV
screening was not systematic and ART remained ‘HBV-blind’, usually
containing lamivudine but not tenofovir. Lamivudine-based ART
was associated with good control of HIV replication but poor HBV
suppression. Emergence of HBV drug-resistance was rapid in patients
with HBV DNA >14 IU/ml and most showed RTM208V/I by week 24 of
ART using NGS. These virological features were associated with a high
prevalence of Elastometry readings >8 kPa.
Conclusions Poorly controlled HBV co-infection poses a threat to the
long-term success of ART programmes in sub-Saharan Africa.
MA03 – Mon 1600
Innate immune regulation in the lung
Tracy Hussell
Manchester Collaborative Centre for Inflammation Research (MCCIR),
Professor of Inflammatory Disease, 2nd floor, Core Technology Facility,
University of Manchester, Oxford Road, Manchester, UK (Email: tracy.
hussell@manchester.ac.uk)
16
Session abstracts
The large number of co-infections detected, usually in more than 10%
of the positive samples, and the potential relation with clinical severity
emphasises the need of implementing broad multiplex technics for the
detection of respiratory viruses.
The activity of innate immunity is influenced by negative regulatory and
immune potentiator pathways. Even in the absence of an antigen, innate
immunity can ‘inflame’ if negative regulators are absent. This resting state
is adaptable and dictated by environmental influences, host genetics and
past infection history. A return to homeostasis post inflammation often
leaves an altered innate immune system with a different threshold of
activity.
Using murine models we show that acute and chronic inflammation
cause long term modifications of the lung microenvironment culminating
in a de-sensitisation to bacterial products, an increase in the myeloid
negative regulator CD200R and a shift in the regulatory miRNA
repertoire, which protect the healing lung from further inflammation, but
may also predisposes to bacterial colonisation. In the extreme this leads
to bacterial pneumonia, sepsis and death. We identify the molecular
mechanisms associated with bacterial susceptibility by showing the
disruption of bacterial recognition by pattern recognition receptors due
to sequestration or diversion of key receptors or downstream signalling
components in the remodelled lung.
A deeper understanding of the consequences arising from innate
immune cell alterations during influenza infection and asthma and the
subsequent development of bacterial complications has important
implications for future drug development.
MA03 – Mon 1630
Viral–bacterial interactions in the upper respiratory tract
Debby Bogaert
Department of Pediatric Immunology and Infectious Diseases, University
Medical Center-Wilhelmina Children’s Hospital, Utrecht, The Netherlands
Respiratory infectious diseases are mainly caused by viruses or bacteria
that often interact with one another. Although their presence is a
prerequisite for subsequent infections, viruses and bacteria may be
present in the nasopharynx without causing any respiratory symptoms.
The upper respiratory tract hosts a vast range of commensals and
potential pathogenic bacteria, which form a complex microbial
community. This community is assumed to be constantly subject to
synergistic and competitive interspecies interactions. Disturbances in the
equilibrium, for instance due to the acquisition of new bacteria or viruses,
may lead to overgrowth and invasion. A better understanding of the
dynamics between commensals and pathogens in the upper respiratory
tract may provide better insight into the pathogenesis of respiratory
diseases. I will discuss the current knowledge regarding bacterial-bacterial
and viral-bacterial interactions that occur in the upper respiratory niche,
and discuss mechanisms by which these interactions might be mediated.
MA03 – Tue 0900
Mixed respiratory virus infections
Florence Morfin, Emilie Frobert, Vanessa Escuret,
Maude Bouscambert, Jean-Sébastien Casalegno, Isabelle Schuffenecker,
Martine Valette, Bruno Lina
Laboratory of Virology, French National Influenza Centre (NIC), French
National Reference Centre for Enteroviruses, EMR, Université Lyon 1,
Hospices Civils de Lyon, Lyon, France
There is an enlarging number of viruses involved in respiratory infections.
Some are known for years, such as influenza, RSV, rhinovirus, adenovirus,
parainfluenza virus, coronavirus. Some others have been recently
discovered or re-discovered : with the new species C rhinoviruses, and
the new coronavirus NL63 and HKU1, and with metapneumovirus and
bocavirus. This large diversity combined with the recent development
of multiplex nucleic acid detection technics for these viruses has led to
a huge improvement in the identification of viruses as an aetiology of
respiratory infections.
We will describe the particular epidemiology of several of these viruses
and present some features of viral co-infections detected in a general
pediatric population and in specific populations.
MA03 – Tue 0930
Co-infections and the host microbiota
A W Walker
Pathogen Genomics Group, Wellcome Trust Sanger Institute, Hinxton, UK
(Email: aw6@sanger.ac.uk; Website: http://www.sanger.ac.uk/research/
projects/pathogengenomics)
The human body is host to an extremely abundant and diverse
collection of microbes, which are collectively called the human-associated
microbiota. Under normal circumstances our resident microbes are
considered to play a number of key roles in the maintenance of human
health. One example is the establishment of ‘colonisation resistance’,
which is driven by our indigenous microbiota effectively inhibiting
colonisation and/or overgrowth by invading ‘foreign’ microbes such as
pathogens. However, although most constituent species are thought to
be harmless or beneficial, the host microbiota can also be the source
of opportunistic pathogenic species, which may contribute to disease
progression and maintenance.
As well limiting invasion by pathogenic microbes, colonisation resistance
also acts to keep potentially pathogenic indigenous species such as
Clostridium difficile under control. However, in the case of C. difficile,
colonisation resistance can be broken by broad-spectrum antibiotic use,
which disrupts the density, composition and activity of the intestinal
microbiota and allows the pathogen to proliferate in the intestine and
cause disease.
To better understand the tripartite relationship between host,
pathogens and indigenous microbial communities we use mouse
models of infection. Using these models, we find that the indigenous
host microbiota can play a number of key roles in disease progression.
Infection with certain strains of C. difficile, for example, results in
prolonged disease and infectiousness, which appears to be maintained
by an inhibited re-establishment of colonisation resistance. In many cases
the inhibition of colonisation resistance is not associated with C. difficile
dominance, but by an overgrowth of a subset of species that may be
derived from within the host’s microbiota. Many of these co-infectionassociated microbiota species have also been detected in humans
suffering from severe C. difficile disease, indicating that the emergence of
pathogenic communities, rather than single pathogenic species, may be
an important factor in some disease cases. Breaking the dominance of
these dysbiotic communities, and restoring colonisation resistance are
key targets for restoration of health. I will therefore describe novel means
of disrupting pathogenic communities and restoring bacterial diversity in
the intestine.
MA03 – Tue 1000
Offered paper Three-dimensional μ-computed tomography to quantify
and diagnose biofilms and thrombus in central venous catheters
W L Niehaus1,2,4,5,7, D A Johnston9, A Page9, M N Mavrogordato8,
L Hall-Stoodley1,4,5, J Webb1,3, P J Thurner7, S C Clarke1,5,6, S N Faust1,4,5,
P Stoodley1,7
1
NIHR Southampton Respiratory Biomedical Research Unit, University of
Southampton and University Hospital Southampton NHS Foundation Trust,
Southampton, UK; 2NIHR Southampton Nutrition Biomedical Research
Unit, University of Southampton and University Hospital Southampton NHS
Foundation Trust, Southampton, UK; 3Academic Unit of Biological Sciences,
Faculty of Natural and Environmental Sciences, University of Southampton,
Southampton, UK; 4NIHR Wellcome Trust Clinical Research Facility, University
of Southampton and University Hospital Southampton NHS Foundation
Trust, Southampton, UK; 5Academic Unit of Clinical & Experimental
Sciences, Faculty of Medicine, University of Southampton, Southampton,
UK; 6Health Protection Agency, Southampton, UK; 7National Centre for
17
Session abstracts
Advanced Tribology, Faculy of Engineering, University of Southampton,
Southampton, UK; 8μ-VIS CT Imaging Centre, Faculy of Engineering, University
of Southampton, Southampton, UK; 9Biomedical Imaging Unit, Faculty of
Medicine, University of Southampton, Southampton, UK
Central venous catheters (CVCs) are used to provide medium to longterm vascular access for chemotherapy, nutrition and dialysis. Bacterial
and fungal infections and thrombus occlusion of CVCs are major
complications. CVC related bloodstream infections (CRBI) are usually
diagnosed by concordant culture of blood and catheter tip. However,
recent studies suggest that culture often fails to detect biofilm bacteria.
Confocal laser scanning microscopy (CLSM), scanning electron
microscopy (SEM) and micro-computed tomography (μCT) were used
to detect biofilms on in vitro and clinical catheters. Various contrast
agents were compared using energy dispersive x-ray analysis (EDAX).
Catheter material and biofilm were segmented and quantified using VG
Studio MAX and Avizo Fire software packages.
CLSM was not conducive to detect biofilms on catheters due to
sectioning, auto-fluorescence and sample curvature. SEM required
extensive sample preparation including sectioning and critical point
drying, which caused biomaterial loss. μCT could detect biofilms in
an in vitro catheter-vein model and ex vivo clinical catheters. Volume
measurements were determined and aggregate distributions were
investigated. μCT results were corroborated with SEM, which indicated
evidence of polymicrobial biofilms.
MA03 – Tue 1015
Offered paper Novel imaging techniques to study the mechanistic basis
for bacterial adhesion during polymicrobial co-infection
David Mason, Jane A McKeating
School of Infection & Immunity, The University of Birmingham, Edgbaston, UK
Microbial infections are one of the leading cause of disease and death in
the UK and worldwide. While viral or bacterial infection alone can cause
disease, the secondary bacterial infection following an initial viral infection
is often much more severe.
It has been suggested that viral pre-infection alters the adhesion of
bacteria to epithelial cell surfaces, promoting enhanced colonisation
and a worsened disease state. Using a range of microorganisms and
cell-systems we are developing imaging techniques with high temporal
and spatial resolution to study the individual adhesion events of bacteria
binding to naive or virus-infected cells.
Characterising the kinetics of these interactions, in the presence or
absence of virus, will provide a better understanding of the receptors and
mechanistic basis underpinning adhesion. When combined with genetic
manipulation of receptor/adhesin pairs, the obligate partners for adhesion
can be elucidated in many different systems.
By developing tools which allow us to better understand host-pathogen
interactions, more specific clinical interventions may be possible, lessening
the severity of polymicrobial coinfection.
MA03 – Tue 1100
Evidence of polymicrobial biofilms in infected surgical repair sutures
and meshes
Paul Stoodley1, Sandeep Kathju2, Luanne Hall-Stoodley3,
Georges Limbert1
1
National Centre for Advanced Tribology at Southampton (nCATS), Engineering
Sciences, Faculty of Engineering and the Environment, University of Southampton,
UK; 2Division of Plastic Surgery, University of Pittsburgh Medical Center,
Pittsburgh, PA, USA; 3University Hospital Southampton NHS Foundation Trust,
Faculty of Medicine, NIHR Respiratory BRU Southampton, UK
Prosthetic mesh and non-dissolvable sutures are routinely used for
hernia repair. In the US approximately 150,000 hernias arise annually
from ~1 million laparotomies. Surgical site infection (SSI) is a major
complication, with an estimated rate between 8 and 15%, equating to
~15,000 patients annually. Bacterial biofilms are hypothesised to play
a role in the frequency and chronicity of these infections. We analysed
mesh and suture samples from a consecutive series of 5 patients with
suspected SSIs associated with prosthetic mesh and 15 patients with SSIs
associated with closure sutures following open gastric bypass surgery,
by confocal microscopy, fluorescence in situ hybridisation (FISH) and
multiprimer PCR-mass spectrometry. All patients had evidence of
biofilms, and polymicrobial biofilms were common. The infections only
resolved after surgical removal of the foreign body. We next examined
the ability of Staphylococcus aureus, Enterococcus faecalis and Enterobacter
cloacae, isolated from an infected polypropylene mesh to form mixed
biofilms in vitro. The different species readily formed biofilms and the
relative abundance of species varied with time. Finally, we used finite
element computer modeling from confocal images to demonstrate that
movement of a suture may produce surface shear stresses sufficient to
trigger the sliding of biofilms over the surface.
MA03 – Tue 1130
Offered paper Sequence-based analysis and microbiological
comparison of the faecal microbiota from healthy individuals and
patients with severe Clostridium difficile disease
B O Adamu1, M D Stares1, A W Walker1, T J Louie2, T D Lawley1
1
Wellcome Trust Sanger Institute, Cambridge, UK, 2University of Calgary,
Alberta, Canada
Clostridium difficile, an anaerobic, spore-forming Gram-positive bacterium,
is the main cause of antibiotic-associated diarrhoea in hospitalised
patients. Current treatments for C. difficile involve the use of antibiotics
such as metronidazole, vancomycin or fidaxomicin. However, 2035% of patients experience recurrent disease, leaving few treatment
options, and many patients become dependent on vancomycin to
remain symptomless. Recurrent disease occurs as a result of a decrease
or elimination of health-associated bacteria by the antibiotics. An
alternative treatment therefore involves the restoration of the indigenous
microbiota with faecal transplants (infusion of homogenised faeces of
a healthy donor). Indeed, >90% success rates have been observed in
patients with recurrent C. difficile. The present study investigated the
differences in microbial diversity between faecal samples from recurrent
C. difficile patients and their respective transplant donors using culturing
and sequence-based analysis of the 16S rRNA gene. Preliminary results
demonstrate that the C. difficile patients harbour more facultative
anaerobes such as Proteobacteria and the Bacilli class of the Firmicutes,
and less Bacteroidetes, Actinobacteria and Clostridia compared to
healthy donors. Isolation of species from healthy individuals may lead to
the rational design of a defined mixture of beneficial bacteria that can be
used to treat recurrent C. difficile infections.
MA03 – Tue 1145
Offered paper Transcriptome and small molecule profiling reveals
the influence of bacterial community structure on the available
metabolites of the cystic fibrosis airway
Kate B Twomey1, Mark Alston3, Michael J Harrison2,
Yvonne McCarthy1, Melanie Febrer3, J Maxwell Dow1, Barry J Plant2,
Robert P Ryan1
1
BIOMERIT Research Centre, Department of Microbiology, Biosciences
Institute, University College Cork, Cork, Ireland; 2Cork Adult Cystic Fibrosis
Centre, Department of Respiratory Medicine, Cork University Hospital,
University College Cork, Cork, Ireland; 3The Genome Analysis Centre, Norwich
Research Park, Norwich, United Kingdom.
Polymicrobial infections of the lung are the foremost cause of morbidity
and mortality in cystic fibrosis (CF) patients. During these infections
the composition of the microbial airway flora alters considerably.
Such changes are usually rapid during patient exacerbation. Although
metagenomic strategies revealed the complexity of the microbiome
they do not necessarily give insight into community function as DNAbased methodologies may detect organisms that are metabolically
inactive. An understanding of which organisms are growing in the CF
18
Session abstracts
Titania-molybdenum surfaces were placed in the brewery environment,
for 3 months, Photoactivity (methylene blue) was retained, and
molybdenum was not lost from the surface.
Findings indicate potential in enhancing hygiene, complementing existing
cleaning and disinfection protocols.
airway is important for antibiotic treatment. We have analysed sputum
samples for small metabolites. In parallel, we examined RNA extracted
from the sputum for the abundance of transcripts of the 16S rRNA
gene by deep-sequencing of cDNA libraries. This analysis revealed that
the population size of all dominant orders of bacteria as measured by
DNA-and RNA-based methods are close, greater discrepancies are
seen with less prevalent organisms, some of which we associate with
CF for the first time. Additionally, we identified a strong relationship
between the abundance of specific anaerobes and fluctuations in several
small metabolites including lactate and putrescine during exacerbation.
This study has identified organisms whose occurrence within the CF
microbiome has been hitherto undetected and revealed potential
biomarkers for exacerbation.
MA03 – Tue 1445
Offered paper Source tracking antimicrobial resistance genes in the
aquatic environment: contribution of animal and human faecal sources
to the environmental resistome
Will Rowe1, David Verner-Jeffreys2, Craig Baker-Austin2,
Duncan Maskell1, Jim Ryan3, Gareth Pearce1
1
University of Cambridge, Cambridge, UK, 2Centre for Environment, Fisheries
and Aquaculture Science, Weymouth, UK, 3GlaxoSmithKline, Ware, UK
Widespread use of antimicrobials in human and veterinary medicine
and in agriculture results in environmental release of resistant bacteria
and associated genetic material. Discharges of faecal sources into
watercourses via sewage treatment facilities and agricultural run-off
are likely to be key pathways in the release and dissemination of
antimicrobial resistance genes (ARGs) throughout the environment.
Persistence and transfer of resistance within bacterial communities poses
significant risks to the sustainable use of antimicrobials. Determining
the relative contributions of human and animal faecal sources to the
environmental resistome will help shape waste management practices to
safeguard the efficacy of antimicrobial drug use.
This study utilised metagenomic sequencing to generate datasets
from river catchment sites rich in animal or human faecal inputs. A
bioinformatics pipeline was established that identified ARGs and mobile
genetic elements (MGEs). Comparison of these datasets showed clear
differences in types of ARGs being contributed to the aquatic system:
quinolone ARGs were exclusively found in the animal source and
vancomycin ARGs were found only in the human source. Diversity
of beta-lactam ARGs was much higher in the animal source, whereas
tetracycline ARG diversity was higher in the human source. Class I
integrons and other MGEs were identified from both sources.
MA03 – Tue 1400
Pseudomonas aeruginosa and fungi in chronic lung disease: molecular
mechanisms and effects on the host
Deborah A. Hogan, Sven Willger, Nora Grahl and Diana Morales
Department of Microbiology and Immunology, Geisel School of Medicine at
Dartmouth, HB 7550, Hanover, NH 03755, USA
Increasing numbers of transplants, medical devices, chemotherapies,
and chronic diseases translate into increasingly complex infections by
opportunistic pathogens. For example, individuals with cystic fibrosis
(CF) are plagued by chronic microbial infections comprised of a
wide variety of bacterial and fungal species. Pseudomonas aeruginosa
and Candida albicans often dominate in the sputum of cystic fibrosis
patients, particularly during periods of disease exacerbation, and coinfections of these two species correlate with worse disease status
indicating that bacterial-fungal interactions might play an important role
in disease severity. Our studies that examine the bacteria and fungi
within longitudinal samples from the lungs of CF patients (micro-and
mycobiome analyses, respectively) in combination with cell culture
models and in vivo expression profiling have allowed us to develop
a model for chemical and metabolic interactions that occur between
microbes in vivo and that negatively impact the host. A deeper
understanding of the intra-and inter-species interactions that occur in
mixed species infections will help us better appreciate the dynamics of
host-associated microbial communities and ultimately will help develop
new and more efficient treatments for complex chronic infections.
MA03 – Tue 1430
Offered paper The potential for the use of photocatalytic surfaces in
the process hygiene in the brewing industry
Joanna Verran1, Peter Kelly1, Soheyla Ostovarpour1, Leanne Fisher1,
Kevin Cooke2, Parnia Navabpour2, Claire Hill3, Carin Tattershall3,
Outi Priha4, Mari Raulio4, Erna Storgards4
1
Manchester Metropolitan University, Manchester, UK, 2Teer Coatings Ltd,
Droitwich, UK, 3Cristal, Lincolnshire, UK, 4VTT Technical Research Centre of
Finland, Finland, Finland
Process hygiene plays a major role in ensuring beer quality. One
approach to the reduction of fouling is the use of photocatalytic surfaces
such as those including titania phase (titanium dioxide), which is active
under UV light. TiO2 coatings can be doped with transition metals to
extend activity under fluorescent light. The aim of this study was to
produce doped coatings on stainless steel and to explore their potential
for use in the brewing industry.
Coatings were produced using magnetron sputtering. Characterisation
included photocatalytic activity via degradation of methylene blue,
and tests for chemical resistance to acid and alkali. Photocatalytic
antimicrobial activity was demonstrated using four brewery isolates and
Escherichia coli (following ISO 22196).
The addition of molybdenum to the coating enhanced activity under
fluorescent (visible) light, but the surface was antimicrobial in the dark.
Tungsten also enhanced photoactivity under visible light, but the surface
was not otherwise antimicrobial.
MA03 – Tue 1500
Bacterial–fungal biofilms (staphylococci and Candida)
Lisa Marie Schlecht1,2, Brian M Peters2,3, Bastiaan P Krom4,
Mary Ann Jabra-Rizk7,8, Mark E Shirtliff,2,8
1
Department of Restorative Dentistry and Periodontology, Ludwig Maximilian
University of Munich, Goethestrasse 70, 80336 Munich, Germany;
2
Department of Microbial Pathogenesis, University of Maryland – Baltimore,
Dental School, 650 W. Baltimore Street, Baltimore, MD 21201, USA;
3
Graduate Program in Life Sciences, Molecular Microbiology and Immunology
Program, University of Maryland – Baltimore, 660 W. Redwood Street,
Baltimore, MD 21201, USA; 4Department of Preventive Dentistry, Academic
Centre for Dentistry Amsterdam (ACTA), University of Amsterdam and Free
University Amsterdam, Gustav Mahlerlaan 3004, 1081 LA Amsterdam,
The Netherlands; 5Department of Immunology, Ruprecht Karls University
Heidelberg, Im Neuenheimer Feld 305, 69120 Heidelberg, Germany;
6
Division of Infectious Diseases, Los Angeles Biomedical Research Institute
at Harbor-UCLA Medical Center, 1124 W. Carson St., Torrance, CA 90502,
USA; 7Department of Oncology and Diagnostic Sciences, Dental School,
University of Maryland – Baltimore, 650 W Baltimore Street, Baltimore,
MD 21201, USA; 8Department of Microbiology and Immunology, School
of Medicine, University of Maryland – Baltimore, 660 W. Redwood Street,
Baltimore, MD 21201, USA
Staphylococcus aureus and Candida albicans are currently ranked the
second and third most commonly isolated bloodstream pathogens.
While systemic staphylococcal infections arise from seeding through
breaks in host epithelial layers, a substantial number of patients have no
documented portal of entry. In this study, we describe a novel strategy
by which S. aureus is able to invade host tissue and disseminate via
19
Session abstracts
adherence to the invasive hyphal elements of C. albicans. We developed
in vitro models and an in vivo murine model of oral co-colonisation to
demonstrate fungal-bacterial interactions. Findings demonstrated that
animals orally co-colonisation with both species exhibited clinical signs
of systemic infection with high morbidity and mortality concomitant
with the recovery of S. aureus from the kidneys, whereas animals
receiving individual species remained healthy. Histopathological and
microscopic analysis of in vitro co-cultures and co-colonisation tongues
revealed extensive invasion of hyphae with S. aureus seen within the
sub-epithelium tissue. Using C. albicans mutant strains, the hyphal adhesin
Als3p was implicated in the adherence of the bacteria to the invasive
hyphae whereas animal co-colonisation with the mutant strain lacking
Als3p did not develop systemic S. aureus infection. Based on the findings
from this study, co-colonisation with C. albicans and S. aureus may allow
hitchhiking of S. aureus with invasive hyphae of C. albicans lead to a
systemic bacterial infection.
MA03 – Tue 1600
Fungal interactions in surgical voice prosthesis infections
Fritz A Mühlschlegel1,5, Mark Baker1, Moira Talpaert2,
Alistair Balfour3, Sarah Stevens4, Campbell Gourlay5
1
Microbiology Service, East Kent University Hospitals NHS Foundation Trust,
Ashford, Kent, UK; 2Pharmacy Service, East Kent University Hospitals NHS
Foundation Trust, Ashford, Kent, UK; 3Head and Neck Service, East Kent
University Hospitals NHS Foundation Trust, Ashford, Kent, UK; 4Speech and
Language Therapy Service, East Kent University Hospitals NHS Foundation
Trust, Ashford, Kent, UK; 5Kent Fungal Group, School of Biosciences, University
of Kent, UK
As part of the regime used to treat patients with laryngeal cancer it is
common to perform a total laryngectomy in which part or the entire
larynx is removed. Following such a procedure the patient will be fitted
with a voice prosthesis that facilitates tracheo‐esophageal speech. A
voice prosthesis is commonly made of silicone rubber and incorporates
a valve that allows air to pass into the esophagus and pharynx for sound
production. A major clinical problem associated with voice prosthesis
is that they can have very short life times. Early voice prosthesis failure
is a result of their tendency to become colonised by micro‐organisms,
and in particular by yeast. To address this problem we have formed a
Voice Prosthesis Infection Management MDT in East Kent. This involves
specialists from Microbiology, ENT and Speech and Language Therapy.
This team considers the evidence‐base but also reaches consensus to
devise a robust clinical care pathway for managing early device failure
associated with yeast infection. Importantly we integrate basic research
into our approach as this will underpin and facilitate clinical practice. In
this presentation we will discuss the clinical picture, introduce the The
Kent Laryngectomy Candida Management Pathway, and discuss fungal
pathogen signal transduction pathways, including the organisms response
to CO2 levels in the patients breath, that may contribute to surgical voice
prosthesis failure.
MA03 – Tue 1630
Building bridges across kingdom divides
Howard F Jenkinson
University of Bristol, Lower Maudlin Street, Bristol, UK
The opportunistic pathogen Candida albicans colonises the human oral
cavity and gastrointestinal tract. Adherence to host cells, extracellular
matrix (ECM), and salivary glycoproteins coating oral surfaces, including
prostheses, is an important pre-requisite for colonisation. In addition,
interactions of C. albicans with oral bacteria such as commensal
streptococci are believed to promote retention and persistence of fungal
cells in mixed-species communities. Building bridges between Candida
and Streptococcus involves interactions of C. albicans hypa-specific surface
proteins e.g. Als3 with streptococcal cell wall anchored surface proteins
e.g. SspB, CshA. Approximately 120 aa residues within the N-terminal
region of Als3p are required for Streptococcus recognition. However, an
amyloid-like adhesive sequence also present in the N-terminal region is
dispensable. Dual kingdom biofilms are more luxuriant than respective
monospecies biofilms, suggesting that there is synergy in colonisation
of the host. Diffusible metabolic signals from Streptococcus, including
competence stimulating peptide (CSP) and autoinducer 2 (AI-2), play
continual roles in modulating microbial cell adherence, eDNA formation
and cell dispersal. Such studies of biologically active polypeptide
sequences and signalling mechanisms provide clues as to how
multicellular biofilms operate and thus how they might be controlled.
Antimicrobial resistance: are
scientists getting the message
right?
MA04
MA04 – Mon 0930
Antibiotic Action – public engagement to stimulate new antibacterial
drug discovery, research and development
Laura J V Piddock
University of Birmingham, UK
Faced with increasing levels of antimicrobial resistance and a paucity
of new antibiotics, in 2009 the British Society for Antimicrobial
Chemotherapy (BSAC) established a working group ‘The Urgent
Need to Regenerate Antibacterial Drug Discovery, Research and
Development’. Reflecting the global nature of the challenges posed,
the working party worked with advisers from the UK, Europe and the
USA, and added to the activities of the IDSA and ReACT. The resulting
report in 2010 indicated that despite the large number of documents by
numerous august bodies in the prior decade, in the main only scientists,
medical microbiologists and infectious diseases physicians were aware
of the issues. One of the recommendations of the working party was
to raise public awareness and stimulate debate amongst all healthcare
professionals, regulators, policy advisers and governments to re-stimulate
interest in antibacterial drug discovery, research and development
and facilitate new, innovative and lasting approaches to the diagnosis,
treatment and prevention of bacterial infections. This resulted in the
launch in November 2011 of ‘Antibiotic Action’ (www.antibioticaction.com) the remit of which is to inform and educate all about
the need for discovery, research and development of new antibacterial
drugs. This talk will cover an overview of the current issues in antibiotic
resistance and the public engagement activities of Antibiotic Action.
MA04 – Mon 1000
Microbiology and the media: experience from the front line
Helen Jamieson
Science Media Centre, 215 Euston Road, London, UK
The worlds of academia and the media couldn’t be further apart –
scientists spend years or even decades on one particular project, while
journalists face hourly deadlines and the need to produce three or four
different stories a day. Given the demands of both environments, and
the challenges of translating complex science between the two, why
should you engage with the media? Opinion polls suggest that the public
get the majority of their information about science from the media, and
microbiology is no stranger to the headlines, so when a story breaks it’s
crucial that journalists hear from the best experts. The Science Media
Centre is an independent press office working on the biggest issues that
make the news, supporting scientists when they find their research in the
eye of the storm.
MA04 – Mon 1100
What are the public’s expectations for antibiotics and how can we
influence them? Developing the TARGET website
Cliodna A M McNulty
Health Protection Agency Primary Care Unit Gloucester, UK
20
Session abstracts
Background The Antimicrobial Stewardship in Primary Care (ASPIC)
collaboration includes the DH, RCGP, HPA, BSAC, CQC, BIA, NPC, IPS
and BPAIIG, RCN, Health Protection Scotland & Public Health Wales
and NI, NHS Information Centre, and interested GPs, pharmacists
& microbiologists. The collaboration working closely with GPs and
medicines managers has assessed current evidence, and materials and
agreed that multifaceted interventions based on the ‘Why’ and ‘How’
proposed by Butler et al should be used to improve antimicrobial
stewardship.
The TARGET toolkit The web-based toolkit was launched on the
Royal college of General Practitioners website www.RCGP.org.
uk/TARGETantibiotics/ on EAAD in 2012, with much media
attention. The toolkit contains a presentation template explaining ‘Why’
antimicrobial stewardship is so important, and ‘How’ the resources can
be used to promote stewardship. The Patient antibiotic leaflet contains
personalised information for patients on how long their illness will last,
when they should return to the doctor and why antibiotics were not
prescribed; empowering patients ,give them confidence to self care for
RTIs. The toolkit also contains the HPA antibiotic guidance template, a
self assessment check list for GPs and commissioners to compare their
antimicrobial stewardship to their peers, audit tools and links to on-line
clinical modules.
Seq analysis using two different protocols. The first was optimised
for mRNA investigations, and proved to be most effective for larger
RNAs, while the second was specific for small RNA species. The two
libraries yielded very different data sets, and collectively provided a
much more comprehensive view of Streptomyces gene expression than
would have been gained by conducting either protocol individually.
We identified hundreds of new small RNAs, the majority of which
appear to be unique to individual Streptomyces species, with only 20%
being conserved in all three species. We also observed considerable
antisense RNA expression, much of which appears to result from the
failure to terminate convergent transcripts. Finally, we detected complex
expression profiles within several antibiotic biosynthetic clusters,
suggesting that there may be novel regulatory mechanisms influencing
antibiotic production.
MA04 – Mon 1130
Antibiotics in an undergraduate curriculum: the good the bad and the
ugly
Laura Bowater
The Norwich Medical School, University of East Anglia, Norwich, UK
The vast majority of undergraduate curricula in the biological sciences
include topics such as molecular and synthetic biology, infection and
immunity and microbiology. The development and usage of antibiotics
has been used to underpin the teaching of key biological theories and
techniques since their discovery in the 20th century. Students are familiar
with the concept of antibiotics as common place tools that are used on
a daily basis in laboratory and clinical settings, but may be less aware of
potential problems and challenges related to their use. Using interactive
discussion groups, this workshop will use the experience and background
knowledge of participants to explore:
• different facets of antibiotics use that undergraduate students
encounter in biological science curricula;
• whether we provide students with clear messages about the
emerging problems of a lack of modern antibiotics and the increasing
number of drug resistant bacteria;
• if undergraduate students receive contradictory messages because
of the way they are taught about antibiotic usage;
• new approaches or suggestions for how we teach undergraduate
students about antibiotic and antibiotic resistance.
A brief report that summarises the findings of this workshop will be
produced and disseminated through appropriate SGM channels.
MA05New approaches to exploit
Streptomyces
MA05 – Mon 1400
RNA tales: comparative transcriptomics in the streptomycetes
Marie Elliot
Department of Biology, McMaster University, 1280 Main Street West,
Hamilton, Ontario, Canada
To begin to understand more about transcription dynamics in the
streptomycetes, and the extent to which non-coding RNA plays a
role, we conducted RNA Seq experiments for three relatively distantly
related Streptomyces species: Streptomyces coelicolor, S. avermitilis and
S. venezuelae. RNA was isolated from cells during vegetative growth,
aerial hyphae formation and sporulation, and was subjected to RNA
MA05 – Mon 1430
Offered paper A novel bifunctional histone protein in Streptomyces:
a candidate for structural coupling between DNA conformation and
transcription during development and stress?
Matthew Aldridge, Paul Facey, Lewis Francis, Sion Bayliss,
Ricardo Del Sol, Paul Dyson
Swansea University, Swansea, UK
Antibiotic-producing Streptomyces are complex bacteria that remodel
global transcription patterns and their nucleoids during development.
Here we describe a novel developmentally-regulated nucleoid-associated
protein, DdbA (Dks-like DNA Binding protein), that consists of an
N-terminal DNA binding histone H1-like domain and a C-terminal
DksA-like domain that can potentially modulate RNA Polymerase activity
in conjunction with ppGpp. Due to its N-terminal domain, the protein
can efficiently bind and condense DNA in vitro. Loss of function of this
DNA binding protein results in changes in both DNA condensation
during development and the ability to adjust DNA supercoiling in
response to osmotic stress. Initial analysis of the DksA-like activity
of DdbA indicates that overexpression of the protein suppresses a
conditional deficiency in antibiotic production of relA mutants that are
unable to synthesise ppGpp, just as DksA overexpression in E. coli
can suppress ppGppO phenotypes. The null mutant is also sensitive to
oxidative stress due to impaired upregulation of transcription of sigR,
encoding an alternative sigma factor. Consequently, we propose this
bifunctional histone-like protein as a candidate that can structurally
couple changes in DNA conformation and transcription during the
streptomycete life-cycle and in response to stress.
MA05 – Mon 1445
Offered paper RNA polymerase distribution, rather than the
transcriptome, better predicts the physiological state of Streptomyces
coelicolor
Colin P Smith1, Emma Laing1, Giselda Bucca1, Huihai Wu1,
Andrzej M Kierzek1, David A Hodgson2
1
University of Surrey, Guildford, UK, 2The University of Warwick, Coventry, UK
Our ability to exploit streptomycetes will be greatly facilitated if we can
accurately model its physiology in silico. We have compared the ability of
genome-wide RNA polymerase distribution data and transcriptome data
to predict the growth state of the model streptomycete, Streptomyces
coelicolor. Surprisingly, we found that RNA polymerase occupancy, but
not the relative transcript abundance, accurately predicted the growth
state of S. coelicolor when the respective data sets were analysed in the
context of the organism’s genome scale metabolic reaction network.
The simulations revealed how the transcription programme governs
metabolic adaptation to the growth phase. In stationary phase key
genes responsible for peptidoglycan biosynthesis and cell division were
down-regulated, accounting for the observed reduction in growth rate.
This integrative analysis also highlighted changes in metabolic fluxes that
were not directly associated with differentially transcribed genes, such
as increased flux through a specific thioredoxin reductase in stationary
21
Session abstracts
phase. We conclude that the positioning of RNA polymerase at
promoter regions, but not the steady state transcript levels, accurately
predicts the growth state of Streptomyces. This reflects the importance of
post-transcriptional control in stationary growth phase.
MA05 – Mon 1500
Offered paper Global and differential RNA-seq analyses of the
transcriptomes of Streptomyces coelicolor and other actinomycetes:
comparison with other methods and data for Escherichia coli
Kenneth McDowall
School of Molecular and Cellular Biology, Faculty of Biological Sciences,
Streptomyces coelicolor serves as a model for studying a group of GC-rich
bacteria that are renowned as the foremost source of clinically useful
antibiotics and many other therapeutics such as anti-helminthics and
anticancer agents. Like many model organisms, its genome has been
sequenced, producing a catalogue of protein-coding and stable-RNA
genes that enables study using ‘omic’ approaches. This has lead to a rapid
expansion in our knowledge of patterns of gene expression and their
dependency on growth conditions, stage of morphological development
and individual genes. However, the underlying networks of gene
regulation are often obscure and may involve the control of steps in
transcription, translation, and RNA processing and degradation. Here we
present a simplified differential RNA-seq approach that greatly expands
the identification of transcriptional start sites of all classes of RNA,
including small regulatory RNAs, and provides excellent coverage of sites
of RNA processing and degradation. Moreover, it is highly sensitive and
has enabled us to detect a background of ‘pervasive’ transcription that
originates from specific sites and the signatures of specialised ribosomes.
Comparison with equivalent data from E. coli and other bacteria is
revealing new difference in, for example, the prevalence and ontology of
leaderless mRNAs. Continuing annotation of the primary and secondary
transcriptome of S. coelicolor will provide systems-level knowledge that
will aid the modelling of antibiotic production as well as the discovery of
pathways that can be manipulated to stimulate secondary metabolism,
much of which is silent under current laboratory conditions.
MA05 – Mon 1515
Offered paper RbpA, a novel transcriptional activator in Streptomyces
coelicolor A3 (2)
Aline Tabib-Salazar1, Richard Lewis1, Steve Matthews2, Mark Paget1
1
University of Sussex, Brighton, East Sussex, UK, 2Imperial College London,
London, UK
RbpA is a RNA polymerase-binding protein that was identified in
Streptomyces coelicolor. It is found in all Actinobacteria, including the
pathogenic agents Mycobacterium tuberculosis, Mycobacterium leprae and
Corynebacterium diphtheriae. Streptomyces strains that have an rbpA
mutation grow at a slower rate than the wild-type and are more sensitive
to the RNA polymerase-targeting antibiotic, rifampicin. Using bacterial
two-hybrid analysis, a number of sigma factors across the different groups
of the σ70 family were tested for their interaction with RbpA and the
exact region of interaction was mapped. Site-directed mutagenesis on
RbpA identified important residues involved in sigma interaction as well
as residues that might be involved in transcriptional activation. Structural
studies on RbpA have resulted in its partial structure to be solved.
MA05 – Mon 1600
Breaking transcriptional locks to discover novel Streptomyces natural
products
Christophe Corre
Chemical Biology Research Laboratory, Department of Chemistry, University
of Warwick, Library Road, Coventry, UK
Mining of actinomycete genomes has revealed the presence of many
cryptic secondary metabolic gene clusters. However, many of these gene
clusters are not expressed in the laboratory environment. Interestingly
pairs of ArpA-like transcriptional regulators are often found within these
gene clusters and are proposed to specifically regulate these pathways. In
addition to hunting for cognate ArpA-like receptor ligands, gene knock-out
of specific repressors have been carried out in Streptomyces strains and
novel metabolites have been identified, purified and characterised. In vitro
studies with recombinant ArpA-like proteins have also been carried out to
investigate DNA-binding sequence specificity and ligand binding specificity.
MA05 – Mon 1630
Offered paper Planosporicin biosynthesis and its regulation in
Planomonospora alba
Emma Sherwood, Mervyn Bibb
John Innes Centre, Norwich, UK
Planosporicin is a lantibiotic produced by the actinomycete
Planomonospora alba that inhibits cell wall biosynthesis in Gram-positive
pathogens.
Genome mining, combined with heterologous expression in
Nonomuraea sp. and deletion analysis in P. alba, were used to identify a
cluster of 15 genes involved in the production of planosporicin, which
occurred upon entry into stationary phase in the natural producer. RTPCR analysis indicated that the cluster contained three operons, while
bioinformatic analysis revealed three putative regulatory genes encoding
a helix-turn-helix DNA binding protein (pspR), an extracytoplasmic
function (ECF) σ factor (pspX) and its likely cognate anti-σ factor
(pspW). Deletion of pspR or pspX abolished planosporicin production,
while deletion of pspW resulted in over-production. Quantitative RTPCR and S1 nuclease protection analyses were used to identify the likely
promoter elements directly controlled by pspX, which, together with
that ofpspR, were confirmed using transcriptional fusions in Streptomyces
coelicolor. We propose a model for the regulation of planosporicin
biosynthesis in which the production of sub-inhibitory concentrations
of the lantibiotic function in a feed-forward mechanism to trigger high
levels of biosynthesis. Thus planosporicin may function not only as an
antibiotic but also as a signaling molecule to coordinate production in the
population as a whole.
MA05 – Mon 1645
Offered paper The discrete acyltransferases of kirromycin biosynthesis
in Streptomyces collinus
Ewa M Musiol1, Andreas Kulik1, Irina Koryakina2, Gavin J Williams2,
Wolfgang Wohlleben1, Tilmann Weber1
1
Interfaculty Institute of Microbiology and Infection Medicine, University of
Tübingen, Tübingen, Germany, 2Department of Chemistry, North Carolina
State University, Raleigh, NC, USA
Kirromycin, an antibiotic produced by Streptomyces collinus Tü 365, is
a potent inhibitor of bacterial protein biosynthesis. Genome mining
methods were used to identify the kirromycin biosynthetic gene cluster
in a genomic library of the producer [1]. The kirromycin biosynthesis
genes were identified on an 82 kb genomic region. A striking feature
of kirromycin biosynthesis is its hybrid trans-AT PKS/cis-AT PKS/NRPS
biosynthetic machinery.
With exception of the ACP of module 5, malonyl-CoA-derived
extender units are loaded to the ACPs of the trans-AT-PKS by the
discrete acyltransferase KirCI. An ethylmalonyl-CoA extender unit is
transferred to the ACP of module 5 of the PKS by a second discrete AT,
KirCII, which also is encoded in the biosynthetic gene cluster [2]. KirCII
currently is the only biochemically characterised discrete AT loading
non-malonate units to ACPs of trans-AT PKS. Interestingly, the enzyme is
also able to transfer un-natural derivatives of ethylmalonyl-CoA to ACPs
in vitro [3]. This makes KirCII an interesting tool for synthetic biology
approaches to manipulate PKS in antibiotic biosynthetic pathways.
References 1. Weber, T., et al. Chem. Biol. 2008, 15, 175-188; 2. Musiol,
E. M. et al., Chem. Biol. 2011, 18, 438-444; 3. Koryakina, I. et al., ACS
Chem. Biol., ASAP, DOI:10.1021/cb3003489
22
Session abstracts
MA05 – Mon 1700
Offered paper Antimycin biosynthesis is regulated by a unique
pathway-specific RNA polymerase sigma factor
Ryan F Seipke, Elaine Patrick, Matthew I Hutchings
University of East Anglia, Norwich, UK
Antimycins are made by Streptomyces bacteria and inhibit the terminal
step in electron transport. They have potent bioactivity against a range
of organisms and are selective inhibitors of the Bcl-2/Bcl-xL family of
anti-apoptotic proteins which are over-produced in drug-resistant
cancers. Although antimycins were discovered more than 60 years ago
the encoding gene cluster and biosynthetic pathway were only recently
discovered and nothing is known about the regulation of antimycin
biosynthesis. We report cloning of the antimycin gene cluster and analysis
of its regulation in Streptomyces albus S4. The antBA, antCDE, antFG
and antHIJKLMNO operons encode antimycin biosynthesis and are only
expressed in substrate mycelium while antimycins are only produced
following differentiation to produce aerial mycelium. antA encodes
the unique RNA polymerase sigma factor σAntA that is only found in
antimycin-producing strains. antFG and antHIJKLMNO, which dictate
biosynthesis of the precursor 3-amino salicylate, are solely dependent on
σAntA and the promoter elements recognised by σAntA are conserved at
the antFG and antHIJKLMNO promoters in all known antimycin clusters,
suggesting a common regulatory mechanism. To our knowledge this is
the first report of this new σ factor sub-family and the first report of a σ
factor controlling antibiotic biosynthesis in Streptomyces.
MA05 – Tue 0900
Analysis and control of Streptomyces morphology in liquid-grown
cultures
Jerre van Veluw, Marloes Petrus, Jacob Gubbens, Gilles van Wezel,
Han Wösten, Dennis Claessen
Molecular Biotechnology, Institute Biology Leiden, Leiden University, Sylviusweg
72, Leiden, The Netherlands
Streptomycetes are filamentous microorganisms that form mycelial
pellets consisting of interconnected hyphae. We have recently used a
flow cytometry approach designed for large particles to show that liquidgrown Streptomyces cultures consist of two distinct populations of pellets
that differ in size. Interestingly, the size of the population of small pellets
is constant, whereas the other population contains larger pellets whose
diameter depends on the strain, the age of the culture and medium
composition. Strikingly, deletion of the genes encoding the conserved
chaplin cell surface proteins leads to disintegration of all pellets and
hyphal death, inferring that these proteins play an instrumental role in
pellet architecture and viability.
MA05 – Tue 0930
Offered paper Inducible gene expression in Streptomyces coelicolor by
means of synthetic riboswitches
Martin M Rudolph, Michael-Paul Vockenhuber, Beatrix Suess
Technical University Darmstadt, Darmstadt, Germany
We applied synthetic riboswitches as genetic control elements in
Streptomyces coelicolor for the conditional control of gene expression.
Natural riboswitches occur within the mRNA -mainly in the 5’UTRs
-and consist of a binding domain (aptamer domain) and an expression
platform. The aptamer domain is able to recognise its particular
ligand with a high binding affinity and specificity. The binding signal
is transmitted to the expression platform and results in altered
gene expression. We utilised synthetic riboswitches to control gene
expression via the interaction between the small molecule theophylline
and the mRNA. Only in the presence of theophylline the sequestration
of the Shine-Dalgarno sequence and the translational start codon is
abolished rendering the translation possible.
Our results indicate, that theophylline-dependent riboswitches
are an appropriate alternative to common used conditional gene
expression systems in S. coelicolor. They are dose dependent and are
combinable with every promoter facilitating the adjustment of desired
gene expression levels. We anticipate that these riboswitches will be
applicable also for other Streptomyces species to express native and
heterologous proteins.
MA05 – Tue 0945
Offered paper Localisation of Streptomyces oriC region reveals
organisation of the tip proximal chromosome by ParAB proteins
Agnieszka Kois-Ostrowska1, Emma Tilley2, Paul Herron2,
Jolanta Zakrzewska-Czerwinska1,3, Dagmara Jakimowicz1,3
1
1-University of Wroclaw, Faculty of Biotechnology, Department of Molecular
Microbiology, Wroclaw, Poland, 22-University of Strathclyde, SIPBS, Glasgow,
UK, 33-Institute of Immunology and Experimental Therapy, Department of
Molecular Microbiology of Microorganism, Wroclaw, Poland
In Streptomyces, like in many other bacteria, the ParAB-parS system
is involved in chromosome segregation. ParAB proteins uniformly
distribute chromosomes along aerial hyphae ensuring each prespore
receives a chromosome. Little is known about ParAB function in
vegetative hyphae, where there is apparently no active chromosome
segregation. Streptomyces ParB protein binds to parS sites around origin
of replication (oriC) region forming large nucleoprotein complexes,
which in vegetative hyphae are observed as a bright focus close to
tip. Here, we report an application of a FROS system to visualise oriC
region, which revealed a novel function of ParAB proteins: organisation
of chromosomes at the tip. Microscopy analysis of parAB deletion strains
using FROS demonstrated that oriC region at the tip is anchored by
ParAB. Moreover, a comprehensive analysis of replisome (DnaN-EGFP)
and segregosome (ParB-EGFP) localisations in strains carrying either
deletion of parA/parB and overexpressing ParA/B suggested that ParAB
proteins are involved in silencing of the replication of the tip proximal
chromosome. On the basis of these results we propose the model of
the organisation of the chromosomes by ParAB proteins in vegetative
mycelium.
MA05 – Tue 1000
Offered paper Studying apical growth to unlock secrets of Streptomyces
development
Neil Holmes1,2, Michael Gillespie1, Richard Leggett1, John Walshaw2,
Gabriella Kelemen1
1
University of East Anglia, Norwich Research Park, Norwich, UK, 2John Innes
Centre, Norwich Research Park, Norwich, UK
The actinomycetes represent some of the first examples of filamentous
growth that is achieved by polar cell wall extension and branching.
As a bacterial model organism for development that is linked to
the production of antibiotics and other secondary metabolites, the
cell biology of Streptomyces coelicolor, a model actinomycete, is of
distinct interest. Streptomyces has a complex life cycle, beginning from
germination of a spore which requires apical growth. Two distinct
mycelia can be produced; first they produce a vegetative mycelium
and secondly aerial hyphae that are capable of forming a linear chain of
spores, completing the life cycle.
A multi-protein complex drives apical growth by positioning cell
wall extension, and sites of new branch formation. Here we present
several approaches that were used to identify the role of one of the
key proteins, Scy, which is essential to correct tip functioning and
morphogenesis. Changing the cellular levels of Scy has a severe effect on
the positioning of the cell wall machinery and new tip emergence. Scy
is also necessary for correct chromosome segregation and cell division.
Understanding the intricacies of apical growth through the use of cell
biology could yield vital information which could help exploitation of the
actinomycetes.
23
Session abstracts
MA05 – Tue 1015
Offered paper The role of phospholipids in growth and development
in Streptomyces coelicolor
John Tiong, Khanungkan Klanbut, Maeidh Alotaibi, Paul Herron
University of Strathclyde, Glasgow, UK
Over the last 50 years many proteins have been identified that are
required for growth and development of streptomycetes. Despite this,
other cellular components, such as membrane phospholipids, have
revealed little attention. Using a combination of genetic, microscopic
and lipidomic techniques we described recently the involvement of the
anionic phospholipid, cardiolipin in tip extension of vegetative hyphae
and erection of aerial hyphae of the model organism, Streptomyces
coelicolor. Cardiolipin localised to tip and branch points and potentially
recruits proteins required for tip extension to negatively curved
membrane regions. More recently we have established the requiremnent
of genes encoding the biosynthesis of other phospholipids for
streptomycetes growth, begun to characterise their patterns of gene
expression and the role of these phospholipids in septal placement.
MA05 – Tue 1100
Offered paper Endophytic actinomycetes act as plant growth
enhancers, pathogen protectors and stress reducers
Arinthip Thamchaipenet
Department of Genetics, Faculty of Science, Kasetsart University, Bangkok,
Thailand
Endophytic actinomycetes were found diversely in various plant species.
Several new genus and species as well as novel bioactive compounds
have been discovered suggesting that they are rich sources for microbial
diversity and new drug discovery. Moreover, these plant dwelling
actinomycetes exhibit PGPB properties (i.e. phosphate solubilisation,
siderophore production, IAA production, ACC deaminase and chitinase
activities) as free-living microorganisms. We have demonstrated that they
help significantly promote plant growth by increase of root and shoot
biomass in rice, mungbean and maize plants. Furthermore, a chitinaseproducing endophytic actinomycete could secure maize plants from
foot rot and wilting diseases infected by Fusarium moniliforme. Recent
investigation on plant-actinomycete interaction was approached by
generating of some single PGPB-trait mutants (e.g. siderophore-deficient
and ACC deaminase-deficient mutants). The siderophore-deficient
mutant clearly demonstrated the effects of this important trait involved
in growth enhancement of rice and mungbean plants with and without
iron. The ACC deaminase-deficient mutant evidently indicated that ACC
deaminase could improve plant resistance to stress conditions including
salinity and flooding. Our results highlight the value of a substantial
understanding of the relationship of the endophytic actinomycetes
towards the plants.
MA05 – Tue 1115
Offered paper 16S phylogeny of the Streptomycetaceae
Alan C Ward1, Nagamani Bora2
1
Newcastle University, Newcastle upon Tyne, UK, 2De Montfort University,
Leicester, UK
Bacterial taxonomy has been a series of paradigm shifts, overturning
previous schemes and changing names. Phylogenetic analysis of 16S is
the basis of the latest Bergey’s manual (1) but is already overtaken by
multi-gene sequencing and whole genome analysis.
Nevertheless, as 16S is comprehensive, universal data, the phylogeny
of the Streptomyces has been recently completed for validly described
species (2,3) providing a reference framework.
However, constant changes can leave taxonomy for taxonomists (4) and
key strains missing from these analyses, many strains in whole genome
sequencing are not validly described species, or described then or
subsequently, and some descriptions are misleading.
7,610 16S sequences from public databases are analysed by standard
phylogenetic methods giving trees and 3D cluster analysis describing the
full diversity of studied strains. It’s application, as a framework for analysis
of antibiotic gene clusters using antiSMASH (5) will be illustrated.
References 1. Bergey’s Manual of Systematic Bacteriology 2nd Edition;
2. Labeda, DP et al. (2012) Antonie van Leeuwenhoek 101, 73-104; 3.
Kämpfer, P. (2012) in Volume 5 The Actinobacteria pp. 1446-1804 of
Bergey’s Manual of Systematic Bacteriology 2nd Edition; 4. Hopwood, DA
(1999) Microbiology-UK, 145, 2183-2202; 5. Medema, MH et al. (2011)
Nucleic Acids Research 39, W339-W346
MA05 – Tue 1130
Offered paper Streptomyces coelicolor Dps proteins; structural and
functional insights
Matthew D Hitchings1, Paul D Facey1, J Hugh Jones1, Ehmke Pohl2,
Paul J Dyson1, Ricardo Del Sol1
1
Swansea University, Swansea, UK, 2The three DNA protection proteins from
starved cells of Streptomyces coelicolor are members of the mini-ferritin
super family that have previously been found to be induced in a stimulus
dependant mann, Durham, UK
The three DNA protection proteins from starved cells of Streptomyces
coelicolor are members of the mini-ferritin super family that have
previously been found to be induced in a stimulus dependant manner.
Found in many prokaryotes, Dps proteins are considered to be of major
importance to stress responses that can aid microbial survival in extreme
conditions. This study investigates the structural and functional properties
of scDps proteins that facilitate their roles within S. coelicolor and finds
scDpsA and scDpsC are capable of oxidising and depositing iron oxide
particles of nano scale within their hallow negatively charged internal
cavities. Interestingly, it was found the major architectural difference
between the proteins came from the geometry of the variable length
amino and carboxylate tail extensions that protrude from the core four
helix bundle of the protein monomer. Assessment of mutant proteins
lacking tails proved them invaluable for oligomeric assembly and stability,
however; these proteins retained their ferroxidase activity capable of
protecting DNA against oxidative damage.
MA05 – Tue 1145
Offered paper The sweet tooth of Streptomyces: elucidating glycogen
synthesis in Streptomyces venezuelae
F Y Miah, M J Buttner, K F Chater, S Bornemann
John Innes Centre, Norwich, Norfolk, UK
Glycogen is a ubiquitous carbon storage sugar found in the
Animalia, Plantae, Fungi and Bacteria kingdoms. A novel glycogen
synthesis pathway, called the GlgE pathway, was discovered by us in
actinobacteria1. The gene encoding the maltosyltransferase GlgE is
essential in Mycobacterium tuberculosis and is currently being explored as
a potential drug target against this deadly human pathogen. Remarkably,
the pathway is relatively wide-spread; 14% of sequenced bacterial
genomes contain all the genes of the pathway2. It is therefore important
to gain a fundamental understanding of how the pathway operates. In
Streptomyces species, the pathway usually co-exists with another long
characterised glycogen biosynthesis pathway. Glycogen has been found in
a wide range of Streptomyces species where it is thought to be converted
to trehalose, which is abundant in spores. We aim to understand
glycogen synthesis in Streptomyces venezuelae, which only has the genes
encoding one glycogen synthesis pathway, the GlgE pathway. We have
taken a mutli-disciplinary approach to explore glycogen metabolism
in S. venezuelae ranging from reverse genetics to biochemistry and
enzymology.
References 1 R. Kalscheuer et al., Nat. Chem. Biol., 6 (2010) 376-384; 2
G. Chandra et al., Microbiology-(UK), 157 (2011) 1565-1572.
24
Session abstracts
MA05 – Tue 1400
RiPPing yarns – tales of actinomycete ribosomally synthesised posttranslationally modified peptide antibiotics
Mervyn Bibb
Department of Molecular Microbiology, John Innes Centre, Norwich, UK
Actinomycetes produce about two-thirds of all known antibiotics of
microbial origin, and remain an important source of potentially clinically
useful compounds. These include ribosomally synthesised and posttranslationally modified peptides (RiPPs). Using genome mining, we have
isolated and characterised gene clusters responsible for the biosynthesis
of the RiPPs cinnamycin, microbisporicin, cypemycin, venezuelin,
bottromycin and planosporicin. This presentation will describe
experiments that have revealed novel enzymology, novel regulatory
mechanisms, a novel mechanism of immunity, and a crucial role for
immunity in regulating the onset of antibiotic biosynthesis; evidence
will also be presented that suggests that at least some RiPPs serve as
signalling molecules to induce their own synthesis.
References Claesen J, Bibb MJ (2010) PNAS 107:16297-16302; Foulston
LC, Bibb MJ (2010) PNAS 107:13461-13466; Foulston LC, Bibb MJ
(2011) J Bacteriol 193:3064–3071; Gomez-Escribano JP, Song L, Bibb MJ,
Challis GL (2012) Chem Sci 3:3522-3525; Goto Y, Li B, Claesen J, Shi
Y, Bibb MJ, van der Donk WA (2010) PLoS Biol, 8: e1000339; Widdick
D, Dodd H, Barraille P, White J, Chater KF, Gasson M, Bibb MJ (2003)
PNAS 100:4316-4321.
attR. Excision is the reaction between attL and attR that regenerates
attP and attB. PhiC31 integrase is a member of the serine recombinase
family of proteins, all of which have a conserved domain that is required
for catalysis and DNA strand exchange. The large C-terminal domain
in phiC31 integrase is characteristic of the serine integrases and is
required for recognition of the different substrates (attP, attB, attL and
attR) and for the control over which substrate pairs recombine. In the
absence of the phiC31 early protein, gp3, integrase only mediates the
integration reaction (attP x attB). If gp3 is present integrase preferentially
performs the excision reaction (attL x attR). This exquisite control over
integration versus excision enables more versatile genome engineering
technologies and the construction of novel organisms that can record
digital information.
MA05 – Tue 1430
Offered paper Understanding the lipoprotein biogenesis pathway: an
essential pathway in Streptomyces?
John T Munnoch, Matthew I Hutchings
University of East Anglia, Norwich, UK
Lipoproteins, a distinct class of lipidated proteins anchored to the
cell membrane, encompass a range of protein functions involved in
interacting with the inter/extra-cellular environment. The Streptomyces
coelicolor lipoprotein biogenesis pathway, unlike in other gram positive
bacteria, is most likely essential. S. coelicolor has three major enzymatic
steps involved: Two Lipoprotein diacylglycerol transferase (Lgt) enzymes
lipidate a conserved cysteine residue at the C-terminal end of the signal
peptide, Lipoprotein signal peptidase (Lsp) then cleaves the N-terminal
signal sequence leaving the lipidated cysteine as the N-terminal amino
acid and two non-essential Lnt enzymes then N-acylate the cysteine
residue to make a triacylated lipoprotein. Each lgt gene has been
successfully deleted individually however a double deletion could not
be isolated. A Δlsp strain was isolated and has a small colony phenotype
which could not be fully complemented. Genome sequencing of the
lsp mutant and its parent strain revealed the insertion of a transposon,
IS21 as well as a variety of SNPs. RNA sequence data revealed the
transposon has inserted into a previously unknown small RNA and stable
lsp mutants can be recovered with much higher frequency in a strain
carrying the IS21 mutation suggesting disruption of this sRNA is an lsp
suppressor.
MA05 – Tue 1445
Offered paper Mechanism and applications of phage-encoded serine
integrases
MAggie Smith1,2, Jane Paget2, Stephen McMahon3, Thanafez Khaleel2,
Andy McEwan2, Jim Naismith3
1
University of York, York, UK, 2University of Aberdeen, Aberdeen, UK,
3
University of St. Andrews, St Andrews, UK
Site-specific recombinases are useful for DNA assembly and genome
engineering. Phage integrases are particularly useful as they are controlled
by accessory proteins, called recombination directionality factors (RDFs).
Integrases act on the phage and host attachment sites, attP and attB
respectively, to integrate the phage genome into the host chromosome
resulting in the prophage flanked by the recombinant sites, attL and
MA05 – Tue 1500
Offered paper sRNA scr5239 controls the expression of the
methionine synthase metE
Michael Vockenhuber, Beatrix Suess
TU Darmstadt, Darmstadt, Germany
We are interested in the identification and characterisation of small noncoding RNAs (sRNAs) in Streptomyces coelicolor. To find such transcripts
we performed deep sequencing of the S. coelicolor transcriptome [1].
One sRNA -a 159 nt transcript called scr5239 -especially attracted
our interest because of its high degree of sequence and structure
conservation. It is constitutively expressed under most conditions tested
and its 2D structure -as validated by enzymatic probing -consists of five
independent stem-loops.
Overexpression or deletion of scr5239 results in distinct macroscopic
phenotypes. The scr5239 overproduction strain does not express
the agarase DagA and therefore cannot use agar as a carbon source
[2]. DagA, however, is not the only target of scr5239. We identified
a second target, the methionine syntase metE. The expression of the
scr5239 is induced by a high methionine level and -according to our
current model -represses metE translation in a feedback loop. Here the
sRNA seems to replace a SAM riboswitch in the regulational network of
metE expression.
Currently, we are characterising the biochemical and genetic features of
this interaction.
References. 1 Vockenhuber M.-P. et al. RNA Biol 2011; 2. Vockenhuber
M.-P., and B. Suess. Microbiology, 2011
MA05 – Tue 1515
Offered paper Streptomyces: the beauty of the beast and its
exploitation for the discovery of novel antimicrobials
G P van Wezel
Leiden University, Leiden, The Netherlands
Central in this talk is Streptomyces, a filamentous soil bacterium that
produces half of all known antibiotics and a range of other natural
products and enzymes. The beauty lies in the organism itself, with its
wonderful development, and in the often colourful natural products it
produces. The treasures that lie hidden in the actinomycete genomes
may well be our final resource in the fight against the rapidly emerging
multi-drug resistant pathogens. Development and antibiotic production
are highly coordinated in this complex microorganism, and we aim to
understand the global regulatory networks that determine the switch
from normal growth to the developmental programme. A major control
system revolves around the nutrient sensory protein DasR, which links
primary metabolism to the control of antibiotic production. Key aspects
of the DasR regulatory network, which is one of the largest regulons
found in bacteria, will be discussed. I will also show how we apply this
knowledge to develop novel approaches to wake up silent antibiotics.
Our NGS-based proteomining technology thereby fascilitates rapid
linkage of novel treasures to the gene clusters responsible for their
production.
25
Session abstracts
MA05 – Tue 1600
The nitric oxide sensor NsrR is required for sporulation in
Streptomyces coelicolor
Matt Hutchings
University of East Anglia, Norwich Research Park, Norwich, UK
Nitric oxide (NO) is a toxic, antimicrobial gas that also functions as a
signalling molecule in eukaryotes. Responses in bacteria include NO
detoxification but more recently NO has also been implicated in the
modulation of more complex processes including antibiotic resistance,
dormancy and multicellular behaviour suggesting NO also acts as a
signalling molecule in bacteria. The most widespread NO sensing
transcription factor in bacteria is NsrR, which senses NO via an iron
sulfur (Fe-S) cluster and responds by modulating the expression of target
genes, including the NO dioxygenase gene hmpA. There is also evidence
to suggest that apo-NsrR regulates a different subset of genes in Bacillus
subtilis. We have very recently shown that deletion of nsrR, in the
multicellular bacterium Streptomyces coelicolor prevents spore formation.
Since the Wbl proteins WhiB and WhiD also contain NO sensitive
Fe-S clusters and are required for spore formation in S. coelicolor we
hypothesise that NO acts as a signalling molecule to regulate sporulation
in streptomycetes.
MA05 – Tue 1630
Offered paper WhiA – an unusual transcription factor required for
sporulation in Streptomyces
Matthew Bush, Maureen Bibb, Govind Chandra, Mark Buttner
The filamentous bacteria Streptomyces has a complex yet fascinating
life-cycle, culminating in the formation of specialised reproductive
structures called aerial hyphae that extend upwards out of the substrate
mycelium into the air. Subsequently, the orderly arrest of aerial growth
and a remarkable synchronous septation event with concomitant
separation of chromosomes occurs to form long chains of 50-100 unigenomic spores. These two key processes require the developmental
master regulator WhiA. Here, I have defined the WhiA-regulon, using
ChIP-seq combined with microarray analysis in Streptomyces venezuelae.
WhiA binding was confirmed in vitro by DNase I footprinting which also
aided my identification of a WhiA-consensus binding motif. Through
this route, I have identified a large number of genes under the direct
control of WhiA, including those clearly linked to roles in sporulation
and as expected, a large number of genes of unknown function. An
example of one such gene is whtA (whiA target A). A ΔwhtA mutant
does not form the characteristic green pigment associated with mature
S.venezuelae spores. Furthermore, Scanning Electron Microscope analysis
reveals a striking irregularity in the placement of the spore septa during
differentiation of the aerial hyphae, suggesting that the protein has a
previously un-described role in cell division.
MA05 – Tue 1645
Offered paper Genomic mining of bioactive marine sponge
Streptomyces strains SM2 and SM8 uncovers novel cryptic clusters with
antimicrobial activity
Jonathan Kennedy1, Lekha Margassary1, Ciaran O’Brien1,
Ian O’Neill1, RuAngelia Edrada-Ebel2, Christina Veigelmann2,
Fergal O’Gara1, John P Morrissey1, Alan D Dobson1
1
University College Cork, Cork, Ireland, 2University of Strathclyde, Glasgow, UK
Marine sponges are a rich source of biologically active compounds, many
of which are produced by microbial sponge endosymbionts. This work
describes the use of a genomics guided approach to exploit some of the
cryptic biosynthetic potential of marine sponge Streptomyces strains.
Two Streptomyces species, SM2 and SM8, were isolated from the sponge
Haliclona simulans. Both exhibited bioactivities including antifungal,
antibacterial, and anti-calcineurin activities1,2. Chemical analyses of these
strains also indicated the presence of novel secondary metabolites.
Draft genomic sequences revealed the presence of multiple potential
secondary metabolism gene clusters for known and unknown
compounds, several of which have been confirmed to be produced by
LC-MS and NMR. Transcriptional analyses have also shown that a large
number of these gene clusters are not expressed. Diverse culturing
conditions, RT-PCR and heterologous expression approaches are
being used to monitor and induce the expression of these cryptic gene
clusters and extend the range of metabolites available for biodiscovery.
One cryptic cluster in Streptomyces SM2 appears to encode a novel
thiopeptide gene cluster which displays antimicrobial activity against
Bacillus subtilis and Pseudomonas aeruginosa.
References 1. Kennedy et al. Marine Biotechnology 2009, 11, (3), 384396; 2. Margassery et al. J Microbiol Methods 2012, 88, (1), 63-6.
MA05 – Tue 1700
Offered paper Streptomyces as microbial tigers and antibiotics as canines
Milind G Watve1, Charushila A Kumbhar1,2
1
Indian Institute of Science Education and Research Pune, Pune, Maharashtra,
India, 2Abasaheb Garware College, Pune,Maharashtra, India
The natural role of antibiotics in the ecology of Streptomyces is debated
and largely unknown. The predatory Myxobacteria and many other
genera of prokaryotic epibiotic and wolfpack predators across different
taxa possess secondary metabolites with antimicrobial action and
these compounds have a role in predation. If all epibiotic predators are
antibiotic producers it is worth testing whether all antibiotic producers
are predators too. We show here that Streptomyces are non-obligate
epibiotic predators of other microorganisms and that predatory abilities
are widespread in this genus. Using time lapse photomicrography we
demonstrate that the growing tips of Streptomyces hyphae lyse cells
of other bacteria in the vicinity and utilise them for growth when no
other source of nutrients is available. Predatory activity is restricted to
surface growth and is not obligately associated with antibiotic production
in conventional culture. However some of the genes crucial to the
regulation of secondary metabolite pathways are differentially expressed
during predatory growth on different prey species as compared to
saprophytic growth. This suggests a role for antibiotics. Our findings
strengthen the association between epibiotic predation and antibiotic
production raising the possibility that predatory growth may be the key
to a diversity of antibiotics not accessed so far.
MA05 – Tue 1715
Offered paper Use of Streptomyces viridosporus cultures to degrade
lignocellulose
Kaylee A Herbert, John N Wardell, Daniel Discoll,
Claudio Avignone-Rossa, Norman Kirkby, Michael Bushell
Microbial and Cellular Sciences, Faculty of Health and Medical Sciences,
University of Surrey, Surrey, UK
Lignocellulose degradation is being explored as part of the process
of bioethanol production from waste products, such as wood flour.
Identified as a natural lignin degrader, Streptomyces viridosporus
is worthy of investigation for lignocellulose degradation, it can also
produce cellulase enzymes. Liquid cultures of S.viridosporus, were
able to liberate glucose from wood flour (mesh size 40). This finding
indicated considerable potential for using this species in lignocellulose
degradation for commercial exploitation. However, S.viridosporus liquid
cultures have a pre-disposition to form pellets (discrete islands of hyphal
growth) which can inhibit the effectiveness of some hyphal species in
biotechnological processes. The pelleting of cultures was reduced by
manipulating inoculum development. Using a continuous (chemostat)
culture and employing carboxymethylcellulose (CMC) as the main
carbon source, five dilution rates were investigated and the effect of
the steady state cultures on the lignocellulose degradation of wood
flour by S.viridosporus analysed. We have obtained conditions whereby
S.viridosporus can successfully degrade lignocellulose, without using
the liberated sugars as a carbon source using at least two of the three
enzymes present in the cellulase enzyme complex.
26
Session abstracts
Next-generation antimicrobials
MA06
MA06 – Wed 0900
Antimicrobial peptide modulation of chemokine and pro-inflammatory
responses
Lauren E Harvey1, Karl Kohlgraf1, Leslie A Mehalick1, Monica Raina1,
Erica N Recker1, Saumya Radhakrishnan2, Samiksha Avinash Prasad2,
Robinson Vidva2, Ann Progulske-Fox3, Joseph E Cavanaugh4,
Shireen Vali2,5, Kim A Brogden6
1
Dows Institute for Dental Research, College of Dentistry, The University
of Iowa, Iowa City, IA 52242, USA; 2Cellworks Research India Pvt. Ltd.,
3rd Floor, West Wing, ‘Neil Rao Tower’ 118, Road #3, EPIP, Whitefield,
Bangalore-560066, India; 3Center for Molecular Microbiology and Department
of Oral Biology, Box 100424, 1395 Center Drive, University of Florida,
Gainesville, FL 32610, USA; 4Department of Biostatistics, College of Public
Health, The University of Iowa, Iowa City, IA 52242, USA; 5Cellworks
Group, Inc., 13962 Pierce Rd., Saratoga, CA 95070, USA; 6Department of
Periodontics and Dows Institute for Dental Research, N423 DSB, College of
Dentistry, 801 Newton Road, The University of Iowa, Iowa City, IA 52242,
USA
Human β defensin 3 (HBD3) is a small, potent antimicrobial host
defense peptide with diverse innate immune activities. It is prominent
throughout the oronasal cavity at concentrations sometimes reaching
upwards to 6.2 µg/ml. The role of HBD3 in regulating chemokine and
pro-inflammatory cytokine responses to specific pro-inflammatory
antigenic exposure is largely unknown. In our recent work, we report
that HBD3 induces a sharp bidirectional modulation of chemokine and
cytokine responses. Our predicted results in a virtual, integrated dendritic
cell model and our observed results in normal human dendritic cells,
show that 0.2 µM HBD3 significantly (p < 0.05) enhances 6 responses,
attenuates 7 responses, and both enhances/attenuates the CXCL1/GRO
and TNFα responses to Porphyromonas gingivalis hemagglutinin B (HagB),
a potent pro-inflammatory stimulus. In murine JAWS II dendritic cells, 0.2
µM HBD3 significantly attenuates, yet rarely enhances, the CXCL2/MIP2, IL-6, and G-CSF responses to 0.02 µM HagB; and in C57/BL6 mice,
0.2 µM HBD3 significantly enhances, yet rarely attenuates, the CXCL1/
KC, G-CSF, and M-CSF responses to intranasal administration of 0.2
µM HagB. Thus, the timing of HBD3 exposure and the concentration
of HBD3 treatment are all important in the vast pliability of an HBD3altered dendritic cell response to a pro-inflammatory stimulus.
MA06 – Wed 0930
Antibiotics from DNA adenine methyltransferase inhibitors
Jenny C. McKelvie1, Jenny Harmer1, Michael D. Maynard-Smith1,
Robert J. Wood1, Peter L. Roach1, Mark I. Richards2, Tim Milne2,
Petra C. Oyston2
1
School of Chemistry, University of Southampton, Southampton, UK;
2
Biomedical Sciences, Defence Science and Technology Laboratory, Salisbury,
UK (Email: plr2@soton.ac.uk)
Multiple antibiotic resistant strains of plague are emerging, driving a need
for the development of novel antibiotics effective against Yersinia pestis.
DNA adenine methylation regulates numerous fundamental processes
in bacteria and alteration of DNA adenine methlytransferase (Dam)
expression is attenuating for several pathogens, including Y. pestis. The
lack of a functionally similar enzyme in humans makes Dam a suitable
target for development of novel therapeutics for plague.
Using a fluorescence based high throughput assay, we have screened
several libraries, including a high diversity set, a fragment set and a
focussed library of substrate analogues. The large number of initial hits
were down-selected by careful counter screening and optimisation to
provide a small number of lead compounds. These lead inhibitors have
been characterised using kinetic analysis, crystallography of Dam:inhibitor
complexes and transcriptional analysis to identify altered expression of
known virulence traits. These data identify modes of action for some of
the inhibitors and indicate they may be active in cultures of Y. pestis.
MA06 – Wed 1000
Is quorum sensing a viable antimicrobial target?
Paul Williams
Centre for Biomolecular Sciences, University of Nottingham, Nottingham, UK
There are two broad strategies for the control of bacterial infections,
either (a) kill the organism or (b) attenuate virulence such the infecting
organism fails to adapt to the host environment and can be cleared
by host defences. Anti-virulence agents offer potential advantages
including expanding the repertoire of bacterial targets, preserving the
host microflora and exerting less selective pressure, which may result
in decreased resistance. In many pathogens, virulence is co-ordinately
controlled via sophisticated global regulatory systems such as quorum
sensing. This is usually defined as cell population density dependent
gene regulation and is mediated via self-generated extracellular
signal molecules. These activate or repress QS target genes once a
critical threshold concentration of signal has been reached. The key
components of any QS ‘module’ are the QS signal synthase, the signal
receptor and the signal molecule. QS systems thus offer multiple targets
for chemical intervention through the blockade of QS signal synthesis,
QS signal molecule degradation or the inhibition of QS signal reception.
Such targets in conjunction with high throughput screens offer multiple
opportunities for the design of synthetic inhibitors and the discovery of
natural products for the treatment of infections caused by multi-antibiotic
resistant bacteria.
MA06 – Wed 1100
The cell envelope as target of novel antimicrobial therapeutics
Hans-Georg Sahl
University of Bonn, Pharmaceutical Microbiology, Meckenheimer Allee 168,
53155 Bonn, Germany
Antibiotic resistance development is continuously eroding our antiinfective treatment options and in spite of considerable investment in
academia and industry sectors, we are not very successful in bringing
new drugs to the market. Over the last two decades, target-based
screening and subsequent hit development frequently produced potent
inhibitors of the chosen target which however could not be developed
into antibiotics. Apparently, for better prediction of suitable targets, a
deeper understanding of functions and of cellular consequences of target
inactivation is necessary. We studied various classes of natural compound
antibiotics and host defense peptides such as glyco-and lipopetides,
lantibiotics and defensins from mammals and invertebrates. Many of
these compounds target bacterial cell wall (CW) biosynthesis, which is an
‘old’ target, but obviously one of best for antibiotic attack. Most of these
inhibitors form complexes with the bactoprenol-bound intermediates
of cell wall biosynthesis, in particular Lipid II, and simultaneously impair
the functional organisation of the interdependent cell wall biosynthesis
and cell division machineries. Some cause massive perturbation of
these machineries without interaction with a defined molecular target.
The acyldepsipeptides block cell division through interference with the
functions of the intercellular protease ClpP. The examples underline the
necessity to learn more about the cellular stresses triggered by antibiotics
and to think beyond mere drug-target interactions for future antibiotic
development; ‘old’ target may have more to offer in this direction.
Schneider T. et al, Science, 328:1168-1172 (2010)
Schneider T. & H.G. Sahl, Int J Med Microbiol. 300:161-169 (2010)
MA06 – Wed 1130
Potent nanoparticulate oligonucleotide antibacterials to treat MRSA
Michael McArthur1,2, Jane Moore2
1
Procarta Biosystems Ltd., Norwich, UK; 2John Innes Centre, Norwich, UK
The alarming ability of pathogenic bacteria to develop resistance to
antibiotics has made imperative the development of new antibacterials.
Transcription Factor Decoys (TFDs) are oligonucleotide therapeutics
that act on novel targets by interfering with transcription of essential
27
Session abstracts
bacterial genetic pathways to prevent infection. TFDs are encapsulated
into nanoparticles that cross the bacterial membrane, triggering a well
coordinated stress response that the oligonucleotides are designed to
block, leading to a rapid and potent inhibition of growth. The efficacy
of the approach has been demonstrated using TFDs that target the
alternative sigma factor, SigB, which is part of the Cell Wall Stress
response, leading to effective treatment of MRSA infections (EMRSA-16)
in vitro at nanomolar concentrations and in vivo using mouse sepsis models.
By demonstrating that genetic regulation is a credible therapeutic target
we anticipate being able to develop a wide range of novel antibacterials
with defined spectra that can be used to tackle current drug-resistant
strains and also be rapidly developed to counter emerging threats.
MA06 – Wed 1400
Offered paper Turnabout is fairplay – use of a bacterial adhesin as an
antimicrobial agent
Anne-Marie Krachler1, Kim Orth2
1
Institute of Microbiology and Infection, The University of Birmingham,
Birmingham, UK, 2UT Southwestern Medical Center, Dallas, Texas, USA
Bacterial pathogens target and manipulate host cells to ensure their
own survival and propagation using an arsenal of toxins and effectorproteins. Delivery of this bacterial weaponry requires intimate contact
between bacteria and host mediated by adhesins. We discovered a novel
class of adhesins (Multivalent adhesion molecules -MAM) employed
by a wide range of bacterial pathogens to ensure tight host cell-binding
during the earliest stages of infection. MAMs consist of six to seven
repetitive domains, which we show is the minimal number required for
competition with other bacterial species. We describe the correlation
between repeat number and binding affinities and dissect this MAMmediated host-pathogen interaction in two discrete binding events:
initial, transient binding to the extracellular matrix protein fibronectin and
subsequent tight binding to the host cell lipid phosphatidic acid. Using
fluorescent bead-coupled adhesins as a minimalist approach to mimic
bacteria, we study how bacteria exploit multivalency both on the level
of individual protein-repeats as well as on the level of MAM surfacedensity to achieve fast and stable host cell-binding and outcompete
other bacterial species. We use this information to engineer MAMs with
improved affinity and avidity and describe the use of these bacteriomimetics as novel approach to fight bacterial infections.
MA06 – Wed 1415
Offered paper Deciphering the secrets of short antimicrobial peptides
for therapeutic use
Ralf Mikut1, Serge Ruden1, Jonas van den Berg1, Christine Wittmann1,
Markus Reischl1, Ilona Schreck1, Frank Breitling1, Clemens Grabher1,
Carsten Weiss1, Rudolf Volkmer2, Kai Hilpert3,1
1
Karlsruhe Institute of Technology (KIT), Karlsruhe, Germany,
2
Universitätsklinikum Charité, Humboldt-Universität zu Berlin, Berlin,
Germany, 3St George’s, University of London, London, UK
Antimicrobial peptides (AMPs) constitute an important part of the innate
immunity fighting invading pathogens. AMPs are capable of killing a broad
range of pathogens, including life-threatening multidrug-resistant bacteria
that challenge the success and achievements of modern medicine.
However, only very few AMPs are in clinical trials or available on the
market. One reason might be the inherent cytotoxicity against human
cells. Here we study how antibacterial activity and cytotoxicity are
connected. To accomplish this, 3,000 different artificial 9mer peptides
were synthesised and for each of those peptides a concentration
dependent killing concentration against Pseudomonas aeruginosa as well
as a hemolytic concentration was determined. Focusing on the most
antibacterial active 30 short AMPs, antibacterial activity was determined
against several pathogenic strains and cytotoxicity was studied in cell
culture experiments, and by performing studies in a very sensitive
whole organism, the zebrafish larvae. This approach led to new peptide
knowledge, which may help accelerate the process of developing new
peptide antibiotics against several types of multidrug-resistant bacteria,
as well as of searching for novel natural peptides with intrinsic antibiotic
activity.
MA06 – Wed 1430
Offered paper Live-cell imaging and mode-of-action of a new
generation of small, synthetic antifungal peptides
Alberto Muñoz1, Akira Alexander1, Dilip M Shah2, Jose F Marcos3,
Nick D Read1
1
The University of Edinburgh, Edinburgh, UK, 2Donald Danforth Plant Science
Center, St Louis, MO, USA, 3Instituto de Agroquímica y Tecnología de
Alimentos-CSIC, Valencia, Spain
The significance of fungal infections has been grossly underestimated,
few drugs are available to treat life-threatening fungal infections, and
resistance against these drugs is rising. Natural and synthetic antifungal
peptides (AFPs) are being actively explored as novel pharmaceuticals.
We are investigating the mode-of-action of various small rationally
designed peptides and synthetic AFPs derived from plant defensins.
For this purpose we are using the fungal model Neurospora crassa
and the human pathogen Aspergillus fumigatus combined with livecell imaging of fluorescently labelled AFPs and other live-cell probes,
inhibitor treatments and mutant analyses. PAF26 is a de novo-designed
hexapeptide possessing two well-defined motifs: N-terminal cationic
and C-terminal hydrophobic regions. We have characterised how each
motif is responsible for PAF26’s dynamic antifungal mechanism of action
involving the electrostatic interaction with cells, cellular internalisation, and
cell killing. PAF26 increases cytosolic free Ca2+ ([Ca2+]c) and several Ca2+
signalling/homeostatic mutants are resistant to the AFP. Different peptide
sequences derived from the γ-core motifs of MsDef1 and MtDef4
defensins inhibit conidial germination and hyphal fusion, and influence
[Ca2+]c, with different potency and specificity. Our results provide new
mechanistic insights into the mode-of-action of AFPs that should help us
design new synthetic AFP-based drugs with improved activity and stability.
MA06 – Wed 1445
Offered paper Synthetic DNA minor groove binding antibiotics
Svetlana Kapis, Svenja Janssen, Fraser Scott, Iain Hunter, Colin Suckling,
Nicholas Tucker
We have evaluated a collection of 49 novel synthetic antibiotic
compounds (sMGBs), which draw inspiration from natural products
such as distamycin -that have the intrinsic ability to bind to the minor
groove of DNA. Some of these compounds are active against Grampositive pathogens including Clostridium difficile, and Staphylococcus aureus
(including MRSA), at comparable levels of activity to vancomycin. There
is a good structure-activity relationship and resistance to sMGBs has not
been an issue in these target organisms. sMGBs appeared less effective
against Gram-negatives such as Pseudomonas aeruginosa. However,
efficacy improved considerably in the presence of an efflux pump
inhibitor. We have screened over 20 efflux pump mutants in order to
identify the relative contributions of different drug efflux systems. Certain
efflux mutants exhibited only 10% growth relative to the wild type P.
aeruginosa strain, demonstrating selectivity between pump and sMGB
substrate and we are currently exploiting this information so that we can
design better anti-pseudomonas drugs. We are also actively investigating
the mode of action against MRSA using a variety of next-generation
sequencing approaches.
MA06 – Wed 1630
The application of defective interfering viruses for the prevention of
influenza
Andrew J Easton
School of Life Sciences, The University of Warwick, Gibbet Hill Road, Coventry,
UK
28
Session abstracts
Influenza A viruses are a continuing threat to people and domestic
livestock, and current anti-flu measures have limitations. The main antiinfluenza drugs are the neuraminidase inhibitors oseltamivir and zanamivir
and antiviral resistance to the more widely used oseltamivir has arisen
relatively rapidly. Defective interfering (DI) influenza virus is a natural
antiviral and we have cloned a highly active on defective interfering (DI)
influenza A virus RNA (244 DI RNA) derived from segment 1. A single
intranasal administration protects both mice and ferrets from lethal
disease caused by a number of different influenza A viruses including the
2009 pandemic strain. In ferrets the amelioration of disease is better than
that provided by repeated doses of Oseltamivir. Surprisingly the 244 DI
RNA also protects against disease caused by the genetically unrelated
influenza B virus and the paramyxovirus pneumonia virus of mice. The
DI RNA acts in three ways: it competes for packaging with the full length
segment 1, it inhibits virus mRNA synthesis in influenza A viruses and the
heterologous protection is achieved by stimulating type I interferon and
possibly other elements of innate immunity. The DI RNA therefore has
potential as a broad spectrum anti influenza treatment.
MA06 – Wed 1700
Forging new paradigms: broad spectrum antivirals against enveloped
viruses
Benhur Lee
Department of Microbiology, Immunology and Molecular Genetics, School of
Medicine at UCLA, USA
Advances in antiviral therapeutics have allowed for effective management
of specific viral infections such as HIV, and recently, HCV. Yet, the
one-bug-one-drug paradigm of drug discovery is insufficient to meet
the looming threat of emerging and re-emerging viral pathogens. This
underscores the need for broad-spectrum antivirals that act on multiple
viruses based on some commonality in their viral life cycle, rather than
on specific viral proteins.
We present recent work on two broad-spectrum antiviral compounds
that act on the virus-cell membrane fusion process, and thus have the
remarkable property of inhibiting the entry of many enveloped viruses.
The first compound (LJ001) acts on the virus and not the cell, and was
hypothesised to exploit the physiological difference between inert viral
membranes and biogenic cellular membranes with reparative capabilities.
We identify the molecular target of LJ001 as unsaturated phospholipids
and present a strong body of evidence that supports a unifying
hypothesis regarding its mechanism of action. The second compound,
25-hydroxycholesterol, is a product of an interferon-stimulated gene
(Ch25h), and also inhibits the entry of many enveloped viruses, but
it acts on the host cell (membrane), not the virus. These two classes
of compounds provide new paradigms that consider the virus-cell
membrane interface as a target for the development of broad-spectrum
antivirals.
MA06 – Thu 0900
Antibiotic inhibition and attenuation in soil
Kalin Vetsigian1, Eric Kelsic2, Roy Kishony2
1
University of Wisconsin-Madison, USA; 2Harvard Medical School, USA
It has been known that bacteria produce antibiotics that inhibit the
growth of other bacteria, causing dense networks of interactions within
natural microbial communities. But it has been unclear how interactions
between antibiotic producing and sensitive bacteria are modulated by
other surrounding species in the community. Are sensitive bacteria
better off or worse off with other bacteria around? Systematically
screening combinations of diverse Streptomyces species, we found that
modulation of inhibitory interactions is very common. Furthermore,
the observed modulation is almost universally not an increase, but
a decrease in inhibition. Similarly, we observed frequent antibiotic
attenuation when an antibiotic producer is replaced with a range of pure
antibiotic compounds. We demonstrate using enzyme inhibitors that
one likely mechanism of antibiotic attenuation is enzymatic degradation.
While we commonly observe species attenuating antibiotics produced
by other species, we, surprisingly, also find bacteria that attenuate
their own antibiotics. The widespread attenuation of antibiotics in
microbial communities suggests that to inhibit the growth of a target
bacterial species the attenuating properties of the surrounding microbial
community as a whole must be subverted.
MA06 – Thu 0930
Rewriting antibiotic clusters: applied synthetic biology
Eriko Takano
Faculty of Life Sciences, Manchester Institute of Biotechnology, University of
Manchester, UK
Streptomyces bacteria are well known for their ability to produce an
immense diversity of secondary metabolites, including many antibiotics.
The underlying biosynthetic machinery is a particularly interesting target
for synthetic biology, due to its inherent modularity at multiple levels1,2.
A treasure trove of antibiotic biosynthesis gene clusters has been
identified by genome sequencing, typically 20–50 per genome3,4. We can
use synthetic biology to re-engineer bacterial genomes to awaken this
multitude of cryptic antibiotic clusters. We have already demonstrated
the potential of this strategy by awakening the cryptic/orphan CPK
gene cluster5, which produces a novel antibacterial compound. We
have developed tools for the refactoring of antibiotic biosynthesis,
which include the in silco analysis of biosynthesis enzymes and parts
for transcriptional and translational control. Generalising this approach
using standardised molecular modules will become a central tool for
discovering new bioactive compounds, ranging from anti-cancer drugs to
antibiotics6.
References 1. Medema MH, Breitling R, Bovenberg RAL, Takano E.
Nature Rev Microbiol (2011) 9:131–137; 2. Medema MH, van Raaphorst
R, Takano E, Breitling R. Nature Rev Microbiol (2012) 10:191-202; 3.
Medema MH, Trefzer A, …, Takano E. Genome Biology and Evolution
(2010) 2:212–224; 4. Medema MH, Balin K, …, Takano E, Breitling R.
Nucl Acids Res (2011) 39:W339–W346; 5. Gottelt M, Gomez-Escribano
JP, Bibb M, Takano E. Microbiology (2010) 156:2343–2353; 6. Medema
MH, Alam MT, Breitling R, Takano E. Bioengineered Bugs (2011) 2:4
MA06 – Thu 1000
Constitutive and differential production of secondary metabolites in
soil and sand microcosms of Streptomyces coelicolor
Geertje Van Keulen
Institute of Life Science, College of Medicine, Swansea University, Swansea,
Wales, UK (Email: g.van.keulen@swansea.ac.uk)
Streptomycetes are soil dwelling filamentous bacteria, known for
their production of bioactive ‘secondary’ metabolites. Genome
sequencing of Streptomyces coelicolor identified 26 known or predicted
(cryptic) secondary metabolite gene clusters ranging from antibiotics
to siderophores, pigments and lantibiotics. Most studies investigating
microbial secondary metabolite production are usually undertaken
in liquid or on solid media. Information is lacking on gene expression
and antibiotics production in situ. The aim of this study was to
investigate primary and secondary metabolism in a model Streptomyces
grown under more natural conditions. The results from proteomic,
transcriptomic, and metabolomic studies of S. coelicolor grown in sand or
soil microcosms with differing carbon, nitrogen and metal content will
be discussed. Interestingly and contrary to the consensus of secondary
metabolism commencing after reduction or cessation of growth, this
study revealed expression of secondary metabolite biosynthetic genes
and proteins at the onset of and during exponential growth in soil and
sand, respectively. Some secondary metabolite genes/proteins were
expressed constitutively which was further supported by metabolic data.
The antibiotic actinorhodin could be detected throughout the growth
phases in sand cosms. Secondary metabolism as term should therefore
be reconsidered under environmental conditions as it may play an
important primary role in nature.
29
Session abstracts
MA06 – Thu 1100
Improved antibody therapies for the treatment of Clostridium difficile
infection
D Humphreys1, M Oxbrow, B MacKenzie, N Fisher, J Compson,
H Hailu, S Cross, L D’Hooghe, K Tyson, D Knight, K Hervé, V O’Dowd,
A Moore, D Lightwood
1
UCB Pharma, 208 Bath Road, Slough, UK (Email: david.humphreys@ucb.com)
Clostridium difficile infections (CDI) remain a major health and economic
burden in care facilities of the developed world in spite of the availability
of effective antibiotic therapies. Potent antibody therapies have the
potential to additionally reduce death rates and reduce the duration
and severity of diarrhoea. Improvement in these aspects would have
a significant positive influence on patients and healthcare providers.
Humanised IgG1 antibodies were generated by animal immunisation
followed by direct rescue of antibody variable regions from B-cells.
Antibodies were tested in a wide range of in vitro and cell culture
assays in order to stratify them on affinity, neutralisation activity, other
potency and biophysical characteristics. UCB Mabs have high affinities
against TcdA and TcdB and demonstrated impressive characteristics in a
range of in vitro and cell culture assays. A mixture of purified antibodies
was tested in a hamster infection protection model. The Mab mixture
conferred very high levels of protection against the acute infection phase
and also for more than 20 days during the chronic / re-infection phase
of hamsters. Mab mixtures such as these may represent one kind of new
treatment for patients for CDI.
MA06 – Thu 1130
Regulatory aspects of phage therapy
David Harper
Chief Scientific Officer, AmpliPhi Biosciences Corporation, Bedford, UK
Phage therapeutics provide a potential solution to combat the crisis in
antibiotic resistance. Regardless of the volume of historical literature
reporting the safety and efficacy of phage, new phage products will
need to be developed and evaluated according to current regulatory
standards. Development of phage products presents unique challenges
due to the biology of the host-phage relationship. It is important to
work with regulators to build consensus on the required path to
approval within existing regulatory frameworks. Phage will need to be
manufactured and characterised according to cGMP, and evaluated for
safety and efficacy in well-controlled clinical trials.
Efforts have been made to persuade authorities to modify regulatory
requirements to facilitate phage product development. These efforts
have been unsuccessful. New legislation may be counter-productive due
to the complexity of setting precedent. We believe that development of
phage therapeutics will be better served using a rationalised approach to
address the requirements of existing regulations. The greatest regulatory
challenges are likely to be in areas of manufacturing and product control
but these hurdles are navigable with support of well-controlled scientific
data. Properly developed, phage therapies could save or improve the
lives of millions battling antibiotic resistant bacterial infections.
MA06 – Thu 1300
Antimicrobials from neglected bacteria
Christian Hertweck
Department of Biomolecular Chemistry, Leibniz Institute for Natural Product
Research and Infection Biology, HKI, 07745 Jena, Germany
Although microbes are the purveyors of a multitude of human
maladies, human medicine and disease treatment also owe a great
deal to microorganisms. This is because microorganism-derived natural
products or their synthetic analogues represent a large proportion of
the antimicrobial therapeutics that are currently in clinical use. Because
they have been pre-evaluated over time by nature for their inherent
biological purpose, natural product antibiotics often come pre-equipped
to bind specifically to their desired cellular targets and are often highly
active. Typical bacterial sources of antibiotics include actinomycetes,
predominantly from the genus Streptomyces, as well as myxobacteria
and cyanobacteria. In the past, many antibiotics were discovered
through antibacterial screening programs and isolating their secondary
metabolites. However, more and more known compounds are rediscovered using this approach.
In this talk some new sources for the discovery of biologically active
natural products will be presented, by providing selected examples of
compounds from bacteria that can be considered ‘neglected producers’.
Genetic and chemical analyses shows that ‘neglected bacteria’, for
example Burkholderia, Janthinobacterium and Clostridium spp., are not only
able to generate structurally intriguing secondary metabolites, but that
these compounds may new leads for antimicrobial development.
MA06 – Thu 1330
Biosynthetic engineering of non-ribosomal lipopeptide and
lipoglycopeptide antibiotics
Jason Micklefield
School of Chemistry & Manchester Institute of Biotechnology, The University
of Manchester, 131 Princess Street, Manchester, UK
The calcium dependant antibiotics (CDA) from Streptomyces coelicolor
belong to the family of nonribosomal lipopeptides that includes
daptomycin, which is now widely used in the treatment of infections
caused by Gram-positive pathogens. Recently we determined the
biosynthetic origins of CDA. For example, the biosynthetic pathways
leading to the non-proteinogenic amino acids in CDA, including
3-phosphohydroxyasparagine, Z-dehydrotryptophan, hydroxyphenyl
glycine and 3-methylglutamic acid as well as the 2,3-epoxyhexanoyl fatty
acid side chain have been elucidated. Using this information, a wide range
of biosynthetic engineering methodology has been developed which
can be applied to alter the structure and modulate the bioactivity of
lipopeptide antibiotics. The methods developed include: mutasynthesis;
auxotrophic precursor directed biosynthesis; active site modifications
of adenylation domains; module or domain swaps of the nonribosomal
peptide synthetase; and modification of the fatty acid synthase (FAS).
Currently we are using these biosynthetic engineering approaches to
engineer new lipoglycopeptide variants of the ramoplanin family of
antibiotics.
MA06 – Thu 1400
Post-translational modifications in natural product biosynthesis
Wilfred A van der Donk
Howard Hughes Medical Institute and University of Illinois at UrbanaChampaign, Urbana, IL 61801 USA
Research in the 20th century identified several major groups of natural
products including terpenoids, alkaloids, polyketides, and non-ribosomal
peptides. More recently, the genome sequencing efforts of the first
decade of the 21st century have revealed that another major class
of natural products is formed by ribosomally synthesised and posttranslationally modified peptides (RiPPs). These molecules are produced
in all three domains of life, their biosynthetic genes are ubiquitous in the
sequenced genomes, and their structural diversity is vast. Lanthipeptides
are examples of this growing class of compounds and many members
are highly effective peptide-derived antimicrobial agents with nanomolar
minimal inhibitory concentrations against pathogens. Lanthipeptides
are post-translationally modified to install multiple cyclic thioethers.
During their biosynthesis, a single enzyme typically breaks 8-16 chemical
bonds and forms 6-10 new bonds with high control over regio-and
chemoselectivity. Until recently, the factors that determine ring topology
were entirely unknown. Use of bioinformatics, phylogenetic, and
stereochemical studies suggest that the substrate peptide may be more
important than previously anticipated to control the ring topology, and
that these enzymes have descended from kinases and phosphoThr
lyases.
30
Session abstracts
MA06 – Thu 1500
Exploiting the Streptomyces and Burkholderia genera to discover new
antibiotics
Gregory L Challis
Department of Chemistry, University of Warwick, Coventry, UK
Bioinformatics analyses have identified gene clusters encoding cryptic
natural product biosynthetic pathways, not associated with the
production of known metabolites, in numerous bacterial genome
sequences. Discovery of the products of such cryptic gene clusters
promises to unearth a hitherto untapped wealth of novel antibiotics.
The discovery of novel antibiotics as the metabolic products of cryptic
polyketide biosynthetic gene clusters in Streptomyces species, via rational
genetic manipulation to increase pathway expression and metabolic
flux, will be described. Efforts to understand the biosynthesis of these
compounds and to exploit this understanding for the production of
novel analogues will be summarised.
Enacyloxins, known antibiotics that target elongation factor Tu, have
been identified as products of a cryptic polyketide biosynthetic
gene cluster in Burkholderia ambifaria. Investigation of the pathway
for enacyloxin biosynthesis has provided new opportunities for the
production of novel analogues that may overcome potential problems
with the clinical application of these natural products.
of the pathway leads to the targeted generation of specific uridyl
peptides in vivo.
Furthermore, we will describe how chemical handles, such as halogens,
can be introduced into natural products through genetic manipulation
rather than semi-synthesis or mutasynthesis. The installed halogen
provides a selectably functionalisable handle enabling synthetic
modification of the natural product using mild cross-coupling conditions
in crude aqueous extracts of the culture broth.
Bacterial–fungal interactions
MA07
MA06 – Thu 1530
The biosynthesis of tropolones in fungi
Jack Davison, Ahmed al-Fahad, Menghao Cai, Zhongshu Song,
Samar Yehia, Colin M Lazarus, Andrew M Bailey, Thomas J Simpson,
Russell J Cox
University of Bristol, School of Chemistry, Cantock’s Close, Bristol, UK
Tropolones occur in plants, bacteria and fungi, but little is known of
their biosynthesis at either genetic, molecular or biochemical levels. The
discovery and structural elucidation of tropolones led to new ideas about
aromaticity and structure and bonding in organic chemistry during the
mid and late 20th century. While feeding studies – notably by Battersby
and Bentley -showed the chemical precursors of compounds such as
colchicine from plants and stipitatic acid from fungi these experiment
could elucidate nothing about the genes, proteins or molecular
mechanisms of tropolone biosynthesis. The biosynthesis of tropolones
thus represents one of the longest-standing mysteries in the field of
biosynthesis. We have discovered two fungal tropolone biosynthetic
gene clusters and used a combination of gene knockout, heterologous
expression and in vitro enzymatic assays to show that fungal tropolones
are formed in two oxidative steps from simple polyketide precursors.
Understanding these steps allows a deeper understanding of the
biosynthesis of a number of other complex fungal metabolites. Further
KO work shows that the pathway is capable of producing libraries of
related compounds.
MA06 – Thu 1600
Chemogenetic generation of pacidamycin analogues
Sabine Grüschow, Rebecca J M Goss
School of Chemistry, University of St Andrews, St Andrews, UK
Natural products represent a vital source and inspiration for the
development of therapeutic agents. Secondary metabolites also play
a significant role in today’s scientific research as molecular tools in the
study of biological systems. In both cases we need to find compounds
with unexplored biological targets and to adapt the existing ones to our
needs if we truly want to use natural products to their full potential. We
are interested in developing methodology to generate natural products
analogues as well as studying chemically unusual metabolites such as the
uridyl peptide antibiotic pacidamycin.
We will discuss how the aminonucleoside pharmacophore is
biosynthesed from uridine, and we will present how genetic manipulation
MA07 – Wed 0900
Genomics-guided discovery of traits contributing to the interactions of
Pseudomonas fluorescens with fungi
Teresa A Kidarsa1, Brenda T Shaffer1, Marcella D Henkels1,
Edward W Davis II1, Jennifer M Clifford1, Karl A Hassan2, Ian T Paulsen2,
Harald Gross3, Joyce E Loper1
1
USDA-Agricultural Research Service, Oregon State University, Corvallis,
OR, USA; 2Macquarie University, Sydney, Australia; 3University of Tübingen,
Germany
The Pseudomonas fluorescens group, composed of more than 50
named species, is ubiquitous in natural habitats and exhibits tremendous
ecological, metabolic, and genomic diversity. Certain strains live on plant
surfaces, protecting them from infection by fungal plant pathogens.
Comparative genomics revealed that gene clusters for the biosynthesis
of anti-fungal metabolites map to lineage-specific regions of the genome,
commensurate with the distinctive biocontrol properties of individual
strains. Our studies focus on Pseudomonas protegens Pf-5, which
produces an array of secondary metabolites toxic to plant pathogenic
fungi and Oomycetes. We identified new anti-fungal metabolites from
orphan gene clusters in the Pf-5 genome and pioneered global-regulatorbased genome mining as an approach to decipher the secondary
metabolome of Pseudomonas spp. We are also gaining a holistic
view of genome expression profiles of Pf-5 living on seed surfaces,
the environment where the bacterium interacts with seed-infecting
pathogens to affect biocontrol. A panel of Pf-5 mutants having deletions
in one or many known or orphan gene clusters were derived and tested
to identify the contribution of individual secondary metabolites to specific
Pf-5 phenotypes. Our studies highlight the enormous heterogeneity of
the P. fluorescens group and the importance of the variable genome in
determining fungal-bacterial interactions.
MA07 – Wed 0930
Investigations on the Pseudomonas fluorescens antifungal metabolite
2,4-diacetylphloroglucinol
John Morrissey1, Danielle Troppens1, Sol Schwartzman1, Nick Read2,
Fergal O’ Gara1
1
University College Cork, Western Road, Cork 1, Ireland; 2The University of
Pseudomonas flurorescens is a rhizosphere-associated soil bacterium that
has been extensively studied because of its plant growth promoting
properties. The capacity to produce metabolites with anti-fungal
activities is arguably the most important of these properties. One of
these metabolites, 2,4-diacetylphloroglucinol (DAPG), has received
special attention as it is an important component of the natural
suppressiveness of certain agricultural soils to fungal diseases of
crops. The genes responsible for the biosynthesis of DAPG have an
interesting evolutionary history as it appears that the biosynthetic and
regulatory genes have been gathered from diverse soil bacteria and
Archaea and assembled into a single locus that is maintained intact in
all DAPG-producing strains. This raises some interesting questions as
to the evolutionary processes underlying construction of clusters of
genes for production of secondary metabolites. The second aspect
31
Session abstracts
of this paper will address the mode of action of DAPG on fungi. We
undertook a genome-wide screen of yeast mutants to identify possible
targets and resistance mechanisms and will present these findings. In
addition, we will present findings from our most recent studies that
were performed in Saccharomyces and Neurospora, where, by the
application of a combination of genetic and physiological approaches, it
was established that DAPG acts as a proton ionophore and dissipates
the proton gradient across the mitochondrial membrane. The uncoupling
of respiration and ATP synthesis ultimately leads to growth inhibition and
is the primary toxic effect of DAPG. Finally, ecological aspects of DAPG
production by P. fluorescens will be discussed.
MA07 – Wed 1030
Comparative genomics of mushroom-pathogenic pseudomonads
David J Studholme1, Peter Burlinson2, Gail M Preston2.
1
Biosciences, University of Exeter, Exeter, UK; 2Department of Plant Sciences,
University of Oxford, South Parks Road, Oxford, UK
Several bacteria of the Pseudomonas fluorescens group cause important
diseases on cultivated mushrooms. We have generated draft complete
genome sequences for eight of these including: Pseudomonas gingeri
NCPPB 3146, Pseudomonas agarici NCPPB 2289, Pseudomonas
fluorescens strains NZ007, NZ011, NZI7, and NZ052, Pseudomonas
tolaasii NCPPB 2192 and PMS117. The ability to infect mushrooms has
evolved several times among pseudomonads and comparison of these
genome sequences suggests a diverse set of virulence strategies. These
genome sequences also encode genes implicated in interactions with
other eukaryotes; for example a cluster of biosynthetic genes in NZI7
specifies the ability to repel predation by nematode worms.
MA07 – Wed 1100
Offered paper Fitness advantage of bacterial cellulose expression in
experimental microcosms and on mushrooms
Andrew J Spiers1, Anna Koza1, Olena Moshynets2
1
The SIMBIOS Centre, Dundee, UK, 2Institute of Molecular Biology and
Genetics of the National Academy of Sciences of Ukraine, Kiev, Ukraine
The ability to form biofilms is an important bacterial colonisation
strategy, though very rarely has it been demonstrated that biofilmformation provides a fitness advantage in natural conditions. Here a
survey of blotch-causing pseudomonads previously recovered from
white mushrooms (Agaricus bisporus) has identified a number producing
cellulose matrix-based biofilms in experimental static liquid microcosms.
Three isolates, together with the reference strains P. fluorescens
SBW25 and P. putida KT2440, were investigated further. The fitness
advantage of cellulose-expression was determined by competitive
fitness assays using pairs of wild-type and cellulose-deficient mutant
strains. Cellulose-expression provided an advantage in static liquid
microcosms where biofilms could form, and on mushrooms where it
was more likely that colonies develop as mushrooms retain little free
water. Survival experiments over 75–100% relative humidity showed
that viable numbers of wild-type strains declined more slowly than the
corresponding cellulose-deficient mutants. Subsequent fitness assays
under the same humidities demonstrated a selective advantage for
the wild-type strains under desiccating conditions. This work suggests
that cellulose-expression provides a fitness advantage in aqueous
environments where it enables biofilm-formation, and in water-limiting
conditions where its hydroscopic nature may retain water within drying
aggregations for longer periods than would normally be the case.
MA07 – Wed 1115
Offered paper Imaging mass spectrometry and genome mining reveal
highly antifungal virulence factor of mushroom soft rot pathogen
Katharina Graupner1, Kirstin Scherlach1, Tom Bretschneider1,
Gerald Lackner1, Martin Roth1, Harald Gross2, Christian Hertweck1
Department of Biomolecular Chemistry, Leibniz Institute for Natural
Product Research and Infection Biology, HKI, Jena, Germany, 2Eberhard Karls
University, Tübingen, Germany
Soft rot diseases caused by various bacteria account for severe losses in
agriculture. In many cases, the chemical mediators of the diseases have
remained elusive, as for the mushroom pathogen Janthinobacterium
agaricidamnosum. We reasoned that knowledge on the causative
agent of soft rot would have a double benefit. Foremost, it could aid
in understanding the pathobiology of the pathogen, which may be a
starting point for protective measures. Second, there is an increasing
need for novel antifungals. We hypothesised that mushroom soft rot
bacteria could excrete antifungal factors, which might also be active
against human pathogens. The discovery and characterisation of a highly
antifungal virulence factor from the soft rot pathogen J. agaricidamnosum,
guided by imaging mass spectrometry and genome mining will be
presented. On agricultural grounds the finding is significant as it helps
understanding the pathobiology of bacteria-induced soft rot. From a
chemical perspective, our study highlights the impact of blending modern
analytics with genetics to unveil cryptic natural products. The approach
of combining imaging mass spectrometry with genome mining holds
promise to be generally applicable to the discovery of cryptic natural
products including chemical mediators such as virulence factors that are
only produced in a particular (patho)biological context.
1
MA07 – Wed 1130
Bacterial biocontrol of chytridiomycosis in amphibians
Reid N Harris
Department of Biology, James Madison University, Harrisonburg, Virginia
22807, USA
Amphibian species are experiencing declines and extinctions due to
the emerging infectious disease chytridiomycosis, which is caused by
the fungal pathogen Batrachochytrium dendrobatidis (Bd). We isolated
species of cutaneous bacteria from amphibians that inhibit Bd in vitro
and postulate that skin microbes contribute to innate skin defenses. We
showed that reducing skin bacteria on salamanders (Plethodon cinereus)
led to greater morbidity after Bd exposure than was observed in
controls. In ‘bacteria-addition’ experiments, we tested the hypothesis that
disease severity decreases when the microbial community of amphibians
is augmented with the anti-Bd bacterial species Janthinobacterium lividum
before infection with Bd. The bacteria produce the anti-Bd metabolite
violacein, which we detected from skins of amphibian species. Bacterial
inoculation of frogs (Rana muscosa) prevented successful colonisation
of Bd, and protected the hosts. Violacein concentration on individuals
inoculated with J. lividum was much higher at week 20 than on control
individuals, implying that J. lividum persisted for 20 weeks and that
violacein was critical in inhibiting Bd. In a field experiment, R. muscosa
were treated with probiotic baths. These individuals survived at a
significantly higher rate than untreated controls. A promising approach
is to search amphibians’ natural microbiota for anti-Bd bacteria as bioaugmentation candidates.
MA07 – Wed 1400
Stability and roles of lichen-associated microbiomes
Martin Grube
Institute of Plant Sciences, Karl-Franzens-University Graz, Austria
Lichens are examples for self-sustained symbioses that can persist under
a wide range of environmental conditions, including the most hostile
habitats for life on Earth. We previously discovered that lichen thalli –
shaped by fungi to contain algae – also host species-specific communities
of bacteria. The roles of the bacteria in lichens could be manifold.
A metaproteomic approach of the ‘lung lichen’ Lobaria pulmonaria
reveals equal amounts of proteins from bacteria and algae in this lichen,
but strikingly different functional contributions. However, a better
understanding of the variation and stability of bacterial communities is
32
Session abstracts
required to understand which functions of bacteria are more commonly
present than others. We found that age and habitat conditions are
modulating the bacterial communities at higher phylogenetic level.
Infections by other fungi rather altered bacterial composition at the strain
level, as by detrended correspondence and profile clustering network
analyses. Lichens represent miniature ecosystems and also serve as a
potent bioressource (e.g., of new compounds, antagonistic strains, and
enzymatic functions) for future biotechnological applications.
MA07 – Wed 1430
Offered paper Revisiting the ‘gut/rumen’ hypothesis for
mycorrhizosphere functioning: a meta-organism paradigm?
Robin Sen
Manchester Metropolitan University, Manchester, UK
Increasing awareness of the central importance of highly intimate
associations between mycorrhizal fungi, bacteria and archaea in relation
to plant and soil productivity highlights the need for more systematic
investigations of these interactions and their functional significance in
plant mycorrhizospheres. The findings and implications of our earlier
studies in ectomycorrhizal Scots pine systems developed in nutrientlimited podzolic forest soils in relation to e.g. primary interaction,
biofilm organisation, spatiality and specificity will be briefly reviewed
together with recent findings from arbuscular mycorrhizal systems
focusing on key nitrogen cycling pathways again in nutrient deficient
soils. Pyrosequencing-based soil bacterial and fungal microbiome
characterisation, we have applied in peatland restoration ecology, will
be highlighted in support of my argument for the need to embrace a
more meta-organism based paradigm to model nutritional relations in
the mycorrhizosphere. I will conclude by re-visiting the mycorrhizosphere
‘gut/rumen’ hypothesis that I originally presented over a decade ago
(New Phyt: 157: 391-394) in an editorial covering of papers from the
centenary New Phytologist Symposium ‘Soil microbes in plant productivity’
held in Helsinki in 2002.
MA07 – Wed 1445
Offered paper Antifungal potential of Lactobacillus amylovorus JG2
against the potato blight pathogen Phytophthora infestans
Jiahui Guo1, Brid Brosnan2, Ambrose Furey2, Elke K Arendt3,
Claudia Axel3, Aidan Coffey3
1
Department of Biological Sciences, Cork Institute of Technology, Bishopstown,
Cork, Ireland, 2Department of Chemistry, Cork Institute of Technology,
Bishopstown, Cork, Ireland, 3Department of Food Science, Food Technology
and Nutrition, National University of Ireland, Cork, Ireland
Phytophthora infestans is the fungus responsible for late blight; the
most devastating disease in potato production today. This study
aimed to assess in vitro antifungal potential of the lactic acid bacteria
Lactobacillus amylovorus JG2 against P. infestans. Five strains which had
initially exhibited strong antifungal activity against Penicillium, Aspergillus
and human dermatophytes were selected and tested against Irish
potato blight P. infestans strains. All strains caused inhibition and the
strongest was Lb. amylovorus JG2. Cell-free culture and freeze-dried
supernatants of Lb. amylovorus JG2 and the non-antifungal Lb. amylovorus
DSM20531 were used to access and compare antifungal activity on agar
and microtitre plate assays. When freeze-dried cell-free supernatant
powder from Lb. amylovorus JG2 was incorporated in culture medium
at concentrations greater than 1%, fungal mycelia radial growth was
inhibited. Additions of 12.5% caused complete inhibition of fungal growth
on the basis of turbidity. Cell-free supernatant of Lb. amylovorus JG2 and
DSM 20531 was analysed by Liquid Chromatography Fourier Transform
Mass Spectrometry (LC-FTMS) using an Accela LC coupled to an
LTQ Orbitrap XL mass spectrometer. Fifteen antifungal metabolites
were detected. This study has demonstrated that biological control,
accomplished by beneficial microorganisms, may be a viable ‘green
approach’ to reducing late blight.
MA07 – Wed 1500
Bacterial–fungal interaction between the plant growth-promoting
bacterium Tsukamurella paurometabola C 924 and Glomus fasciculatum
or Glomus clarum in the lettuce rhizosphere
Marieta Marin Bruzos, Jesús Mena Campos, Ramon Franco Rodríguez,
Eulogio Pimentel Vazquez, Alain Moreira Rubio, Ileana Sánchez Ortiz
Centre for Genetic Engineering and Biotechnology. Circunvalación Norte y
Avenida Finlay. POBox 387 CP 70100 Camaguey, Cuba (Email: marieta.
marin@cigb.edu.cu)
The study of fungal-bacterial interactions in soils is not only interesting
from a basic point of view but has also yielded findings of societal and
economical relevance, such as in the application of biological controls
of plant diseases. This study evaluated the effect of Tsukamurella
paurometabola C 924, a plant growth promoting bacterium with
nematocidal action isolated from banana rhizosphere, as single
inoculant or combined with arbuscular mycorrhizal fungi (AMF) Glomus
fasciculatum or Glomus clarum in lettuce (Lactuca sativa L.). Controls
included non-bacteria non-AMF, and each AMF species alone. Five
replicates were used. AMF did not show any influence, neither in
T. paurometabola C 924 c.f.u. counts in soil, nor over its phenotypic
nematocidal characters. On the other hand, the bacterium stimulated
AM colonisation for both fungi species as well as an early infection.
Combined inoculation improved significantly fresh weight of plants as
compared with the microorganisms separately or the non-inoculated
control.
Keywords: Tsukamurella, Glomus, arbuscular mycorrhiza, fungal-bacterial
interaction, growth promotion, lettuce
MA07 – Wed 1600
The mycorrhiza helper bacteria: a model for the study of fungal–
bacterial interactions
P Frey-Klett1, A Deveau1, P Burlinson1,3, A Cusano1,4, S Uroz1,
J Garbaye1, A Sarniguet2, G Preston3
1
INRA Nancy, France; 2INRA Rennes, France; 3University of Oxford, UK ;
4
IIT, Naples, Italy (Email: klett@nancy.inra.fr)
The tree-soil interface in boreal and temperate forest ecosystems consists
in a diverse assemblage of ectomycorrhizas closely associated with
bacterial communities, which are selected by the ectomycorrhizal fungi
from the soil reservoir (Frey et al, AEM, 1997; Uroz et al, AEM, 2012).
This multitrophic ectomycorrhizal complex plays a central role in gross
production and nutrient cycling (Frey-Klett & Garbaye, New Phytol, 2005;
Uroz et al, Trends Microbiol, 2009). So far, its functioning has been poorly
documented. However, recent studies have stressed the importance
of physical, metabolic and functional interactions between bacteria and
ectomycorrhizal mycelia. Many mycorrhizosphere bacteria live in contact
with ectomycorrhizal fungi, forming biofilms on the hyphal surface or
even colonising the intracellular compartment. Among the bacterial
communities associated with the ectomycorrhizal complex, the so-called
‘ Mycorrhiza Helper Bacteria (MHB)’ (Frey-Klett et al, New Phytol, 2007)
significantly promote the establishment and/or the functioning of the
ectomycorrhizal symbiosis. The present knowledge about this particular
functional group of bacteria will be discussed focusing on the model
MHB strain Pseudomonas fluorescens BBc6R8, with regards to the rapidly
developing scientific field of fungal-bacterial interactions (Tarkka et al, Curr
Genet, 2009 ; Frey-Klett et al, MMBR, 2011).
MA07 – Wed 1630
Toxin biosynthesis in fungal–bacterial interactions
Christian Hertweck
Department of Biomolecular Chemistry, Leibniz Institute for Natural Product
Research and Infection Biology, HKI, 07745 Jena, Germany
This talk highlights the significance of toxin-producing, fungus-associated
bacteria in the areas of ecology, medicine, and nutrition. By combining
genome mining, bioinformatics analyses and chemical analytical
33
Session abstracts
techniques we uncovered the biosynthesis of a number of virulence
factors and toxins. Rhizoxin is the causative agent of the rice seedling
blight fungus Rhizopus microsporus. The phytotoxin efficiently binds to
rice beta-tubulin, which results in inhibition of mitosis and cell cycle
arrest. Surprisingly, rhizoxin is not biosynthesised by the fungus itself, but
by endosymbiotic bacteria of the genus Burkholderia. Our unexpected
findings unveil a remarkably complex symbiotic-pathogenic alliance that
extends the fungus–plant interaction to a third, bacterial key player.
Furthermore, we identified the molecular basis for the biosynthesis of
a highly toxic polyketide produced by Burkholderia gladioli, a common
contaminant of the food fermentation fungus Rhizopus oligosporus.
Bongkrekic acid is a respiratory toxin that efficiently inhibits adenine
nucleotide translocase. Through sequencing of the bacterial genome
and functional analyses of the biosynthetic genes new insights into the
complex polyketide assembly were gained. These studies not only
facilitate the understanding of complex ecological processes, but also
open the possibility to develop new drug candidates and potential biocontrol agents against crop diseases.
MA07 – Thu 0900
Bacterial–fungal interactions in soil – how bacteria take profit of fungal
counterparts
Jan Dirk van Elsas
Department of Microbial Ecology, Centre for Ecological and Evolutionary
Studies, University of Groningen, The Netherlands
Given the fact that the bacteria and fungi comprise considerable and
often the main biomass in soil, outnumbering other soil biota, studying
their putative interactions is important. In a range of studies, in our
laboratory as well as in that of others, evidence has emerged for the
specific capability of particular soil bacteria to (actively) interact with
soil fungi (Laccaria, Lyophillum and others). A main driving force may
be the need of soil bacteria to colonise available niches and obtain
nutrients from the latter. In the lecture, achievements so far obtained
will be discussed, as well as the potential future avenues to explore this
emerging area of research. Briefly, in a range of experiments, we took as
criteria (1) the prevalence of particular bacterial types in the mycosphere
of (mycorrhizal) fungi in the field and (2) the capacity of such bacteria
to migrate with developing fungal hyphae through soil. Consumption of
glycerol released by the host fungus Lyophillum strain Karsten was a main
driver of the bacteria. We also obtained evidence for the involvement
in the interaction of particular bacterial systems, such as the type three
secretion system, migratory capability and biofilm formation. Moreover,
we detected an effect of modulation of the pH surrounding fungal
hyphae. Early evidence for an effect of bacteria on fungal physiology was
also found. In terms of the selected bacterial taxa, Burkholderia terrae like
types were very proficient colonisers of fungal hyphae, migrators as well
as migration helpers. These also exerted a so-called migration helper
effect on other bacteria. In summary, different bacterial capabilities seem
to allow bacteria to find and colonise hospitable niches at soil fungi.
MA07 – Thu 0930
Bacterial and fungal geomicrobiology: a problem with communities?
Geoffrey M Gadd
Division of Molecular Microbiology, College of Life Sciences, University of
Dundee, Dundee, Scotland, UK
All kinds of organisms in aerobic and anaerobic parts of the
biosphere are of wide significance in effecting and affecting global
biogeochemical processes, including element cycling and metal and
mineral transformations. However, researchers tend to work on
bacteria or on fungi and rarely on both, even when considering
aerobic terrestrial locations. A broad appreciation of fungi as agents
of biogeochemical change is still lacking, and they are frequently
neglected, in contrast to bacteria, in terrestrial, aquatic and subsurface
ecosystems. This presentation will discuss some important roles of
fungi in geologic processes (geomycology) with emphasis on metal
and mineral transformations and the examples outlined will include
roles in degradation and transformations of rocks and metal-containing
minerals, metallic lead, depleted uranium, uranium and manganese
oxides, and fungal biodeterioration of concrete, the latter being of
general biodeteriorative significance regarding built structures as well as
radionuclide containment and storage.
MA07 – Thu 1030
Arbuscular mycorrhizal fungi (AMF) – a new cog in the nitrogen cycle?
Angela Hodge
Department of Biology, University of York, Wentworth Way, York, UK
The arbuscular mycorrhiza fungi (AMF) form the most widespread and
ancient type of mycorrhizal symbiosis. Yet, we know little about the
fundamental biology of this key fungal phylum given their obligate biotrophic
nature and the inability to culture these fungi in the absence of a host plant.
AMF may enhance the rate of organic material decomposition. Because they
are not saprotrophs, these fungi probably influence decomposition indirectly
through their interactions with other microorganisms. AMF can alter the
bacterial community while complex microbial communities can suppress
AMF hyphal growth and reduce the effectiveness of the symbiosis. These
interactions may play a large part in the biology of AM fungi, which appear
to rely heavily on organic materials for their substantial nitrogen (N) demand.
We have recently demonstrated that AMF proliferated extensively in organic
materials even when carbon supply to the fungus was restricted, and the
patches provided an important N source for the AMF, suggesting the AMF
successfully competed with other microorganisms for released N. AMF with
access to an organic patch were also better able to colonise a new host plant
demonstrating a fungal growth response. These findings demonstrate AMF
play a previously unappreciated role in the global N cycle.
MA07 – Thu 1100
Offered paper Microbial community dynamics and desulfonating bacterial
abundance in the mycorrhizosphere of bi-compartmental microcosms
respond to inoculation with arbuscular mycorrhizal fungi
Jacinta M Gahan, Achim Schmalenberger
University of Limerick, Limerick, Ireland
About 95% of soil sulfur (S) is organically bound, predominantly as
sulfonates, and is not directly plant available. Specific bacteria in both
the soil and rhizosphere mobilise sulfonate-S but very little is known
about these bacteria in the mycorrhizosphere. Since mycorrhizal fungi
support growth of most land plants, desulfonating bacteria in the
mycorrhizosphere may be of substantial benefit to the plant host. This
study analysed the effect of arbuscular mycorrhiza (AM) inoculation with
Glomus intraradices (G) and a mix of 6 AM species (M) on microbial
communities and desulfonating bacteria with Lolium perenne, Agrostis
stolonifera and Plantago lanceolata as plant hosts in bi-compartmental
microcosms. AM inoculation significantly increased percentage root
colonisation over non-inoculated control systems (C) and the quantity
of cultivable desulfonating bacteria in the mycorrhizosphere over preinoculated soil for all plants. Mycorrhizosphere community analysis via
Denaturing Gradient Gel Electrophoresis revealed significantly different
bacterial and fungal communities following AM inoculation for all plants.
The results demonstrate that increased AM root colonisation impacts
bacterial and fungal community dynamics in the mycorrhizosphere and
corresponding increased abundance of desulfonating bacteria that may
be beneficial for plant-S supply.
MA07 – Thu 1115
Offered paper Identification of the gene clusters involved in the
biogenesis of the broad spectrum plant pathogenic antifungal and antioomycete haterumalide compound, oocydin A
Miguel A Matilla1, Finian J Leeper2, George P C Salmond1
1
Department of Biochemistry, University of Cambridge, Cambridge, UK,
2
Department of Chemistry, University of Cambridge, Cambridge, UK
Haterumalides are halogenated macrolides with a wide range of biological
34
Session abstracts
properties, including strong antitumor, antifungal and anti-oomycete
activities. The potent haterumalide oocydin A (ooc) was identified from
two plant-associated bacteria through its high bioactivity against plantpathogenic fungi and oomycetes. In this study we describe the ooc
biosynthetic gene cluster which was identified by genome sequencing,
comparative genomics and chemical analysis in four plant-associated
enterobacteria belonging to Serratia and Dickeya genera. Disruption of
the ooc gene cluster abolished oocydin A production, and therefore,
bioactivity against plant pathogenic fungi and oomycetes. The ooc gene
clusters spans between 77-and 80-kb and consists of 23 ORFs organised
in three transcriptional units. Five of the ooc genes encode multimodular
polyketide synthase (PKS) proteins. The absence of integrated
acyltransferase (AT) domains in all the biosynthetic modules of these five
PKS proteins and the presence of two free-standing AT proteins classifies
the oocydin A gene cluster within the growing class of trans-AT PKSs.
Based on the structure of the gene clusters and extensive in silico analysis,
we propose a biosynthetic model for the production of oocydin A. An
understanding of oocydin biosynthesis could enable the production of
modified derivatives with novel utility in agriculture and medicine.
This work aimed to identify indigenous fungi from commercial green
Coffea arabica and Coffea robusta coffee beans, and to evaluate lactic
acid bacteria (LAB) for their anti-fungal effects against the isolated
strains. Fungi were isolated using standard techniques and identified by
DNA-sequencing and morphological analyses. Fungal isolates generally
belonged to Aspergillus, Penicillium, Fusarium, Rhizopus, and Mucor
genera. LAB or crude mixtures of LAB-CFS (cell free supernatant) were
investigated to control/inhibit fungal growth of coffee bean isolates. The
in vitro antagonistic LAB plate test was done on bacterial agar containing
pre-cultured LAB spots with subsequent application of fungal sporecontaining agar. The plates were then incubated aerobically at 25°C for
7 days. The LAB-CFS test was done by incorporating CFS-agar into MRS
agar, with subsequent application of fungal spore-containing agar. The
plates were incubated as for the LAB plate test. The efficacy of the in
vitro approaches were evaluated based on visual determination of fungal
growth and measurement of fungal-free halos surrounding LAB spots in
the LAB plate tests, or measuring% plate covered by fungal mycelia in
the LAB-CFS plate tests. The use of LAB was successful in the inhibition
of a broad range of fungal species from diverse geographical locations.
MA07 – Thu 1130
Close encounters of the dirt kind: the battle between bacteria and
fungi in soil
Wietse de Boer
Netherlands Institute of Ecology, Dept. Microbial Ecology, Wageningen,
The Netherlands
Bacteria and fungi play a major role in the decomposition of organic
compounds in soil. Although there is differentiation between these microbial
groups in the ability to degrade specific types of organic compounds, this
differentiation is far from complete. Confrontation of soil bacteria and
fungi with overlapping metabolic abilities occurs in many situations. In this
presentation, I will highlight some of these confrontations with emphasis
on antagonistic interactions. In addition to direct antagonistic interactions
between bacteria and fungi, side effects of bacteria-bacteria competition
on fungi will be addressed. Both direct and indirect antagonistic interactions
between soil bacteria and fungi can form the basis for the development of
novel biocontrol strategies and the discovery of new antibiotics.
MA07 – Thu 1345
Offered paper Innate immunity in Fusarium graminearum
Simon Ipcho1, Gitte Erbs1, Thomas Sundelin1, Peter K Busk2,
Mari-Anne Newman1, Stefan Olsson1
1
Department of Plant and Environmental Sciences, University of Copenhagen,
Copenhagen, Denmark, 2Department of Biotechnology, Aalborg University,
Copenhagen, Denmark
Fungi are often victims of bacterial parasitism but little is known about
fungal defense mechanisms. The potential existence of fungal innate
immunity was studied using Fusarium graminearum as model organism
and bacterial flagellin. The presence of flagellin triggered an initial
mitochondrial and cell membrane hyperpolarisation which was detected
using the florescent dye DiOC7(3) followed by the production Nitric
Oxide (NO), common to innate immunity signaling in eukaryotes. NO
appears to be produced by an inducible enzyme that is regulated by
complex mechanisms but centrally modulated by Calcium/Calmodulin.
Inhibition studies suggest the presence of a Nitric Oxide Synthase
(NOS), but no typical arginine utilising NOS was identified within
the F. graminearum’s genome by homology search. Genes resembling
the archetypal NOS, as well as argininosuccinate lyase were deleted.
However, the mutants still produced NO. The presence of alternative
pathways contributing towards the production of NO was investigated
by adding a variety of potential substrates to challenged cultures. Various
reactions were observed suggesting that several pathways are present.
In conclusion: F. graminearum reacts strongly to the presence of the
bacterial Mirobial Associated Molecular Pattern (MAMP) flagellin with
an upregulation of NO production showing the presence of innate
immunity like responses also in fungi.
MA07 – Thu 1300
Characterising antifungal-producing bacteria in the nests of the leafcutter ant Acromyrmex octospinosus
Matt Hutchings
University of East Anglia, Norwich Research Park, Norwich, UK
The leaf-cutter ant Acromyrmex octospinosus harvests fresh vegetation
and uses a symbiotic fungus to convert them into an edible food source.
These ants also live in symbiosis with antibiotic-producing actinomycete
bacteria which protect the ants and their fungal cultivar against bacterial
and fungal infections. We have identified two key symbionts in a single
colony of A. octospinosus ants and identified the antifungal compounds
made by these Pseudonocardia and Streptomyces strains using genome
mining and activity-guided purification. The application of chemical and
genetic tools to identify and unlock these pathways revealed the presence
of multiple antifungal compounds made by these two strains, which might
explain the lack of drug resistance in co-evolved fungal pathogens. Longer
term, the construction of mutant symbiont strains which cannot make
antifungals will allow us to test whether the ants can distinguish between
beneficial and non beneficial actinomycete strains during the formation of
their external microbiome.
MA07 – Thu 1330
Offered paper Identification of the fungal microflora of coffee beans
from different origins and evaluation of lactic acid bacteria fungal
antagonism
Deborah M Waters1, Alice Moroni2, Elke K Arendt1
1
University College Cork, Cork, Ireland, 2Nestlé Research Centre, Nestec LTD.,
Vers-Chez-les-Blancs, Switzerland
MA07 – Thu 1400
Communication between bacteria and fungi leads to activation of
fungal silent gene clusters
Axel A Brakhage1,2
1
Department of Molecular and Applied Microbiology, Leibniz Institute
for Natural Product Research and Infection Biology (HKI) Jena, Germany,
2
Institute of Microbiology, Friedrich Schiller University Jena, Germany (Email:
axel.brakhage@hki-jena.de)
We have discovered that communication between microorganisms
represents a physiological trigger for activation of silent fungal gene
clusters. The physical interaction of the important model fungus
Aspergillus nidulans with a distinct soil-dwelling bacterium Streptomyces
rapamycinicus, identified from a collection of 58 species of actinomycetes,
led to the selective activation of the silent polyketide synthase gene
cluster encoding orsellinic acid biosynthesis. This reprogramming of
the fungus by the bacterium requires the histone acetylase GcnE of A.
35
Session abstracts
nidulans, which is part of the Saga/Ada complex. GcnE was shown to
specifically increase the K9 acetylation of histone 3 at genes belonging
to the orsellinic acid biosynthesis gene clusters after co-incubation with
S. rapamycinicus. Interestingly, S. rapamycinicus also has the potential to
trigger the activation of a silent gene cluster in A. fumigatus, which led
to the discovery of a novel compound. Knowledge of these regulatory
interactions will ultimately lead to a better understanding of the
physiological and ecological function of secondary metabolites and pave
the way for a novel avenue to drug discovery through targeted activation
of silent gene clusters.
References Schroeckh et al. (2009) PNAS; Nützmann et al. (2011) PNAS;
Brakhage (2013) Nature Rev. Microbiol.
MA07 – Thu 1500
Antifungal lactic acid bacteria and yeasts as biopreservatives
Johan Schnürer
Swedish University of Agricultural Sciences (SLU), Uppsala, Sweden
There is a need to reduce postharvest spoilage of food and animal feed
by mycotoxin forming moulds. Lactic acid bacteria (LAB) are traditionally
used to ferment and preserve food and feed, e g dairy products and
grass silage. The antimicrobial activity of LAB is due to the synthesis
of small organic acids such as lactic, acetic, and formic acids and other
substances such as 2,3-butadione, reuterin (3-hydroxypropionaldehyde).
LAB strains MiLAB14 and MiLAB393 produced 3-phenyllactic acid, several
3-hydroxy fatty acids, and a series of diketopiperazines. The 3-hydroxy
fatty acids were most potent, with inhibitory concentrations against yeasts
and moulds of 10 to 100 µg per ml, whereas 3-phenyl-lactic acid and
diketopiperazines were less active (milligrams per ml). New antifungal
compounds such as benzene-acetic acid, 2-propenyl ester and peptides
ranging from 9-55 amino acids have recently been described. Few
antifungal lactic acid bacteria have actually been put into practical use,
Lactobacillus plantarum MiLAB393, sold globally as FeedTech F3000, being
one exception. The yeast Pichia anomala improves stability of moist grain
in airtight storage systems, inhibiting both moulds and bacteria. Recent
findings also suggest strong synergistic effects on inhibition of mould bread
spoilage by combining this yeast and an antifungal L. plantarum.
MA07 – Thu 1530
Lactic acid bacteria producing antifungal compounds: from plant
protection to cereal product
Elke K Arendt, Emanuele Zannini
School of Food and Nutritional Sciences, University College Cork, College
Road, Cork, Ireland (Email: e.arendt@ucc.ie)
Fungal food spoilage plays a pivotal role in the deterioration of food and
feed systems and some of them are also able to produce toxic compounds
for humans and animals. The mycotoxins produced by fungi can cause
serious health hazards, including cancerogenic, immunotoxic, teratogenic,
neurotoxic, nephrotoxic and hepatotoxic effects and Kashin-Beck disease. In
addition to this, fungal spoilage/pathogens are causing losses of marketable
quality and hygiene of foodstuffs, resulting in major economic problem
throughout the world. Nowadays, food spoilage can be prevented using
physical and chemical methods, but no efficient strategy has been proposed
so far to reduce the microbial growth ensuring public health. Therefore,
lactic acid bacteria (LAB) can play an important role as natural preservatives.
The protection of food products using LAB is mainly due to the production
of antifungal compounds such as carboxylic acids, fatty acids, ethanol, carbon
dioxide, hydrogen peroxide and bacteriocins. In addition to this, LAB can
also positively contribute to the flavour, texture and nutritional value of
food products. This presentation focuses on the use of LAB for plant
protection and food preservation given their extensive industrial application
in a wide range of foods and feeds.
MA07 – Thu 1600
Bacterial–fungal interactions in foods
MArco Gobbetti, Carlo Giuseppe Rizzello, Rossana Coda
Department of Soil, Plant and Food Science, University of Bari Aldo Moro,
Bari, Italy
Bacteria and fungi cohabit several food ecosystems. In some cases,
commensalism occurs between bacteria and fungi, which populate
bacterial surface-ripened cheeses or between bacteria and yeasts, which
colonise kefir grains. Lactic acid bacteria and yeasts also constitute the
prevailing biota of several fermented vegetables as well as interactions,
which are mainly based on the carbohydrate and nitrogen metabolisms,
take place during sourdough fermentation. Fungi represent the
main cause of microbial spoilage of leavened baked goods, causing
considerable economic losses for the industry and decreased shelf life
of the products. In addition to chemical preservatives, mainly low sized
peptides and organic acids, which are synthesised by lactic acid bacteria
during sourdough fermentation, alone or in combination with natural
extracts from vegetables have the capacity to inhibit a large spectrum of
fungi and to considerably extend the shelf life of leavened baked goods.
RNA – so much more than just a
genome
MA08
MA08 – Thu 0900
Modulation of stability of hepatitis C viral RNA and poly(A) site usage
in Insig1 mRNA by microRNA 122
Peter Sarnow, Erica Machlin, Kara Norman, Selena Sagan
Department of Microbiology and Immunology, Stanford University School of
Medicine, Stanford, CA 94305, USA
In general, microRNAs interact with sites residing in 3’ noncoding
regions in target mRNAs, leading to posttranscriptional downregulation
of mRNA expression. In contrast, liver-specific microRNA miR-122 is
known to bind at two adjacent sites that are close to the 5’ end of the
hepatitis C virus (HCV) RNA genome, resulting in upregulation of viral
RNA abundance. Curiously, defined mutations at distinct locations in
miR-122 abolished upregulation of viral RNA, yet functioned well in
microRNA-dependent downregulation of reporter mRNA expression.
Extensive analyses have shown that miR-122 regulates the turnover of
HCV RNA, with only minimal effects on viral mRNA translation or rates
of replication. Several studies have shown that sequestration of miR122 lowers the abundance of cholesterol and downregulated fatty acid
biosynthesis. We have discovered that a major target for miR-122 is Insig1
mRNA. Insig 1 functions as an inhibitor of liver-specific transcription factor
SREBP that modulates the transcription of many genes in the cholesterol
biosynthesis pathway. Specifically, miR-122 downregulates the synthesis
of an Insig1 mRNA isoform that is used for Insig1 protein synthesis. Thus,
sequestration of miR-122 offers an attractive strategy for the temporal
downregulation of HCV and cholesterol in infected livers.
MA08 – Thu 0930
Translational recoding by RNA viruses: ribosome ‘skipping’
Martin Ryan
School of Biology, University of St Andrews, Biomolecular Sciences Research
Complex, North Haugh, St Andrews, Fife, UK
Picornaviruses possess a single, long, ORF encoding a polyprotein which
undergoes ‘processing’. A universal theme amongst picornaviruses is
the rapid separation of the polyprotein domain comprising the capsid
proteins from those downstream, comprising the replication proteins.
In many genera this separation is mediated by a translational recoding
event: ribosome ‘skipping’. Here, an oligopeptide sequence (‘2A’)
interacts with the ribosome exit tunnel inducing changes within the
peptidyl-transferase centre to inhibit peptide bond formation. The stalled
translation complex is ‘rescued’ by the activity of release (termination)
factors 1 and 3 (eRF1/3), normally acting at termination codons to
release completed polypeptides. In ribosome skipping, however, eRF1/3
act at a sense codon (mid-ORF) to release the nascent polypeptide and
hence release the ‘brake’ on polypeptide elongation. Two alternative
36
Session abstracts
outcomes may ensue: either translation is terminated, or, the ribosome
re-enters the elongation cycle to synthesise the downstream (replication)
proteins. Active ‘2A-like’ sequences are found in the genomes of different
types of RNA viruses: in some cases they are functionally analogous to
picornavirus 2As, in other cases they apparently are used to acquire
(‘bolt-on’) extra functions. Recently we have characterised many active
2As within non-LTR retrotransposons and structural genes from a wide
range of organisms.
MA08 – Thu 1000
Manipulating protein–protein interactions with RNA aptamers: novel
experimental tools with therapeutic potential
Nicola J Stonehouse
RNA aptamers are produced by the process of in vitro selection (SELEX)
and bind to target proteins with high specificity and affinity. They
have applications as therapeutic/diagnostic agents and can be used as
molecular tools to interfere with protein-protein interactions in vitro and
in cells. An introduction to this technology will be presented, together
with recent data to demonstrate the utility of aptamers as molecular
tools in studies with two viruses, human papillomavirus 16 (HPV16)
and foot-and-mouth disease virus (FMDV). HPV 16 is associated
with the development of cervical cancer, mediated by the E6 and E7
oncoproteins, both of which interact with a large number of cellular
proteins. In this study, RNA aptamers selected to the E7 oncoprotein
resulted in the degradation of E7 and induction of apoptosis in cells
derived from a cervical carcinoma (that are actively expressing E7).
FMDV causes a livestock disease of significant economic importance. The
genome replication of this virus can be very rapid but a persistent state
can also occur when very little replication occurs. Key to the replication
process is a virally-encoded RNA-dependent-RNA polymerase enzyme.
Aptamers selected to this enzyme were shown to effectively inhibit its
activity with an IC50 of 20 nM.
MA08 – Thu 1100
The k-turn: a key element in RNA architecture
David M J Lilley
CR-UK Nucleic Acid Structure Group, University of Dundee, Dundee, UK
(Email: d.m.j.lilley@dundee.ac.uk)
The kink-turn (k-turn) is a widespread and important element in the
architecture of RNA. It generates a local kink in an RNA helix with an
included angle of 50°, thus mediating long-range interactions in folded
RNA. k-turns are important in RNA species involved in translation, RNA
modification and splicing, and control of gene expression. The k-turn
may be induced to fold into its kinked conformation by a number of
processes. Folding can occur in response to the addition of metal ions
in a two-state folding process, or because of tertiary contacts. Many
k-turns are binding sites for specific proteins, and binding can also
induce the formation of k-turn structure. We have used single-molecule
experiments to show that this occurs by conformational selection.
k-turns play a key role in RNA folding in a delicate interplay involving
sequence variation, protein binding and tertiary contacts. k-turns exist in
two structural classes that differ in key hydrogen bonding patterns in the
core of the structure. Moreover, the same sequence can adopt either
structure depending upon its environment. We have determined the
crystal structure of Kt-7 in a variety of situations, showing the variation in
geometry as a function of protein binding and tertiary contacts.
MA08 – Thu 1130
Advances in imaging plant virus infection
Karl Oparka, Olga Linnik, Karen Bell, Jens Tilsner1
Institute of Molecular Plant Sciences, University of Edinburgh, Mayfield Road,
Edinburgh, UK; 1Biomedical Sciences Research Complex, University of St
Andrews, BMS Building, North Haugh, St Andrews, Fife, UK
Imaging has played a key role in understanding the complex interplay
between plant viruses and their hosts. In this talk, I will describe methods
for imaging the location of viral gene products and viral RNA during the
infection cycle. In particular, I will stress the role that super-resolution
imaging methods have in unraveling the cell-cell movement strategies
of a number of viruses. We have been using potato virus X (PVX) as a
model system for studying the role of movement proteins in facilitating
the interaction with plasmodesmata (PD) that allows the viral genome
to traffic from cell to cell. I will present evidence, based on detailed
imaging studies and mutational analysis of virally encoded proteins, that
replication and movement functions are closely linked at the entrances of
plasmodesmata.
MA08 – Thu 1200
RNA virus micro-evolution and subpopulations within the infected host
Marco Vignuzzi
Institut Pasteur, Paris, France
RNA viruses present extreme genetic diversity from which adaptive
mutations are selected in the host that results in genetic drift, that over
time can lead to the emergence of new strains. To address the role of
diversity in adaptation, we have generated mutator and antimutator
fidelity variants of a number of RNA viruses and perform in vivo
infections with these variants and their wild type counterparts. We
perform deep sequence characterisation of within host subpopulations
to address whether short-term in vivo evolutionary experiments can
predict such longer term emergence events. Using chikungunya virus
infection of mosquitoes as an example, we show that such approaches
could have identified the 2005/6 emergence resulting in the Indian
Ocean epidemic and that continued evolution of this strain identifies
novel mutations that increase dissemination and transmission in both
insect and mammalian hosts. The manipulation of genetic diversity as an
antiviral/vaccine approach and the potential to predict the evolutionary
trajectory of current circulating strains with epidemic potential will be
illustrated in this work.
MA08 – Thu 1330
Viral RNA elements – their role in translational control of gene
expression
Lisa O Roberts
Faculty of Health and Medical Sciences, University of Surrey, Guildford, Surrey, UK
The genomes of positive strand RNA viruses act as both template
for replication and also as mRNAs for the synthesis of viral proteins.
Their genomes often contain complex structures involved in directing
translation of the viral mRNAs. For example, the 5’ untranslated region
(UTR) of all picornavirus genomes contains an internal ribosome entry
site (IRES) element that directs cap-independent internal initiation of
protein synthesis.To date, four distinct classes of picornavirus IRES element
have been described. The enteroviruses and rhinoviruses (e.g. poliovirus)
share one type of element while the cardioviruses and aphthoviruses
(e.g. encephalomyocarditis virus) share a second type. Hepatitis A virus
has a third distinct class of IRES. It has been found recently that certain
picornaviruses contain a fourth class of IRES element which is closely
related to the IRES found in hepatitis C virus (HCV, a hepacivirus within
the Flaviviridae). We have recently found that the picornavirus Seneca
Valley Virus contains an IRES with striking similarity to the classical swine
fever virus (CSFV) IRES, another member of the Flaviviridae. Our current
work is focused on the study of these HCV-like IRESs within picornavirus
genomes and identification of structures within them that are important
for both translation and replication.
MA08 – Thu 1400
Viral RNA the multifunctional shapeshifter
Andrew M L Lever
University of Cambridge, Department of Medicine, Addenbrooke’s Hospital,
Hills Road, Cambridge, UK
37
Session abstracts
Apart from prions all living things use RNA. Of the 24 families of viruses
in the seven groups of the Baltimore classification 16 use RNA as their
genome and two have reverse transcriptase and rely on RNA for
genomic information. RNA is used functionally at all stages of protein
production but it also has intrinsic structural and enzymatic capabilities
as well as its increasingly understood role in gene regulation. All of these
functions are represented in viral RNA. The versatility of this molecule
lies in its use of a very limited number of basic chemical building blocks
to form an enormous variety of different shapes, often more than one
from the same sequence, together with its reactive 2’ hydroxyl group.
Conformational plasticity is a feature of one particular process in RNA
viruses, that of encapsidation of their genome within the virion. The
structural basis of this is starting to be understood in greater detail for
some viruses such as retroviruses. By contrast for a segmented virus like
rotavirus the accurate selection of genomic segments for packaging, while
it must be structure based, is still obscure.
MA08 – Thu 1430
When the genome is not enough: how hepatitis C virus expands its
proteome
Craig E Cameron
Department of Biochemistry and Molecular Biology, The Pennsylvania State
University, University Park, PA 16802, USA
Hepatitis C virus (HCV) is an RNA virus that establishes a persistent
infection in humans. In spite of the small size of its genome, HCV is
capable of hijacking numerous cellular pathways and thwarting host
defenses for decades. The ability of HCV to so effectively control its host
requires more functions than can be encoded in the genome by relying
solely on conventional mechanisms. Non-structural protein 5A (NS5A)
contains both a structured domain and an intrinsically disordered domain
(IDD). The structured domain represents a novel class of GU-rich RNAbindings protein that not only recognises sequences in viral RNA but also
in cellular RNA, thus facilitating replication and manipulation of host gene
expression. The IDD of NS5A is phosphorylated by a variety of kinases.
We suggest the existence of a phosphorylation code that expands the
functional proteome of the virus. We will present examples of specific
phosphorylation events imparting unique structure, dynamics and/or
functions to the IDD of NS5A. We conclude that both the NS5A RNAbinding activity and the NS5A phosphoproteome represent important
yet underexplored targets of drug development.
Virology workshop:
Vaccines and antivirals
MA09
MA09 – Wed 0900
Offered paper Use of synthetic btv reassortants to investigate
immunogenic determinants and develop simple vaccine platforms
Sandro F Nunes1, Claude Hamers2, Andrew Shaw1, Maxime
Ratinier1, Matthew Golder1, Pascal Hudelet2, Massimo Palmarini1
1
Centre for Virus Research, Glasgow, UK, 2Merial Animal Health, Lyon, France
Bluetongue virus (BTV) causes a major infectious disease of ruminants.
There are at least 26 distinct serotypes of BTV (BTV-1 to BTV-26) and
several of them have been circulating in Europe in recent years. In this
study, we generated by reverse genetics a panel of synthetic BTV (sBTV)
reassortants containing the ‘backbone’ of BTV-1 and heterologous outer
core proteins in order to (i) investigate the major viral immunogenic
determinants and (ii) develop simple vaccine platforms. Currently, we
rescued several sBTVs containing the VP2/VP5 of different BTV serotypes.
Replication kinetics of these sBTV were similar to the wild type parental
BTV-1. Using reassortants between BTV-1 and BTV-8, we confirmed that
neutralising antibodies in sheep are induced only by VP2. Interestingly,
rgBTV reassortants containing chimeric VP2 proteins derived by both
BTV-1 and BTV-8 were neutralised by both BTV-1 and BTV-8 antisera
unlike wild type BTV-1 and -8. Finally, we derived inactivated BTV
vaccines based on our BTV-1/BTV-8 rgBTV and showed that they are
fully protective against wild type BTV infection in sheep. In conclusion, we
have shown that synthetic rgBTV viruses can be successfully engineered in
order to further understand BTV infection and to improve vaccine design
and production in the future.
MA09 – Wed 0912
Offered paper Development of virus-like particle vaccines for
epizootic hemorrhagic disease virus
Kinda Al-Shaikhahmed, Polly Roy
London School of Hygiene & Tropical Medicine, London, UK
Epizootic Haemorrhagic Disease Virus (EHDV) predominantly infects
deer. However, it may also infect and cause disease in cattle as
exemplified by recent outbreaks in Europe and USA. Currently, there is
no safe vaccine is available to control the virus spread. EHDV is a member
of Orbivirus genus, Reoviridae family, which consists of three layers of
proteins organised into two capsids, an outer capsid (VP2 and VP5) and
an inner capsid (VP7 and VP3) that encloses the polymerase complex
and 10-segmented dsRNA genome. Here we aimed to generate
recombinant virus-like particles (VLPs) vaccines for EHDV-1, -2, the most
endemic of the 7 serotypes. Since assembly of Orbivirus VLPs requires
four major proteins (VP2, VP3, VP5 and VP7), we first cloned, sequenced
and expressed each protein individually as a recombinant baculovirus
(BV). The authenticity of each was assessed. Each of the four genes was
then cloned into transfer vectors containing bipartite selection cassettes
under different BV promoters which facilitate insertion of EHDV genes at
different loci of the genome (p10, odv-e56, egt and ph). Using this system
VLPs of different combination of EHDV proteins have been generated
by a single BV. Assembly of these particles are examined biochemically,
immunologically and by EM.
MA09 – Wed 0924
Offered paper Contribution of the gag gene to variation in
susceptibility to protease inhibitors between different strains of HIV-1
Katherine A Sutherland1, Jean L Mbisa1, Patricia A Cane1,
Deenan Pillay2, Chris M Parry1,3
1
Health Protection Agency, London, UK, 2University College London Medical
School, London, UK, 3MRC/UVRI Uganda Research Unit on AIDS, Entebbe,
Uganda
Background Resistance to HIV-1 protease inhibitors (PIs) develops by
the accumulation of mutations in protease. We have previously shown
that variability in Gag, the protease substrate, may contribute directly to
reduced susceptibility. Here, we describe natural variation in Gag that
may contribute to differences in susceptibility of wild type viruses to PIs.
Methods A single-cycle, phenotypic drug susceptibility assay was used
to assess the variation in PI susceptibility and contribution of Gag to
susceptibility of six HIV subtype B molecular clones.
Results Significant differences in PI susceptibility between the viruses
were observed with some strains displaying up to 9-fold decrease in PI
susceptibility compared to a wild type control. For two molecular clones,
JRFL and YU2, the reduced susceptibility of full-length Gag-protease was
caused solely by the gag gene. This reduction in susceptibility is conferred
solely by the N terminal 240 amino acids of Gag, and changes at positions
30 and 102 within Gag contributed to the reduced susceptibility.
Conclusions Variation in Gag of HIV-1 contributes to different
susceptibilities to PIs. This study provides further evidence of the direct
role of Gag in PI susceptibility, and that Gag and protease should be
considered together when investigating PI susceptibility.
MA09 – Wed 0936
Offered paper Intradermal immunisation with SIV Gag-based vaccines
alone inhibits acquisition of SIVmac251
Neil Almond1, Richard Stebbings1, Mark Page1, Bo Li1, Neil Berry1,
Claire Ham1, Debbie Ferguson1, Nicola Rose1, Edward Mee1,
Christiane Stahl-Hennig2, George Dickson3, Takis Athanasopoulos4,
38
Session abstracts
Adel Benlahrech5, Shan Herath5, Andrea Meiser5, Steve Patterson5
1
NIBSC, Potters Bar, Hertfordshire, UK, 2DPZ, Gottingen, Germany, 3Royal
Holloway, University of London, London, UK, 4University of Wolverhampton,
Wolverhampton, UK, 5Imperial College, London, UK
Effective anti-viral cell mediated immunity depends both on the frequency
of antigen specific T cells and their quality. We evaluated a vaccine
protocol involving DNA prime and Adenoviral vector boost to deliver SIV
gag to Cynomolgus macaques (Macaca fascicularis). Intra-dermal or intramuscular delivery of 100μg plasmid DNA expressing SIVmac239 derived
gag on weeks 0,4, 8 boosted with 10e7 infectious units of recombinant
Adenovirus 5 vectors expressing the same SIV Gag, on week 19, elicited
both CD8 and CD4 T cell responses. Approximately 50% of antigen
specific CD4+ T cells expressed the mucosal homing marker alpha4
beta7. When vaccinated macaques were exposed to uncloned SIVmac251
by repeated low dose, atraumatic challenge via the intra-rectal route, from
week 23 of the study, a significant delay in acquisition of SIV was obtained
amongst vaccinated macaques compared with naive controls challenged
in a similar manner. Furthermore, peak viral loads amongst vaccinated
macaques were significantly lower than challenge controls. Modification
of the SIV gag , to ubiquinate it at the amino terminus or fragment the
antigens expressed, did not enhance anti-viral T cell responses, nor
improve vaccine protection. We are investigating whether the CD4 or
CD8 T cell responses were crucial to the vaccine’s success.
MA09 – Wed 0948
Offered paper The human mitochondrial RNA polymerase is an off
target for antiviral ribonucleosides
Jamie J Arnold1, Suresh D Sharma1, Joy Y Feng2, Adrian S.Ray2,
Eric D Smidansky1, Maria L Kireeva3, Aesop Cho2, Jason Perry2,
Jennifer E Vela2, Yeojin Park2, Yili Xu2, Yang Tian2, Darius Babusis2,
Blake R Peterson4, Averell Gnatt5, Mikhail Kashlev3, Weidong Zhong2,
Craig E Cameron1
1
The Pennsylvania State University, University Park, PA, USA, 2Gilead Sciences,
Inc., Foster City, CA, USA, 3NCI-Frederick, NIH, Center for Cancer Research,
Frederick, Maryland, USA, 4The University of Kansas, Lawrence, Kansas, USA,
5
University of Maryland School of Medicine, Baltimore MD, USA
There is an intensifying effort to develop antiviral ribonucleosides for
treatment of hepatitis C virus (HCV) infection. Many ribonucleosides
with anti-HCV activity have entered early-stage, clinical trials but most
have failed to advance to phase III because of toxicity of unknown
(or unreported) origin. Here we present evidence that anti-HCV
ribonucleosides are substrates for the human mitochondrial RNA
polymerase (POLRMT) in vitro and in cells. Ribonucleosides containing
2’-C-methyl, 4’-methyl and 4’-azido substituents that are non-obligate
chain terminators of HCV RNA polymerase (HCV NS5B) also inhibit
RNA elongation by POLRMT. In addition, the efficiency of incorporation
in vitro predicted outcomes in cells when normalised for intracellular
metabolism of the antiviral ribonucleoside to the triphosphorylated
form. Together, the biochemical and cellular assays described here
will: 1) facilitate identification of compounds with potential for causing
adverse effects during preclinical studies; and 2) permit discovery and
development of ribose substituents that diminish utilisation by POLRMT
without decreasing utilisation by viral RdRps. We suggest the possibility
that the small fraction of patients exhibiting adverse effects during
clinical trials may be more sensitive to mitochondrial insults as a result
of undiagnosed mitochondrial disease(s). The paradigm reported here
should facilitate development of efficacious, safe antiviral ribonucleosides.
MA09 – Wed 1000
Offered paper Enhancing the immunogenicity and vaccine efficacy of
vaccinia virus through the removal of innate immunomodulatory genes
Rebecca P Sumner, Hongwei Ren, Geoffrey L Smith
Vectors based on vaccinia virus (VACV), the vaccine used to eradicate
smallpox, are popular candidates for vaccination against numerous
infectious diseases including malaria and AIDS. Although VACV induces
robust cellular and humoral responses, enhancing the safety and efficacy
of these vectors remains an important area of research. VACV expresses
a plethora of proteins that inhibit the host innate immune response and
recent work has demonstrated that the removal of some of these genes
from the virus enhances vaccine efficacy. One example is a recombinant
VACV Western Reserve (WR) strain lacking the C6L gene (vΔC6). C6L
encodes an immunomodulatory protein that inhibits interferon regulatory
factor 3 activation. Intradermal vaccination of mice with vΔC6 provided
better protection against challenge with VACV WR compared to wild-type
virus, despite attenuation. Increased protection was not due to improved
humoral responses, but instead correlated with enhanced cytotoxicity of T
cells one month post-vaccination. Current research is focussed on gaining
a mechanistic understanding of how removing innate immune modulatory
genes positively impacts the formation of immune memory, with the hope
of not only improving the safety and efficacy of VACV as a vaccine vector,
but also allowing us to identify ways to design more immunogenic vaccines.
MA09 – Wed 1012
Offered paper Structure-guided design of a virus-free polio vaccine
Clare Nicol1, Andrew J Macadam2, Luigi De Colibus3,
Sarah Knowlson2, Joseph Newman4, Elizabeth E Fry3,
David I Stuart3, James M Hogle5, Phillip D Minor2, Tobias J Tuthill4,
Nicola J Stonehouse1, David J Rowlands1
1
University of Leeds, Leeds, West Yorkshire, UK, 2National Institute for
Biological Standards and Control, South Mimms, Hertfordshire, UK, 3Oxford
University, Oxford, Oxfordshire, UK, 4Institute for Animal Health, Pirbright,
Surrey, UK, 5Harvard Medical School, Boston, Massachusetts, USA
With the global eradication of wild poliovirus moving ever closer to
completion, strategies for continued vaccination in a poliovirus-free
world need to be considered. Both of the current vaccines in use have
drawbacks which make them less suitable for use in a post-eradication
environment. The oral poliovirus vaccine (OPV) can give rise to vaccineassociated paralytic poliomyelitis (VAPP) and circulating vaccine-derived
poliovirus (cVDPV). The inactivated poliovirus vaccine (IPV) requires
large-scale culture of infectious virus, which in a post-poliovirus era is
likely to be subject to highly stringent regulations. One alternative is the
use of poliovirus-like particles (polio-VLPs) as vaccines. Unfortunately,
polio-VLPs are prone to conformational changes leading to inappropriate
antigenic forms at physiological temperatures. Informed by structural
data, this project aims to identify stabilising mutations that could be
introduced into the poliovirus capsid to produce a thermostable polioVLP with appropriate antigenic and immunogenic characteristics. Using
molecular dynamics simulations and protein modelling, a number of
potentially stabilising mutations have been identified. For example,
introduction of disulphide bonds across the inter-pentamer interfaces
resulted in poliovirus empty particles with improved thermostability over
wild-type empty particles while retaining wild-type antigenicity.
MA09 – Wed 1100
Offered paper The lyssavirus glycoprotein: how significant are the
currently characterised antigenic sites in the induction of neutralising
antibodies?
Jennifer S Evans1,2, Edward Wright3, Andrew J Easton2,
Anthony R Fooks1, Ashley C Banyard1
1
Animal Health and Veterinary Laboratories Agency, Weybridge, Surrey, UK,
2
The University of Warwick, Coventry, West Midlands, UK, 3University of
Westminster, London, UK
Rabies, the archetypal lyssavirus, is one of the most feared viruses
known to man and causes >50,000 deaths per year. Other members
of the lyssavirus genus cause clinical disease consistent with rabies. The
lyssavirus glycoprotein is the sole target for virus neutralising antibodies
and several amino acid epitopes have been linked to virus neutralisation.
Lyssaviruses are segregated into phylogroups that indicate the level of
protection afforded by current vaccines. It is generally accepted that an
39
Session abstracts
antibody response to rabies vaccines affords protection against all viruses
that are categorised into phylogroup I. However, this antibody response
does not protect against lyssavirus species within phylogroups II and
III. Indeed, experimental data has shown that the antibody repertoire
induced by rabies virus vaccines is completely unable to neutralise
viruses in these phylogroups. In this study we have generated lentivirus
pseudotypes containing chimeric lyssavirus glycoproteins that have had
their antigenic sites swapped between phylogroup I and II viruses. With
these we have assessed neutralisation of both wildtype and chimeric
pseudotype particles with both phylogroup I and phylogroup II specific
hyperimmune sera. We have used chimeric pseudotype viruses to
analyse the role of defined antigenic domains in development of
phylogroup specific immune responses.
MA09 – Wed 1112
Offered paper A novel approach to determining influenza virus
resistance to oseltamivir
Mojca Zelnikar, Samantha J Lycett, Andrew J Leigh Brown
The University of Edinburgh, The Institute of Evolutionary Biology, Edinburgh,
UK
Influenza viruses cause annual epidemics in many countries and occasional
worldwide pandemics. Currently, there are two classes of antiviral drugs
for preventing and treating influenza infections but the resistance to both
has risen drastically in the last few years. The increasing emergence of
influenza viruses resistant to oseltamivir is concern because of its use in
immunosuppressed patients therefore understanding the determinants
of resistance is important. While the drug resistance-conferring mutations
in neuraminidase (NA) are well known, their interaction with the
haemagglutinin (HA) sequence has been neglected. The aim of our study
was to test for association of amino acid variants in HA with resistance to
oseltamivir. We used classification trees and random forests. On a basis
of a large sample size (542 matched HA and NA sequences) we show
the importance of mutations on three amino acid sites in HA (188, 191,
192; H3 numbering) with high specificity and sensitivity for predicting the
resistant phenotype of influenza viruses. For classification trees analysis the
sensitivity was 71.3% and specificity was 98.6%. Random forest analysis
yielded higher sensitivity (73.4%) and higher specificity (99.4%) revealing
a strong association between resistance in NA and the sequence of the
unlinked HA segment.
MA09 – Wed 1124
Offered paper The study of heterosubtypic neutralising antibody
responses against H5N1 influenza viruses in human subjects using a
comparative serology approach
Eleonora Molesti1, Francesca Ferrara1, Emanuele Montomoli2,
Nigel Temperton1
1
Viral Pseudotype Unit, University of Kent, Chatham Maritime, Kent, UK,
2
Department of Physiopathology, Experimental Medicine and Public Health,
University of Siena, Siena, Italy, Italy
The continuous rapid genetic and antigenic evolution of H5 subtype
influenza viruses has major implications for the sensitivity of serological
assays and can limit the efficacy of pre-pandemic human vaccines and the
ability to undertake effective sero-surveillance in susceptible populations.
A panel of serum samples collected from the Italian population between
1992 and 2007 and previously found to be positive for antibodies against
H5N1 as determined by SRH, were evaluated using pseudotype based
neutralisation assays in combination with haemagglutination-inhibition
assays using a clade 1 and a clade 2 H5N1 serological antigen. From
the results obtained it can be concluded that the pseudotype assay
measures cross-reactive antibody responses that are not picked up with
the HI assay. Both assays measure antibodies with different (functional)
specificities contributing to a comprehensive analysis of humoral
immunity to influenza viruses.
MA09 – Wed 1136
Offered paper Humoral response and antiviral cytokine expression
following vaccination of thoroughbred weanlings – a comparison of
commercially available vaccines
Sarah Gildea1, Michelle Quinlivan1, Barbara Murphy2, Ann Cullinane1
1
Virology Unit, The Irish Equine Centre, Johnstown, Naas, Co.Kildare, Ireland,
2
School of Agriculture, Food Science and Veterinary Medicine, University
College Dublin, Belfield, Dublin 4, Ireland
This study compared humoral and cytokine responses following
vaccination with an ISCOM based (Equilis Prequenza TE), a canarypox
recombinant (ProteqFlu-Te) and an inactivated whole virus vaccine
(Duvaxyn IE-T Plus). Forty-four seronegative Thoroughbred weanlings
were vaccinated (V1/V2) 29 days apart. Antibody response was
monitored for three weeks post V2 by single radial haemolysis. The
pattern of antibody response was similar for all vaccines but significantly
higher in weanlings vaccinated with Duvaxyn IE-T Plus. In this study
39% of weanlings failed to seroconvert following V1. PAXgene blood
samples were collected on days 0, 2, 7 and 14 post V1. Gene expression
levels of IFN-γ (marker for cell-mediated immune response), IL-1β
(pro-inflammatory cytokine) and IL-4 (B cell stimulating cytokine) were
measured using RT-PCR. Mean gene expression levels of IL-1β and
IL-4 peaked on day 14 post V1. IL-1β gene expression in weanlings
vaccinated with Duvaxyn IE-T Plus was significantly higher than in those
vaccinated with the other two products. Vaccination with all three
vaccines resulted in an increase in IFN-γ gene expression which peaked
on day 7 post V1. There was no significant difference in IFN-γ gene
expression between the whole inactivated, the subunit and the canarypox recombinant vaccines included in this study.
MA09 – Wed 1148
Offered paper Immunomodulatory effects of the novel kinase inhibitor
RV1088 in response to influenza virus infections in primary respiratory
cell cultures
Jonathan W Ashcroft1, Daniel Brookes2, Kazuhiro Ito2,
Wendy S Barclay1
1
Section of Virology, Imperial College London, UK, 2RespiVert Ltd., London, UK
Respiratory infections caused by influenza viruses are responsible for
significant morbidity and mortality world-wide. Given the emergence
of strains resistant to licensed antiviral drugs that include adamantanes
and neuraminidase inhibitors, it is imperative that novel compounds be
identified to treat influenza virus infections. Severe outcome has been
linked to the over induction of the host innate immune response resulting
a ‘cytokine storm.’ In certain patient populations, a therapeutic approach
to help control over induction of the innate immune response may be of
benefit. However concerns have been expressed that suppression of the
host immune response might also lead to an increase in virus replication
and directly enhance viral induced pathology. A small molecule compound
(RV1088, RespiVert) developed for the treatment of respiratory disorders
linked with a strong inflammatory response significantly inhibited the
induction of an array of human cytokines elicited by both type A and
type B influenza strains in primary human airway epithelium cultures.
Furthermore, at low multiplicities of infection, RV1088 also reduced viral
replication, an effect which was augmented by concurrent treatment with
Zanamivir. This or similar molecules, may represent a new generation of
compounds suitable for the treatment of respiratory virus infections.
Virology workshop:
The virome and viromics
MA10
MA10 – Wed 0900
Offered paper Frequent detection of virus-like sequences in reagents
commonly used for viral metagenome preparation
William Gregory, Zen Lu, Sinead Lyons, Peter Simmonds, Colin Sharp
The Roslin Institute, Midlothian, UK
40
Session abstracts
There are now numerous methods for the enrichment and amplification
of viral sequences for metagenomic analysis and novel virus discovery.
Following viral enrichment, amplification procedures by sequenceindependent methods involving either Taq or phi-29 polymerases are
generally required to produce sufficient DNA for high throughput
sequencing. We generated sequence libraries for both Roche454 and
Illumina Hi-seq platforms by sequence-independent PCR from various
biological sources (serum, a cell line and faecal supernatants) that had
been enriched for viral nucleic acids in parallel with control libraries with
no nucleic acid added. Despite the use of fresh reagents and preparation
of libraries making every possible effort to eliminate exogenous DNA
contamination, negative control reactions did produce amplicons. Analysis
of the resultant sequences by homology search showed abundant viral
sequences in all the libraries but the majority that were present in the
sample libraries had closely related sequences in the negative controls,
many of which were homologous to virus recently discovered using similar
methods. Further PCR analysis of selected sequences confirmed the
presence of these nucleic acids in the reagents used for library generation.
This work highlights the crucial need for inclusion and reporting of negative
control samples when producing viral metagenomic data.
MA10 – Wed 0912
Offered paper Proteomics informed by transcriptomics – everything
you ever wanted to know, in one go
David Matthews
University of Bristol, Bristol, UK
We have been developing ways of integrating high throughput
quantitative proteomics with deep sequencing of the virus infected cells.
This approach, called Proteomics Informed by Transcriptomics (PIT)
analysis was recently validated using human adenovirus infection of human
cells. We have shown it is possible to simultaneously monitor changes in
mRNA abundance and protein abundance for several thousand genes
and proteins simultaneously. We will demonstrate how this can be used
to selectively identify novel targets of virus induced proteolytic destruction.
In the case of adenovirus infection we present evidence that POLDIP3
and CLU are newly identified targets of the adenovirus induced ubiquitin
ligase complex. We will illustrate how this approach can be applied to any
virus in any host cell even if the cells are derived from a non model animal
species. This is particularly important as high throughput approaches to
gene and protein identification frequently require access to high quality
gene annotation files in order to maximise our data return.
MA10 – Wed 0924
Offered paper Diverse reassortment patterns of avian influenza
viruses among different subtypes in internal segments
Lu Lu, Andrew J Leigh Brown, Samantha J Lycett
The University of Edinburgh, Edinburgh, UK
Sixteen HA and nine NA subtypes of influenza A virus exist in wild
birds and at least 103 of the possible 144 type A influenza virus HANA combinations have been found, which indicates a high frequency of
reassortment among HA and NA subtypes, although some restrictions on
patterns of combination may exist. Here, we quantify the reassortment
patterns among subtypes in the Eurasian avian viral pool by reconstructing
ancestral subtypes on time-scaled phylogenies of the internal protein
coding segments. Reassortment rates of certain subtypes were estimated
using Bayesian and maximum likelihood methods. The rates showed
significant diversity among different subtypes. Specifically, H5, H6, H7
and H9 of HA subtype present significantly lower reassortment rates
than those of subtype H1, H3 and H4. N1 and N2 subtypes of NA have
lower reassortment rates compared to those of N3, N5, N6, N7 and N9.
The lower rates observed for H5N1 and H9N2 may be explained by the
large proportion of strains derived from domestic poultry populations.
In contrast, the higher rates observed in the H1N1, H3N8 and H4N6
subtypes could be due to their primary origin as infections of wild birds
with multiple low pathogenicity strains in the large avian reservoir.
MA10 – Wed 0936
Offered paper Genetic variability and the classification of hepatitis E
virus
Donald B Smith1, Michael Purdy2, Peter Simmonds1
1
The University of Edinburgh, Edinburgh, Scotland, UK, 2Centers for Disease
Control, Atlanta, Georgia, USA
The classification of hepatitis E virus (HEV) variants is currently in
transition without agreed definitions for genotypes and subtypes, or
for deeper taxonomic groupings into species and genera that could
incorporate more recently characterised viruses infecting birds, bats,
rodents and fish that have been assigned to the Hepeviridae family. We
have performed an extensive analysis of available sequences to assist
in the re-classification of this virus family based on genetic relationships.
Synonymous substitutions were saturated in comparisons between
and within virus genotypes. Consequently, concatenated ORF1/ORF2
amino acid sequences (excluding the HVR) provided a clearer division of
HEV variants into six genotypes (types 1-4, infecting humans) and two
additional genotypes (from wild boar). Variants isolated from rabbits,
closely related to genotype 3, occupied an intermediate position. No
consistent criteria could be defined for the assignment of virus subtypes.
Comparison with conserved amino acid sequences from the more
divergent variants from chickens, bats, and rodents supported a division
into 4 proposed species each with specific host associations: A) humans,
rabbits and pigs, (B) rats and ferrets, (C) bats and (D) chickens. The
much more divergent HEV-like virus from fish (cutthroat trout virus)
should be assigned to a second genus.
MA10 – Wed 0948
Offered paper Wild-type EBV genome sequence and genome diversity
of EBV and KSHV from multiple tumour types
Anne L Palser1, Robert White2, John Arrand3, Martin Allday2,
Lawrence Young4, Paul Murray3, Paul Farrell2, Paul Kellam1
1
Wellcome Trust Sanger Institute, Hinxton, Cambridge, UK, 2Imperial College,
London, UK, 3University of Birmingham, Birmingham, UK, 4The University of
Warwick, Warwick, UK, 5University College London, London, UK
Epstein–Barr virus (EBV) and Kaposi’s sarcoma-associated herpesvirus
(KSHV) are oncogenic human herpesviruses. EBV is associated with
several different tumour types. Many of these tumours show striking
geographic variations in prevalence and in contrast to high seropositivity,
only a small proportion of people infected develop tumours. At the
whole genome level, understanding of EBV and KSHV sequence diversity
is limited. Currently there is no published wild-type EBV genome
sequence from a healthy individual and it is unclear whether specific virus
strains or polymorphisms are associated with different tumours.
We have adapted target enrichment methods used for exome
sequencing to selectively pull out virus DNA from host DNA and then
deep sequenced the captured virus DNA. We were able to sequence
and assemble 100 EBV and KSHV genomes from infected cell lines,
tumours, blood and saliva using this approach. Samples were taken from
different locations worldwide and preliminary analysis shows a mainly
geographic clustering of EBV strains in phylogenetic trees. We can look
more closely at individual SNPs to investigate disease-specific genetic
changes and polymorphisms in known tumour-associated genes. It also
allows an estimation of inter-genome recombination and open reading
frame-specific rates of mutation, which may highlight genomic regions
important for pathogenesis.
MA10 – Wed 1000
Offered paper Development of a single-cell viral sequencing technique
and its application in analysing HCV diversity at the cellular level
Carol Leitch1, John McLauchlan1, Daniel Haydon2
1
MRC-University of Glasgow-Centre for Virus Research, Glasgow, UK, 2Institute
of Biodiversity, Animal Health and Comparative Medicine, Glasgow, UK
Single-cell genomics is becoming an important tool in the field of cellular
41
Session abstracts
biology to analyse between-cell differences. We developed for the first
time a system to analyse viruses at the cellular level and used these
techniques to investigate HCV quasispecies diversity within individual
cells. We hypothesised that evolution proceeds independently within
each discrete cellular compartment. RNA extracted from individual Huh7
cells constitutively expressing sub-genomic HCV JFH-1 replicons was
used for high fidelity nested RT-PCR of NS5B. Assay validation indicated
a detection limit of 3 copies/cell. Six cellular populations were cloned
and sequenced, and population diversity was investigated by median
joining networks. Each cell contained a unique quasispecies, suggesting
different within-cell evolutionary trajectories, and lower diversity than the
total cell population. The predominant variants displayed less replicative
fitness than the wild type replicon. Real time RT-PCR performed on
each cellular population indicated a range of 84-160 replicon copies/
cell. This study introduces the novel strategy of analysing viruses at the
cellular level and subsequently applies it to the assessment of within-cell
HCV quasispecies diversity. Potentially the technique has numerous
applications in virus research including the evolution of antiviral and
immune escape variants, host/virus interactions and correlations between
viral diversity and disease progression.
MA10 – Wed 1012
Offered paper Potential role of next-generation sequencing in the
diagnostic algorithm of acute HCV infection
Tamer Abdelrahman1, Joseph Hughes1, Janice Main1,2,
John McLauchlan1, Emma Thomson1,2
1
MRC Centre for Virus research, University of Glasgow, Glasgow, UK,
2
Department of Hepatology and infectious diseases, Imperial College NHS
trust, London, UK
In hepatitis C virus (HCV) infection, the virus circulates as a mixture
of closely related but distinct genomes called quasispecies. The
hypervariable region-1 (HVR-1) is an excellent target for sequence
analysis to distinguish between different variants. We studied the
complexity of viral sequences in samples taken before treatment from
patients who subsequently failed standard of care therapy in an HIV/
acute HCV cohort. The (HVR-1) region was amplified using nested RTPCR. PCR products were sequenced by direct sequencing (DS), clonal
analysis (CA) and next generation sequencing (NGS). Phylogenetic trees
were constructed using the maximum likelihood (ML) method. In the 16
patients that failed treatment, CA and NGS results revealed that 56% of
patients had mixed infection with either more than one subtype or one
genotype two or more genotypes that were not detected by DS. The
average number of sequences generated from each sample was 26000
using NGS compared to 20 sequences by CA. NGS was superior to
CA in detecting different variants in 25% of patients. The development
of screening tools to detect viral sequence complexity at baseline, in
order to detect mixed infections, particularly in the context of treatment
decisions involving genotype-specific direct-acting antiviral agents, and
associated resistance mutations.
MA10 – Wed 1100
Offered paper Next-generation sequencing of RNA viral genomes
from clinical and cultured samples: application in discovery of a novel
lyssavirus in Africa
Denise A Marston1,2, Daniel L Horton1, Lorraine M McElhinney1,3,
Richard J Ellis1, Emma L Wise1, Stacey L Leech1, Chanasa Ngeleja5, Tiziana
Lembo4, Sarah Cleaveland4, Xavier de Lamballerie2, Anthony R Fooks1,3
1
Animal Health & Veterinary Laboratories Agency (AHVLA), Weybridge,
Surrey, UK, 2Aix-Marseille University, Marseille, France, 3National Consortium
for Zoonosis Research, Leahurst, Wirrel, UK, 4University of Glasgow, Glasgow,
UK, 5Central Veterinary Laboratory, Dar es Salaam, Tanzania
With the advent of Next-generation sequencing (NGS) technologies,
the ability to generate large amounts of sequence data has revolutionised
the genomics field. Viruses have relatively small genomes in comparison
to other organisms and as such, would appear to be an obvious
success story for the use of NGS technologies. However, due to the
relatively low abundance of viral RNA in relation to host RNA, RNA
viruses have proved relatively difficult to detect and sequence using
NGS technologies. We have developed a simple, robust methodology,
without the use of ultra-centrifugation, filtration or viral enrichment
protocols, to prepare RNA from diagnostic tissue samples, cell
monolayers and tissue culture supernatant, which were subsequently
sequenced on the Roche 454 platform. Full genome sequence of
multiple lyssaviruses, including a novel species, (Ikoma lyssavirus (IKOV))
were successfully obtained from both cultured material and clinical
samples without reference sequences. Analysis of the viral genome
sequence of IKOV, derived from the brain of an African civet in Tanzania,
raised the possibility of incomplete vaccine protection, which was
subsequently assessed in-vitro and in-vivo. The approaches reported can
be universally applied to discovering and obtaining consensus genome
sequences of RNA viruses from a variety of sources.
MA10 – Wed 1112
Offered paper Modelling mutational and selection pressures on
dinucleotides in eukaryotic phyla and RNA viruses that infect them
– identification of selection pressures against CpG and UpA in
cytoplasmically expressed mRNA sequences and in RNA viruses
Wenjun Xia, Kenneth Baillie, Ken McKinnon, Peter Simmonds
Loss of CpG dinucleotides in genomic DNA through methylationinduced mutation is characteristic of chordates and plants and viruses
infecting these phyla although these show other dinucleotide frequency
biases with uncharacterised underlying mutational or selection
mechanisms. We developed a parameterised Markov process to
identify what dinucleotide context dependent mutations best accounted
for compositional patterns in genomic and cytoplasmically expressed
mRNA sequences of different vertebrates, other chordates, plants,
ecdyzoa and RNA viruses. Increased frequencies of C→T upstream
of G (C→T,G) best modeled dinucleotide frequencies in mammalian
and plant genomic DNA. Different context-dependent mutations were
modeled in other eukaryotes with non-methylated genomes DNA.
Dinucleotide frequencies in mRNA sequences from all organisms were
dominated by mutations that eliminated UpA dinucleotides consistent
with cytoplasmically driven selection for mRNA stability. Surprisingly,
mRNA sequences from organisms with methylated genomes showed
evidence for additional selection against CpG. Similar selection processes
were identified in mammalian and plant RNA viruses and which
potentially account for their previously described but unexplained
under-representations of CpG and UpA dinucleotides. Methods we
have developed identify mutational processes and selection pressures
in organisms and viruses that provide new insights into nucleotide
compositional constraints and a wealth of biochemical and evolutionarily
testable predictions for the future.
MA10 – Wed 1124
Offered paper Changes in deformed wing virus (DWV) population
diversity and the honeybee (Apis mellifera) global transcriptome
resulting from infestation with the mite Varroa destructor
Eugene V. Ryabov1, Graham Wood2, Jessica M Fannon1,
Jonathan Moore2, James C Bull1, Dave Chandler1, Andrew Mead1,
Nigel Burroughs2, David J Evans1
1
School of Life Sciences, The University of Warwick, Coventry, CV4 7AL, UK,
2
Warwick Systems Biology Centre, The University of Warwick, Coventry, CV4
7AL, UK
DWV causes asymptomatic infection and accumulates to low levels in
the honeybee (Apis mellifera) in the absence of the parasitic mite Varroa
destructor. Conversely, mite-infested pupae have high levels of DWV and
can develop overt symptoms. Using brood frame-transfer we subjected
Varroa-free larvae to exposure to viruses, both mite-and feedingtransferred, from a Varroa-infested hive.
42
Session abstracts
The virus populations in individual bee pupae and the associated mites
were analysed using qPCR and deep sequencing. DWV levels increased
slightly in bees infected per os but about 1000-fold in mite-infested bees,
and DWV populations in these pupae were distinct from those in the
Varroa-free colony. The increase of DWV levels in the Varroa-infested
pupae was accompanied by a marked decrease in DWV diversity,
although in the associated mites virus diversity remained high.
Gene expression was analysed in the bee pupae using whole genome
microarrays. Mite-free pupae infected with DWV per os exhibited
changes in expression of 4% of genes; this response possibly suppressed
orally delivered DWV. In mite-infested pupae with high DWV, 9% of
genes were differentially expressed, including a number of immunerelated genes.
Virology workshop:
Assembly and structure
MA11
MA11 – Wed 0900
Offered paper Picornavirus uncoating – another piece of the jigsaw
David I. Stuart1,5, Jingshan Ren1, Xiangxi Wang2, Zhongyu Hu3,
Gao Qiang4, Yao Sun2, Xumei Li2, Claudine Porta1, Thomas S. Walter1,
Robert J. Gilbert1, Yuguang Zhao1, Danny Axford5, Mark Williams5,
Katherine McAuley5, David J. Rowlands6, Weidong Yin4, Junzhi Wang3,
Zihe Rao2,7, Elizabeth Fry1
1
Division of Structural Biology, University of Oxford, Oxford, UK, 2National
Laboratory of Macromolecules, Institute of Biophysics, Beijing, China,
3
National Institute of Food and Drug Control, Beijing, China, 4Sinovac Biotech
Company, Beijing, China, 5Diamond Light Source, Didcot, UK, 6Institute of
Molecular and Cellular Biology, University of Leeds, Leeds, UK, 7Laboratory of
Structural Biology, Tsinghua University, Beijing, China
Enveloped viruses fuse virus and host cell membranes using a protein
machine to allow cell entry, but it is much less clear how the fragile
genomes of non-enveloped viruses are transferred into the host
cell. Picornaviruses are small, icosahedral animal RNA viruses and
are well-established models for non-enveloped viruses. Within this
family the enteroviruses have a surface depression (canyon) which
often contains the receptor binding site. Binding here dislodges a fatty
acid molecule from within the hydrophobic β-barrel core of VP1,
resulting in a cascade of structural changes, leading to the release of the
N-terminus of VP1 and VP4, to form the expanded 135S intermediate
or A-particle. This particle is endocytosed and latterly engages fully
with the vesicle membrane, to deliver the RNA to the cell. Recent
EM and crystallographic studies have visualised expanded capsids
revealing channels through which internal capsid proteins and the viral
genome might exit the particle. The atomic structure of an uncoating
intermediate of CAV16 reveals the VP1 protein partly extruded from
the capsid. Together with recent EM results, this allows us to propose
a detailed, evidence-based hypothesis for a further stage in picornavirus
uncoating, addressing the puzzle of how non-enveloped viruses efficiently
infect cells.
MA11 – Wed 0912
Offered paper Recombinant VP4 of human rhinovirus induces
membrane permeability by formation of a size-selective multimeric
pore
Anusha Panjwani1, Dave Rowlands2, Nicola Stonehouse2,
James M Hogle3, James Chou3, Toby Tuthill1
1
Pirbright Institute, Pirbright Laboratory, Surrey, GU24 0NF, UK, 2University
of Leeds, Leeds, LS2 9JT, UK, 3Harvard Medical School, Boston, MA 02115,
USA
In contrast to enveloped viruses, non-enveloped viruses cannot use
membrane fusion to enter cells and must therefore penetrate the
cell membrane using alternative mechanisms which remain poorly
defined. The picornavirus family of non-enveloped viruses includes
significant pathogens such as poliovirus, human rhinovirus (HRV) and
foot-and-mouth disease virus. Picornavirus assembly culminates in a
maturation cleavage of capsid component VP0 into VP4 and VP2. During
picornavirus cell entry, the capsid protein VP4 is released, interacts with
the cell membrane and is implicated in membrane penetration. We have
produced recombinant histidine-tagged HRV VP4 and demonstrated
that both recombinant VP4 and virus particles induce membrane
permeability in liposome model membranes. Chemical crosslinking
and dextran size-exclusion studies demonstrated that VP4 forms a
multimeric (pentameric/hexameric) membrane pore with a channel size
limit consistent with transfer of the RNA genome. Putative VP4 pore
structures were visualised by negative staining electron microscopy.
Circular dichroism demonstrated predominantly alpha helical secondary
structure. Membrane permeability induced by recombinant VP4 was
influenced by temperature, pH and lipid composition, with maximum
VP4 activity observed under conditions most physiologically relevant
to natural infection. This study presents a model system for defining
molecular details of VP4 membrane penetration by this important family
of pathogens.
MA11 – Wed 0924
Offered paper A conserved determinant in VP1-2 homologues from all
herpesviruses required for capsid routing to the nuclear pore via the
MTOC
Thomas Hennig, Fernando Abaitua, Peter O’Hare
The large structural protein VP1-2 of HSV (UL36 gene) is conserved
across the herpesvirus family and plays a key role in several early events
leading to nuclear entry. We previously identified an N-terminal, essential
nuclear localisation signal (NLS) at position 475 in herpes simplex
virus which is involved in capsid routing to the nuclear pore via the
microtubule organising centre (MTOC). We have now examined this
function in VP1-2 homologues from all herpesvirus families, including
HCMV, EBV and KSHV. The basic motif is present, but exhibits subfamily specific organisational differences. Nevertheless each of these
motifs confers nuclear localisation to a heterologous test protein and in
HSV-1 VP1-2 chimeras. Importantly, the motifs from HCMV, VZV and
EBV can rescue with variable efficiencies the defect in K.VP1-2DNLS, an
HSV mutant which, while it can enter and be transported within cells,
shows a defect after reaching the MTOC but prior to nuclear pore
docking. These results therefore indicate that in the viruses tested, and
likely in all herpesviruses, this determinant is essential and required for
early capsid routing to the nuclear pore to initiate genome transport and
expression.
MA11 – Wed 0936
Offered paper The intracellular trafficking of HSV-1 envelope proteins
during assembly
Sheung-Yee Kathy Lau
During Herpes simplex virus 1 (HSV-1) assembly, sixteen different viral
envelope proteins are targeted to viral assembly sites in order for their
incorporation into virions. How these envelope proteins localise to such
cytoplasmic membrane compartments still remains unclear. Following
protein biosynthesis, the default trafficking route for membrane proteins
is to the plasma membrane unless they contain specific targeting
information. Some HSV-1 envelope proteins, such as glycoprotein M
(gM), encode specific endocytic motifs allowing for their internalisation
and targeting to subcellular compartments. However, other HSV-1
envelope proteins, including the essential entry proteins gD and gH/L,
contain no evident trafficking motif and remain on the plasma membrane
in the absence of other viral proteins. Previously, we have demonstrated
that gM targets gH/L to intracellular compartments for incorporation
into assembling virions. We now demonstrate that in addition to gM,
the HSV-1 envelope proteins gK/pUL20 and pUL43 mediate the
43
Session abstracts
subcellular targeting of gD and gH/L. We have investigated the roles
of gM, gK/pUL20 and pUL43 in HSV-1 glycoprotein localisation during
infection using a series of deletion viruses. Our observations suggest that
HSV-1 possesses at least three redundant mechanisms to mediate the
intracellular trafficking of envelope proteins to the sites of virus assembly.
MA11 – Wed 0948
Offered paper Human immunodeficiency virus types 1 and 2 assemble
at different cellular locations
Michela Marongiu, Justine E Alford, Emma C Anderson
School of Life Sciences, The University of Warwick, Coventry, UK
Assembly of human immunodeficiency virus (HIV) particles is thought
to begin with the oligomerisation of membrane-associated Gag
polyproteins. HIV-1 Gag molecules interact with viral RNA via their
nucleocapsid domains in a perinuclear region and are then trafficked
predominantly to the plasma membrane where they interact with the
membrane via their N-terminal myristylated matrix domain. It is assumed
that HIV-2 Gag behaves in a similar manner. We have studied the
localisation of HIV-1 and HIV-2 Gag within cells and found the two to
be quite different; HIV-1 Gag was found throughout the cytoplasm and
at the plasma membrane whereas HIV-2 Gag was concentrated within
intracellular compartments. Electron microscopy showed the assembly
and budding of HIV-1 particles at the plasma membrane whereas HIV2 particles could be seen budding into intracellular membrane-bound
compartments. These were seen to contain both immature and mature
virus particles. We have demonstrated that the HIV-2 matrix domain
alone confers this localisation. Further investigation of the mechanism by
which HIV-1 and HIV-2 matrix domains determine the site of particle
assembly has shown that the two viruses interact differently with the
clathrin adaptor proteins 1 and 3.
MA11 – Wed 1000
Offered paper Epstein–Barr virus genome packaging factors converge
in inner genome storerooms of BMRF1 cores within viral replication
compartments
Tatsuya Tsurumi, Atsuko Sugimoto
Aichi Cancer Center Research Institute, Nagoya, Japan
Productive replication of the Epstein–Barr virus (EBV) occurs in discrete
sites in nuclei, called replication compartments, which include subnuclear
domains, designated BMRF1-cores, which are highly enriched in the
BMRF1 protein. During viral lytic replication, newly synthesised viral DNA
genomes are organised around BMRF1-cores and then stored inner
subdomains. We have used three dimensional surface reconstruction
imaging to examine spatial arrangements of the sites of encapsidation in
EBV replication compartments. Viral factors required for DNA packaging
were located in innermost subdomains within the viral genome
storerooms, while capsid structural proteins were found both outside
and inside. Based on these observations, we propose a model that capsid
maturation and DNA encapsidation occur inside in innermost areas
of BMRF1-cores. This is the first report of capsid assembly sites in EBV
genome manufacturing plants.
MA11 – Wed 1012
Offered paper An in vitro assembly system for bluetongue virus: a tool
to investigate RNA packaging
Po-yu Sung, Polly Roy
London School of Hygiene and Tropical Medicine, London, UK
Bluetongue virus (BTV) is a vector-borne, non-enveloped virus with a
genome containing 10 segments of double stranded RNA. BTV has an
outer capsid and inner capsid (core). The BTV core has two layers; an outer
layer which comprises VP7 and an inner layer (subcore) of VP3. The interior
of the core hosts replication complexes which are constituted of VP1, VP4,
and VP6, as well as BTV genomic RNA. Although much is known about
BTV core structure, the order and mechanism of BTV genome packaging
are still to be investigated. An in vitro assay mimicking BTV core assembly
was newly developed and has been proven to reconstitute infectious BTV
cores. Here, we used it as a tool to investigate the order of BTV core
assembly, the order of segmented RNA genome packaging and the possible
sequences that govern such order. Assembly was estimated with RT-PCR,
electron-microscopy and infectivity assay. We found that smaller segments
are more important for core assembly and the untranslated regions play
an important role. This technique opens a door for studying how different
RNA segments are selectively packaged in BTV and other members of the
family Reoviridae.
MA11 – Wed 1100
Offered paper Genome segment selection in the Reoviridae
MArk G Boyce, Malcolm McCrae
The Pirbright Institute, Pirbright, Surrey, UK
The process by which the correct complement of genomic RNA
segments are selected and encapsidated by members of the Reoviridae
remains is perhaps the most intriguing molecular mystery of this virus
family. Very early studies in orthoreoviruses provided experimental
evidence that the selection process operates on the single stranded viral
mRNAs produced by the virion associated RNA polymerase and this has
been assumed to also be the case for other viral genera although little or
no experimental data exists to confirm this assumption. In the absence
of a mechanism to account for the correct packaging of a full genome
complement, it seems reasonable to expect that cis-acting sequences
within the segments act to i) identify the viral RNA over cellular RNAs
and ii) strongly favor the packaging of complete sets of viral RNA rather
than partial sets. The development of efficient helper independent gene
rescue protocols for some genera of the Reoviridae has provided a
tractable experimental system with which to search for these genome
segment selection signals. Using the RNA transcript based rescue system
developed for orbiviruses, results obtained using a novel screening assay
aimed at identifying specific nucleotides involved in genome segment
selection/packaging will be described.
MA11 – Wed 1112
Offered paper Phospholipase D2, rab11 and influenza A virus budding
Amanda D Stuart1, Emily A Bruce1, Helen M Wise2, David Brown1,
Paul Digard2
1
Division of Virology, Department of Pathology, University of Cambridge,
Cambridge, UK, 2Roslin Institute, The University of Edinburgh, Edinburgh, UK
The cellular rab11 pathway is involved in influenza A virus (IAV)
assembly and budding. Phospholipase D2 (PLD2) and its product
phosphatidic acid (PA) are involved in regulation of rab11 trafficking.
We have shown that during IAV infection plasma membrane (PM) levels
of PA were elevated and that this elevation was due to the activity of
PLD2. Inhibition of PLD2 resulted in 1000 fold inhibition of IAV. At later
stages during infection we observed aberrant genome trafficking and
budding. At this stage in infection the virus genome normally colocalises
with rab11 in large aggregates under the PM. After PLD2 depletion
the genome failed to traffic to the PM and was found in a dispersed
cytoplasmic pattern or retained in the nucleus with a large perinuclear
accumulation colocalising with rab11. Thin section electron microscopy
revealed defective budding with virions of similar morphology to that we
reported after rab11 depletion. Class I rab11 FIPs are targeted to the
PM by interaction with PA. Depletion of individual FIPs had no effect on
infection however depletion multiple FIPs inhibited virus infection and
resulted in similar patterns of aberrant genome trafficking indicating the
PLD2 effects were in part due to defects in class I FIP function.
MA11 – Wed 1124
Offered paper Virus wrapping by endocytic tubules – a general model
of alphaherpesvirus envelopment
Ben Bleasdale1, Michael Hollinshead1, Meleri Jones2, Judith Breuer2,
Gillian Elliott1
44
Session abstracts
Imperial College London, London, UK, 2University College London, London,
UK
The process of alphaherpesvirus envelopment is highly complex. Recent
work from our group has proposed a novel mechanism for the final
envelopment of herpes simplex virus 1 (HSV1), whereby the virus utilises
abundant endocytic tubules derived from the plasma membrane, rather
than the previously defined trans-Golgi network (TGN) compartment, as
its source of envelope.
To determine if such a mechanism applies to other alphaherpesviruses,
we have now carried out detailed ultrastructural studies on the human
pathogen varicella zoster virus (VZV) and the animal pathogen bovine
herpes virus 1 (BHV-1). VZV-infected human keratinocytes and
BHV1-infected bovine epithelial cells were labelled with the fluid phase
endocytic marker horseradish peroxidase and examined by electron
microscopy. For both viruses, labelled endocytic tubules recently derived
from the plasma membrane were observed to enwrap capsids located
throughout the cytoplasm. Moreover, serial sectioning revealed that
such tubules often fused into laminar structures, topologically suited to
envelopment. Importantly, neither virus was seen to envelope at the
TGN.
Overall, our findings suggest that the newly proposed HSV-1
envelopment mechanism is paralleled in BHV-1 and VZV infections,
supporting a broader application of this mechanism across the
alphaherpesviruses, and revealing a new mechanism for virus exploitation
of the endocytic pathway.
1
MA11 – Wed 1136
Offered paper The lipidome of rotavirus (RV) infected cells
Eleanor R Gaunt, Qifeng Zhang, Winsome Cheung,
Michael Wakelam, Andrew M L Lever, Ulrich Desselberger
Lipid droplets (LDs) are subcellular storage depots for neutral fats
and triacylglycerols, and are dynamic and motile organelles with
compositional plasticity. RVs establish replication complexes in
viroplasms (‘viral factories’) with which LDs colocalise. Infected and
uninfected cells were subjected to equilibrium ultracentrifugation and
subcellular fractionation. Fractions containing viroplasms were identified
by densitometry, western blotting for virus-and LD-specific proteins,
and RNA visualisation. Subsequently, the lipidomes were characterised
by mass spectrometry. Fourteen different classes of lipids were
differentiated. The concentrations of virtually all lipids were elevated in
RV-infected cells, with the exception of triacylglycerols. Fractions of low
density (1.11-1.15 g/ml), in which peaks of the RV dsRNA genome and
viral and LD proteins were observed, also contained peak increases of
lipids. This confirmed the close interaction of LDs with viroplasms. An
increase in the ratio of the amounts of surface to internal components
of LDs upon RV infection suggested that the LD/viroplasm complexes
became enlarged. The data further support the validity of disturbing LD
metabolism as a therapeutic approach for RV disease.
MA11 – Wed 1148
Offered paper Crystal structures of the nucleocapsid protein from the
Bunyamwera and Schmallenberg viruses
Antonio Ariza, Thomas A Edwards, John N Barr
The family Bunyaviridae comprises virus species that infect humans,
animals, plants and insects, many of which cause devastating disease.
The family is separated into five genera of which Orthobunyavirus is
the largest genus, comprising over 50 named viruses within at least 16
serological groups. A recent addition to this genus is Schmallenberg
virus, which has caused widespread disease in cattle, sheep and goats
in many regions of Europe. All orthobunyaviruses possess genomes
that comprise three separate strands of negative, single-stranded RNA
that are encapsidated with a virus-encoded RNA-binding nucleocapsid
protein (N). We have solved the crystal structure of N from the
Schmallenberg virus (Smbv-N) and the prototypic Orthobunyavirus
member Bunyamwera virus (Bunv-N). The structures were solved
from selenomethionine-substituted Smbv-N expressed in E. coli and
purified including a non-enzymatic RNA removal step. The structure
of the selenomethionine-derivative of Smbv-N was solved via the
Single Anomalous Data (SAD) method and was then used to solve the
structures from higher-resolution data obtained from native Bunv-N and
Smbv-N by applying the Molecular Replacement (MR) method. The data
allowed us to perform a structural, functional and evolutionary analysis of
Orthobunyavirus nucleocapsids, which provided mechanistic insight into
the assembly and packaging of the Orthobunyavirus RNA genome.
Virology workshop:
Innate immunity
MA12
MA12 – Wed 0900
Offered paper Genome-wide RNA structure in RNA viruses and host
sensing
Jeroen Witteveldt1, Richard J Blundell1, Nora McFadden1,
David J Evans2, Ian G Goodfellow3, Peter Simmonds1
1
Infection and Immunity Division, Roslin Institute, The University of Edinburgh,
Edinburgh, UK, 2School of Life Sciences, The University of Warwick, Coventry,
UK, 3Calicivirus Research Group, University of Cambridge, Cambridge, UK
Bioinformatic analysis has shown the presence of a huge variation in the
predicted RNA structure throughout the genomes of positive-stranded
RNA viruses. Genome-scale ordered RNA structure (GORS) correlates
with the ability of viruses to persist in its natural host, suggesting a link
between RNA structure and innate immune system. We investigated the
host response to GORS (structured) and non-GORS (unstructured) viral
RNA in a non-replicating model and found a clear inverse correlation
between the predicted level of RNA secondary structure formation
and level of Interferon-β induction. The attenuation in host response by
GORS RNA was based on the evasion of host sensors, as non-GORS
RNA recognition is not inhibited by GORS RNA. Non-GORS RNA
showed a negative effect on cell viability and induced formation of
antiviral stress granules into which the viral RNA co-localises. Induction of
interferon was dependent on IRF3, but independent of endosomal TLR
sensors. By gene knockdown and (chemical) inhibitors we showed that
the cytosolic sensors RIG-I and PKR, but not MDA-5 played essential
roles in recognition of non-GORS RNA. Overall, the data suggest a
novel immune evasion method whereby large-scale RNA structure in
genomic RNA enables viruses to evade detection by the host innate
immune system.
MA12 – Wed 0912
Offered paper Defective genomes of parainfluenza virus 5 and their
role in interferon induction
MArian Killip1, Dan Young1, Derek Gatherer2, Craig Ross3,
John Short1, Andrew Davison2, Steve Goodbourn3, Richard Randall1
1
School of Biology, Centre for Biomolecular Sciences, University of St Andrews,
St Andrews, Fife, UK, 2MRC-University of Glasgow Centre for Virus Research,
Glasgow, UK, 3Division of Basic Medical Sciences, St George’s, University of
London, London, UK
Parainfluenza virus 5 (PIV5) preparations generated by high multiplicity
passage are potent activators of the interferon (IFN) induction
cascade due to the accumulation of defective interfering (DI) viruses.
Nucleocapsid RNA from these virus preparations was analysed by deep
sequencing to examine the range of DIs present, and to approximately
quantify the ratio of defective to non-defective genomes. Genome
sequence coverage of IFN-inducing PIV5 wt and PIV5-VΔC (which lacks
a functional V protein) was enriched in the trailer region, consistent
with an accumulation of trailer copyback (TrCB) genomes. We used
bioinformatics to identify the join points associated with the different
TrCB species present and to determine their relative frequencies. We
45
Session abstracts
also identified potential leader copyback and internal deletion genomes,
although these species did not contribute significantly to IFN induction.
DI-rich PIV5 wt preparations strongly activate the IFN induction cascade
despite encoding the V protein, an efficient inhibitor of the IFN response.
We demonstrate that non-defective PIV5 wt cannot prevent activation
of the IFN response by co-infecting TrCBs due to the interfering effects
of TrCBs on non-defective virus protein expression. Consequently,
TrCBs rapidly activate the IFN induction cascade prior to the expression
of detectable levels of V by co-infecting non-defective virus.
MA12 – Wed 0924
Offered paper Poxvirus virulence factor A49 targets E3 ligase β-TrCP
to subvert NF-κB activation
Carlos Maluquer de Motes1,2, Daniel S Mansur1, Leonie
Unterlholzner3, Rebecca P Sumner1,2, Brian J Ferguson1,2, Hongwei Ren1,2,
Pavla Strnadova1,2, Andrew G Bowie3, Geoffrey L Smith1,2
1
Imperial College London, London, UK, 2University of Cambridge, Cambridge,
UK, 3Trinity College Dublin, Dublin, UK
Transcription factor NF-κB is essential for immune responses against
pathogens. Its activation requires the phosphorylation, ubiquitination
and proteasomal degradation of IκBα. Many viruses including poxviruses
modulate NF-κB activity. Here we describe a novel inhibitor of NF-κB
from vaccinia virus (VACV) called A49 that blocks NF-κB activation by
molecular mimicry. A49 contains a motif conserved in IκBα which, in
IκBα, is phosphorylated by upstream kinases causing its ubiquitination
and degradation. Like IκBα, A49 binds the C-terminal domain of E3
ligase β-TrCP, but it is not ubiquitinated. By targeting β-TrCP, A49
prevents ubiquitination and degradation of IκBα, and in consequence
NF-κB is retained in the cytpoplasm. Serine-to-alanine mutagenesis
within the IκBα-like motif of A49 abolished β-TrCP binding, stabilisation
of phosphorylated IκBα and inhibition of NF-κB activation. Given the
central role of β-TrCP, A49 inhibited NF-κB activation deriving from
activation of Toll-like receptors, interleukin-1 receptor and tumour
necrosis factor receptor. Furthermore, a VACV lacking A49 showed
reduced virulence in vivo. Mechanistically, poxvirus A49 shares similarity
with the HIV protein Vpu, and given its conservation in variola virus, this
work reveals a common strategy for suppression of host innate immunity
by the viruses that cause smallpox and AIDS.
MA12 – Wed 0936
Offered paper Exogenous expression of PIAS4 inhibits host-cell
intrinsic antiviral immunity
Kyle Grant2, Peter Wasson1, Lily Tong1, Steven McFarlane1,
Chris Boutell1
1
University of Glasgow Centre for Virus Research, Glasgow, UK, 2North
Carolina State University, Raleigh, North Carolina, USA
Viral infection represents a dynamic interaction between host defence
mechanisms and viral countermeasures that modulate the intracellular
environment in favour of viral replication. Promyelocytic Leukemia
Nuclear Bodies (PML-NBs) are discrete nuclear sub-structures implicated
in the regulation of an intrinsic immune response that restricts many
nuclear replicating viruses. During herpesvirus infection, PML-NBs
localise to sites associated with viral genomes, generating a repressive
environment that impedes viral replication. PML-NB formation and
recruitment is dependent on the ability of its constituent proteins to be
modified by the Small Ubiquitin-like MOdifier (SUMO) proteins. We
report that ectopic expression of the SUMO ligase PIAS4 can deplete
the intracellular pool of endogenous SUMO-2/3, disrupting PMLNBs and inhibiting viral genome recruitment via a dominant-negative
constriction in PML and Sp100 SUMO-modification. This activity partially
relieves host-cell restriction observed during an ICP0-null mutant
HSV-1 infection and the establishment of latency of the HSV-1 mutant
in1374. Furthermore, HCMV immediate-early gene expression and
plaque formation efficiency is enhanced in cells ectopically expressing
PIAS4 or depleted of the E2-SUMO conjugating enzyme Ubc9. Our
data demonstrates the importance of the SUMO pathway in mediating
intrinsic immunity and highlights the need for caution when interpreting
ectopic expression studies in relation to SUMO ligases.
MA12 – Wed 0948
Offered paper Investigating tick cell innate defences against arbovirus
infection
Claudia Rückert1,3, Rennos Fragkoudis1, Gerald Barry2, Finn Grey3,
Alain Kohl4, Lesley Bell-Sakyi1, John K Fazakerley1
1
The Pirbright Institute, Pirbright, Surrey, UK, 2MRC -University of Glasgow,
Centre for Virus Research, Institute of Infection, Immunity and Inflammation,
Glasgow, Scotland, UK, 3The Roslin Institute and Royal (Dick) School of
Veterinary Studies,The University of Edinburgh, Midlothian, Scotland, UK, 4MRC
-University of Glasgow, Centre for Virus Research, Glasgow, Scotland, UK
Arthropod-borne viruses (arboviruses) have the ability to replicate in
both vertebrates and invertebrates. Ticks are important vectors of many
highly pathogenic arboviruses, including tick-borne encephalitis virus
and Crimean-Congo haemorrhagic fever virus. While the principally
mosquito-borne alphaviruses (Togaviridae) are not known to be
transmitted by ticks, Semliki Forest virus (SFV) does have the ability to
infect and replicate in tick cell lines derived from many different species.
The aim of our study is to characterise the innate antiviral defences of
tick cells. Genes involved in innate immunity pathways including Toll and
JAK/STAT were identified in the Ixodes scapularis genome by BLAST
search and expression of identified genes was detected in tick cells by
RT-PCR. Using transient knockdown of gene expression and subsequent
infection with SFV we begun to to characterise the role of the identified
genes in virus replication. For instance, we showed that knockdown of
STAT had no effect on SFV replication, whereas knockdown of cactus,
an orthologue of mammalian IκB, led to a two-fold increase in virus
replication suggesting SFV benefits from activation of the Toll pathway.
MA12 – Wed 1000
Offered paper Intracellular detection of antibody-bound viruses and
bacteria by TRIM21
W A McEwan, J C H Tam, R E Watkinson, S R Bidgood, F Hauler,
D L Mallery, L C James
MRC Laboratory of Molecular Biology, Hills Road, Cambridge, UK
Antibodies are a key component of the adaptive immune response
but their activity has been thought to be limited to the extracellular
environment. We have recently demonstrated that pathogens traffic
surface-bound antibodies to the intracellular compartment where
they are recognised by the high affinity cytosolic Fc receptor TRIM21.
We have shown that antibody-bound particles are rapidly degraded,
resulting in a highly efficient process of neutralisation. In this study we
demonstrate that following detection of pathogen-antibody complexes,
TRIM21 is able to synthesise K63-linked ubiquitin chains resulting in
signalling via NF-kB, AP-1 and IRF3/5/7 pathways. Detection by TRIM21
is sufficient to induce the production of cytokines including CXCL10,
IL-6 and IFN-Beta. Furthermore a wide range of intracellular pathogens,
including Salmonella, non-enveloped DNA viruses and RNA viruses can
be detected by TRIM21. TRIM21 therefore provides context-dependent
detection of intracellular antibody, mediating potent neutralisation and
signalling functions.
MA12 – Wed 1012
Offered paper Obesity and susceptibility to severe outcomes following
pandemic 2009 H1N1 infection may be linked by an aberrant innate
immune response in the obese host
MArk H Almond, Michael R Edwards, Sebastian L Johnston,
Wendy S Barclay
During the 2009 H1N1 influenza pandemic, obesity was convincingly
identified as a novel, independent risk factor for multiple markers of
46
Session abstracts
gene expression in latently infected monocytes and potentially amplify
viral reactivation and HCMV dissemination.
disease severity, including hospitalisation, intensive care unit admission
and death following infection. Given that 1.46 billion adults worldwide
are currently overweight and that pH1N1/09 resulted in approximately
59 million cases of severe illness in the US alone, disturbingly little is
known about the mechanisms underpinning susceptibility to severe
outcomes following respiratory viral infection in obesity. Studies in dietinduced obese mice have evaluated the effects of obesity on mortality,
lung injury and the cytokine milieu however, to date, no human studies
have been performed. We hypothesise that obesity attenuates type I
and III interferon responses secondary to leptin-induced upregulation of
suppressor of cytokine signaling (SOCS) molecules. To test this we are
evaluating the effects of leptin and other clinically relevant adipokines
on viral replication and cytokine production in human cells following
infection with a prototypic strain of pH1N1 2009.
MA12 – Wed 1100
Offered paper Mosquito innate immune responses in control of
chikungunya virus infection
Melanie McFarlane1, Estelle Martin2, Camilo Arias-Goeta2,
Claire L Donald1, Zoe Laing1, Stephanie M Graham1,
Laurence Mousson2, Esther Schnettler1, Alain Kohl1, Anna-Bella Failloux2
1
MRC-University of Glasgow Centre for Virus Research, Glasgow, UK, 2Institut
Pasteur, Paris, France
Arboviruses are viruses which are transmitted to susceptible vertebrate
hosts by the bite of an arthropod vector. Chikungunya virus (CHIKV) is a
re-emerging arbovirus that is endemic in tropical and sub-tropical regions.
The areas where CHIKV is present are becoming more widespread due
to the spread of the insect vector. The major vector for CHIKV is Aedes
aegypti mosquitoes. The competence of the mosquito vector to transmit
CHIKV may also be determined by the interaction of the vector immune
system and the virus. Arthropods rely on their innate immune system
to limit virus replication and thus virus transmission. There are a number
of innate immune pathways in the mosquito that could potentially
be involved in virus control. In this study we have utilised Ae. aegypti
-derived cell lines to investigate the response of RNA interference
(RNAi) as well as the IMD, TOLL and JAK/STAT signalling pathways to
CHIKV infection. In vivo studies have also been performed in Ae. aegypti
mosquitoes to investigate the control of CHIKV infection by the RNAi
pathway. The TOLL and RNAi pathways seem to be the most important
antiviral responses against chikungunya virus in the mosquito vector.
MA12 – Wed 1112
Offered paper Latent infection of monocytes by human
cytomegalovirus results in a cell secretome which profoundly impacts
on natural killer cell responses
C-C Kevin Chen, Emma Poole, Gavin Mason, John Sinclair, Mark Wills
Following primary infection, human cytomegalovirus (HCMV) is not
cleared by the host, at least in part, due to the establishment of latency
in the myeloid lineage cells. We have previously shown that HCMV
modifies the secretome of latently infected CD34+ cells; it inducing
CCL8 which causes chemotaxis of both monocytes and CD4+ T
cells, and also induces IL-10 and TGf-beta which inhibits T-cell effector
function. CD34+ progenitor cells are primarily bone marrow resident
but give rise to latently infected CD14+ monocytes which migrate into
the circulation and peripheral tissues. In contrast to CD34+ cells it is not
known what effect latent HCMV infection has on the CD14+ monocyte
secretome
We have, therefore, analysed latency-induced changes in the secretome
of CD14+ monocytes. Our results show that, unlike the CD34+ cells,
the CD14+ latent secretome does not induce CCL8 or monocytic cell
migration. In contrast, latently infected CD14+ monocytes secrete the
chemokines CXCL10 and IL-10 which causes chemotaxis of CXCR3
positive NK cells and NK activation, respectively. We also demonstrate
that these activated NK cells are able to induce viral immediate early
MA12 – Wed 1124
Offered paper An analysis of the NSs protein of the newly emerged
Schmallenberg virus
Gerald Barry1, Mariana Varela1, Maxime Ratinier1, Ilaria Piras1,3,
Marco Caporale1,2, Alain Kohl1, Massimo Palmarini1
1
MRC Centre for Virus Research, Institute of Infection, Immunity and
Inflammation, College of Medical, Veterinary and Life Sciences, University of
Glasgow, Glasgow, UK, 2Istituto G. Caporale, Teramo, Italy, 3Dipartimento di
Patologia e Clinica Veterinaria, Universita’ degli Studi di Sassari, Sassari, Italy
Schmallenberg virus (SBV) is an orthobunyavirus that emerged in Europe
in late 2011. It causes a mild disease in adult cattle and sheep but causes
congenital defects in offspring, often leading to abortion of the foetus.
SBV infection was first reported in the Netherlands and Germany but
it then spread very rapidly to several European countries including the
UK. The NSs protein of many bunyaviruses is known to antagonise the
cellular innate immune system by blocking transcription in the cell. Each
NSs protein studied appears to use a different mechanism to achieve
this. We have recently shown that SBV NSs blocks IFN production from
infected cells and SBV lacking NSs is attenuated in wild type mice but is
not attenuated in IFN-/-mice.
We have studied different functions of the SBV NSs protein. We show
that the NSs protein inhibits RNA polymerase II function, thus blocking
cellular transcription and translation. The SBV NSs protein also interacts
with and manipulates apoptotic pathways that may reduce the spread of
the virus. Collectively these data suggest that the SBV NSs protein has
similar functions to other bunyaviruses although important differences do
exist.
MA12 – Wed 1136
Offered paper VF1 required for murine norovirus persistence in vivo
Tina Christodoulou1, Dalan Bailey2, Ian Goodfellow1
1
University of Cambridge, Cambridge, UK, 2The Pirbright Institute, Surrey, UK
Human noroviruses cause 90% of non-bacterial gastroenteritis
and infection can be troublesome for the elderly and the
immunocompromised, suggesting an important role of the innate
immune system in controlling the infection. The recently identified
Virulence Factor 1 (VF1) protein encoded by the open reading frame 4
of MNV was shown to be an accessory protein that increases replication
efficiency in the host yet dispensible in vitro. Lack of VF1 expression
led to increased, rapid expression of antiviral genes that includes
interferon-β, and increased caspase 3/7 activation, suggesting that VF1
plays a role in delaying apoptosis and antagonising the innate immune
response. This study aims to further elucidate the mechanism of action
of VF1.
Here, a comprehensive animal study was undertaken where C57BL/6
mice were inoculated with the persistent strain MNV3. Through the use
of PCR mutagenesis and reverse genetics, MNV3-M1 was engineered to
lack VF1 expression and successfully recovered. Results show significantly
reduced viral shedding when compared to the wildtype virus, and VF1
expression is restored by day 5. In addition, the differences in viral load
between MNV3-M1 and the wildtype virus subside after reversion,
suggesting a selective pressure in vivo for VF1 expression.
MA12 – Wed 1148
Offered paper Polarised cell migration during cell to cell transmission
of herpes simplex virus in human skin keratinocytes
Fernando Abaitua, Rabiya Zia, Mike Hollinshead, Peter O’Hare
In addition to transmission involving extracellular free particles, virus
propagation, especially in the presence of extracellular neutralising
antibody, can occur via intercellular transmission at cell junctions or
47
Session abstracts
polarised contacts. This concept underpins analysis of virus spread,
plaque size and viral and host functions involved in transmission. Here
we demonstrate a novel process involved in cell-to-cell transmission of
herpes simplex virus (HSV) in human skin cells that has not previously
been appreciated. We show using time lapse microscopy that infection
induces the polarised migration of skin cells into the site of infection.
The uninfected skin cells then migrate over infected cells, becoming
infected in a spatially confined cluster containing hundreds of cells. The
cells within this cluster do not undergo cytocidal cell lysis but harbour
abundant enveloped particles within cells and within interstitial regions
below the cluster surface. Cells at the base and outside the cluster were
generally negative for virus immediate-early expression. We also show
that at least one component of this induced migration is the paracrine
stimulation of a cytotactic response from infected cells to uninfected
cells. The existence of this process radically changes our concept of HSV
transmission and the potential functions, virus and host factors involved.
Virology workshop:
genome
MA13
MA13 – Wed 1348
Offered paper A 5´ stem–loop within the non-primate hepacivirus
5ÚTR inhibits IRES activity
Hazel Stewart1, Dale Jones1, Michaela Conley1, Sinead Lyons2,
Peter Simmonds2, Mark Harris1
1
School of Molecular & Cellular Biology, Faculty of Biological Sciences,
University of Leeds, Leeds, UK, 2The Roslin Institute, The University of
The non-primate hepacivirus is a homologue of HCV which has been
recently isolated from domestic horses and dogs. NPHV has not been
observed to cause disease in its natural hosts. Despite the high sequence
identity between NPHV and HCV, there are significant differences in
the untranslated regions. This virus therefore provides a unique model
for identifying the contribution of these regions to the pathogenicity of
HCV. The NPHV 5’ UTR is predicted to have a similar overall structure
to HCV and is therefore assumed to possess IRES activity, although this
has not yet been experimentally confirmed. Intriguingly, the NPHV 5’
UTR also contains an additional predicted 5’ stem-loop which is not
conserved within the other hepaciviridae. To assess the IRES activity
of the NPHV 5’UTR and the function of the additional stem-loop, we
utilised bicistronic vectors in which renilla luciferase was translated in
a cap-dependent fashion and the 5’UTR mediated cap-independent
translation of firefly luciferase. Our results confirm that the NPHV
5’ UTR possessed IRES activity to similar levels as the HCV 5’UTR.
Deletion of the additional stem-loop enhanced IRES activity, indicating
this structure inhibits hepacivirus IRES function, and may contribute to
the reduced pathogenicity of this newly identified virus.
MA13 – Wed 1400
Offered paper Characterising the function of murine norovirus
proteins using epitope-tagged viruses
Lucy Thorne, Jia Lu, Luis Urena, Surender Vashist, Edward Emmott,
Ian Goodfellow
Human noroviruses are a major cause of viral gastroenteritis worldwide,
yet understanding of norovirus replication lags behind that of other
RNA viruses due to the inability to culture human norovirus. Murine
norovirus (MNV), which can be propagated in permissive cells, now
provides a system to study the functions of the norovirus proteins. Here,
the FLAG epitope tag was inserted into the unknown MNV proteins
NS1-2, NS4 and VP2, at previously identified tolerated positions. This
allowed recovery of the first infectious epitope-tagged noroviruses, with
wild-type growth characteristics. Using the FLAG tag, all three proteins
were found to localise to the replication complex in infected cells. Viral
and cellular interaction partners of NS1-2 and NS4 were then identified
by immunoprecipitation coupled with quantitative proteomics based
on stable isotope labelling by amino acids in cell culture (SILAC). As
validation, the interaction between NS1-2 and the cellular protein DDX3
was confirmed by immunoblotting. We recently showed that DDX3 is
required for efficient MNV replication, perhaps mediated by interactions
with both NS1-2 and the viral RNA, as RNA inhibition-mediated
knockdown of DDX3 inhibited MNV replication. Characterisation of
the other interaction partners of NS1-2 and NS4 should reveal their
functions and new intracellular targets of MNV.
MA13 – Wed 1412
Offered paper Control of a bacterial toxin by an antitoxic RNA
pseudoknot involves selective, RNA-driven self-assembly of an inactive
protein–RNA complex
Francesca L Short1, Xue Y Pei1, Tim R Blower1, Shue-Li Ong1,
Peter C Fineran2, Ben F Luisi1, George P C Salmond1
1
University of Cambridge, Cambridge, UK, 2University of Otago, Dunedin,
New Zealand
Bacteria have evolved many mechanisms to protect themselves
from bacteriophage, including the ToxIN system of Pectobacterium
atrosepticum. ToxINPa is a Type III toxin-antitoxin system that comprises
a toxic ribonuclease (ToxNPa) and an antitoxic RNA (ToxIPa), which
together form an inactive complex. Phage infection triggers the release
of ToxNPa, causing cell death before the phage can replicate and thereby
protecting the clonal population. The success of this antiviral strategy
therefore depends on the strong inhibition of ToxNPa by the RNA,
ToxIPa, under normal conditions. In this study we aimed to define how
ToxI recognises and inhibits its toxin partner.
We show, through cross-inhibition experiments with a second ToxIN
system (ToxINBt from Bacillus thuringiensis), that ToxI RNAs are highly
selective inhibitors. Processed and unprocessed forms of ToxIPa can
inhibit ToxNPa in vitro, without any cellular factors or exogenous energy.
We show that inhibition is linked to the self-assembly of a trimeric
ToxINPa complex previously observed by crystallography. Finally, we
explain the basis for ToxI antitoxin selectivity through the crystal
structure of the ToxINBt complex. Our results show how a small,
processed RNA acts as a highly selective and robust inhibitor of a
potentially lethal protein.
Reference Short et al (2012). PNAS DOI:10.1073
MA13 – Wed 1424
Offered paper Investigating the interactions between human
papillomavirus oncoproteins and their cellular targets using RNA
aptamers
Özlem Cesur, Clare Nicol, G Eric Blair, Nicola J Stonehouse
School of Molecular and Cellular Biology, Leeds, University of Leeds, UK
High-risk human papillomaviruses are responsible for approximately
90% of anogenital and oropharyngeal cancers, due to the actions of the
viral oncoproteins E6 and E7. The most well-characterised interaction
of HPV16 E7 is with the cell cycle control protein pRb, promoting pRb
degradation and resulting in cell cycle misregulation. We have selected
and characterised RNA aptamers to E7 in order to study this further.
Aptamers are single-stranded oligonucleotides that fold into complex
structures and bind target molecules in a conformation-dependent
manner. The E7 aptamers have been stabilised by the inclusion of
modified pyrimidines. Several aptamers were able to induce apoptosis
in an HPV16-transformed cervical carcinoma cell line (SiHa) that actively
expresses both E6 and E7. Of particular interest is the E7 aptamer A2.
Treatment of cells with A2 resulted in a loss of the E7 oncoprotein
and a rise in cellular pRb levels. A control aptamer selected to an
unrelated protein had no effect. Our studies are continuing to explore
the therapeutic potential of A2, to further characterise both E6 and E7
aptamers and to deliver these molecules to primary cells.
48
Session abstracts
MA13 – Wed 1436
Offered paper Effects of modifying CpG and UpA dinucleotide
frequencies on replication fitness of RNA viruses
Nicky J Atkinson1, Inga Dry1, Helen Wise1, Paul Digard1,
David J Evans2
1
The University of Edinburgh, Edinburgh, UK, 2The University of Warwick,
Coventry, UK
Suppression of CpG and UpA dinucleotide frequencies is widely
observed in mammalian and plant RNA virus genomes but the mutational
or selection pressures driving this compositional change are unknown. To
investigate this, echovirus 7 (EV7), Theiler’s virus (TMEV) and influenza
A virus (IAV) mutants were produced in which frequencies of CpG and
UpA dinucleotides were increased in two 1kb regions or whole segments
(IAV). Replicative fitness of CpG-high EV7 mutants was severely reduced
with viral yields approximately 10,000-fold lower than wild type. Elevation
of UpA attenuated virus replication but to a lower extent (10-fold).
Similar results were observed in TMEV and IAV, with all three viruses
showing a small plaque phenotype and increased RNA to infectivity ratios.
Although no IFN-β or upregulated ISG expression could be detected in
response to EV7 or TMEV, CpG-induced attenuation was at least partially
mediated through PKR, and preliminary evidence suggests an association
with induction of antiviral stress granules. This is consistent with a role for
cytoplasmic RNA sensors in orchestrating the response to foreign RNA
(with potentially different mechanisms for CpG and UpA). These findings
demonstrate a previously unsuspected role of dinucleotide composition in
recognition of RNA viruses by the innate immune system.
MA13 – Wed 1448
Offered paper Influence of genome-scale RNA structure disruption on
the replication of murine norovirus – identical replication kinetics in
cell culture but attenuation of viral fitness in vivo
Nora McFadden1,2, Armando Arias2, Inga Dry1, Dalan Bailey2, Jeroen
Witteveldt1, David J Evans3, Ian Goodfellow2, Peter Simmonds1
1
The University of Edinburgh, Edinburgh, UK, 2University of Cambridge,
Cambridge, UK, 3The University of Warwick, Coventry, UK
Mechanisms by which certain RNA viruses, such as HCV, establish
persistent infections and cause chronic disease are of fundamental
importance in viral pathogenesis. Mammalian positive-stranded RNA
viruses establishing persistence possess genome-scale ordered RNA
secondary structure (GORS) in their genomes. Murine norovirus (MNV)
persists in immunocompetent mice and provides an experimental model
to functionally characterise GORS. Deletion mutants were constructed
with coding sequences in NS3/4-and NS6/7-coding regions replaced
with sequences with identical coding and (di )nucleotide composition
but disrupted RNA secondary structure (F1, F2, F1/F2 mutants).
Mutants replicated with similar kinetics to wild type (WT) MNV3 in
RAW264.7 cells and primary macrophages, exhibited similar (highly
restricted) induction and susceptibility to interferon-coupled cellular
responses and equal replication fitness by serial passaging of co-cultures.
In vivo, both WT and F1/F2 mutant viruses persistently infected mice
although F1, F2 and F1/F2 mutant viruses were rapidly eliminated 1-7
days post-inoculation in competition experiments with WT. F1/F2
mutants recovered from tissues at 8 months showed higher synonymous
substitution rates than WT and nucleotide substitutions that led to
partial restoration of RNA secondary structure. GORS plays no role in
basic replication of MNV but has a determining role in viral fitness and
persistence in vivo.
MA13 – Wed 1515
Offered paper Components of the human interactome of influenza A
virus ribonucleoproteins revealed by RNA tagging and proteomics
Ashley York, Ervin Fodor
University of Oxford, Oxford, UK
Influenza A viruses encode for a limited number of proteins and are
therefore highly reliant on numerous host cellular functions to support
their replication cycles. Here we report an RNA tagging and proteomic
approach being employed to identify cellular factors that interact with
viral ribonucleoproteins during the course of the virus life-cycle. During
infection, genomic single-stranded viral RNA (vRNA) is replicated via a
complementary (cRNA) intermediate, both of which are encapsidated
in viral nucleoprotein and bound by the heterotrimeric RNAdependent RNA polymerase to form viral ribonucleoprotein (vRNP)
and complementary viral ribonucleoprotein (cRNP) complexes. The
heterotrimeric polymerase is also responsible for viral mRNA synthesis
via a cap-snatching mechanism. Recombinant influenza A/WSN/33
viruses have been generated by reverse genetics to contain an RNA tag
which is bound by the Pseudomonas aeruginosa phage 7 coat protein,
the interaction of which is exploited for the RNA affinity-purification of
influenza A ribonucleoproteins from infected cells with high specificity.
Identification of cellular factors co-purifying with viral RNPs by liquid
chromatography-tandem mass spectrometry has revealed a number
of candidate proteins that interact with viral ribonucleoproteins, which
may help us to understand the role of the host cellular machinery during
influenza A virus infection.
MA13 – Wed 1527
Offered paper Global analysis of human cytomegalovirus microrna
targets identify novel host–virus interactions
Jon A Pavelin1, Natalie Reynolds1, Rebecca Tirabassi1,2, Jay Nelson1,2,
Finn Grey1
1
Roslin Institute, Edinburgh, UK, 2Vaccine and Gene Therapy Institute, Oregon
Health & Science University, Portland, Oregon, USA
Human cytomegalovirus (HCMV) encodes 22 microRNAs, the functions
of which are largely unknown. Using a biochemical technique (RNA
induced silencing complex immunoprecipitation) we have identified
hundreds of putative host transcript targets of all HCMV microRNAs
expressed during infection. Using microRNA knockout viruses and
microRNA mimics we have resolved specific interactions between
HCMV microRNAs and the top candidate target transcripts from
our initial screen, and validated their down-regulation by western blot
analysis. The majority of the targets represent novel virus cell interactions
and include genes involved in: vacuolar acidification and endosome
maturation (ATP6V0C); essential amino acid degradation (BCKDHA);
cell cycle progression (CCNE2); and viral epitope binding (LGALS3).
Furthermore, the regulation of these targets plays important roles in
HCMV biology, with siRNA knockdown resulting in a marked effect on
virus replication. The down-regulation of these targets either inhibits
replication, as is the case for ATP6V0C, or promotes replication as
is the case for BCKDHA, and LGALS3. These studies demonstrate
the effectiveness of identifying novel host-virus interactions through
microRNA target identification.
MA13 – Wed 1539
Offered paper Novel approaches to investigate cellular targets of
Epstein–Barr-encoded RNAs
Elizabeth Boulden, Rachel Bosshard, Goran Gregorovic,
Paul J Farrell
EBV produces two noncoding RNAs, EBERs (Epstein–Barr encoded
RNAs) 1 and 2, which are abundantly expressed in latent infection and
in several EBV-associated cancers. The EBERs have been reported to
contribute to oncogenesis in EBV-positive Burkitt’s lymphoma and to
tumour development in SCID mice. We have confirmed the known
binding of EBER1 to L22 and found the distribution of L22 to differ
between lymphoblastoid cell lines (LCLs) and tumour cell lines. No
binding partners have yet been identified for EBER2. Using recombinant
EBV strains with deletion of either EBER1 or EBER2, we identified gene
expression changes in LCLs correlated with absence of EBER1 or EBER2.
To be able to investigate EBER function directly, we have now adapted a
second generation lentiviral vector system for delivery of either EBER1 or
49
Session abstracts
EBER2 co-expressed with GFP. We are now using this system and the
recombinant EBV strains to explore EBER functions.
MA13 – Wed 1551
Offered paper Analysis of viral miRNA expression and function during
human cytomegalovirus latency in the myeloid lineage
Betty Lau1, Emma Poole1, Georgina Okecha1, Eain Murphy2,
Mark Wills1, John Sinclair1
1
University of Cambridge, Cambridge, UK, 2Lerner Research Institute,
Cleveland, USA
Human cytomegalovirus (HCMV) encodes 24 miRNAs which are
expressed during productive infection and it is emerging that these
miRNAs play an important role modulating viral and cellular functions to
optimise infection.
However, whether viral miRNAs have any role in latency is unknown.
Consequently, we have profiled HCMV miRNA expression in
experimentally latent CD34+ cells and monocytes, and routinely observe
expression of viral UL112-1. miR-UL112-1 has previously been shown
to down-regulate the immediate early transcript IE72. As IE72 is essential
for initiating lytic infection, it is possible that the inhibition of IE72 by
miR-UL112-1 aids latent carriage. Consequently, we have analysed the
effect on latency of deleting the miR-UL112-1 target site within the IE72
RNA. We find that latent infection with the deletion mutant results in an
aberrantly higher level of IE72 transcripts.
Surprisingly, this uncontrolled expression of IE72 appears to have
little effect on the maintenance of, or reactivation from, latency in
myeloid cells. However, as opposed to normally latent monoctytes, our
preliminary results show that latent monocytes expressing increased IE72
become more efficient targets for IE72-specific CD8+T cells.
Thus, we hypotheses that miR-UL112-1 inhibition of IE72 during latency
aids efficient evasion of T cell recognition of latently infected cells.
MA13 – Wed 1603
Offered paper A novel method to examine individual RNA structures
within a mixed population reveals insights into the HIV-1 RNA
dimerisation process
Julia C Kenyon, Liam J Prestwood, Andrew M L Lever
University of Cambridge Department of Medicine, Cambridge, UK
The 5’ end of the HIV-1 RNA genome contains sequences and
structures that regulate many steps in the viral lifecycle. Retroviruses
package two copies of each genome, hence dimerisation and packaging
are linked, and these processes appear to be controlled by structural
switches within the RNA. To study these switches, we have developed
a new technique known as ‘in-gel SHAPE (selective 2’OH acylation
analysed by primer extension)’. Individual RNA structures within a
mixed structural population are isolated by electrophoresis under native
conditions, and secondary structural probing takes place within the
gel. This makes it possible to examine the structures of different RNA
conformers under native conditions, without the need to stabilise each
structure by mutagenesis or by using non-physiological buffers. We have
validated the technique using a well-characterised RNA structure, and
show it to be accurate and highly reproducible. We have then used it
to show that the HIV-1 RNA monomer contains a pseudoknot, where
the dimerisation initiation site interacts with the U5 region, and that the
native dimer structure corresponds closely with previously proposed
structures of the monomeric RNA. These two structures highlight the
areas involved in the RNA structural switch and those that remain static.
MA13 – Wed 1615
Offered paper Viral RNAs have evolved for efficient, two-stage
packaging
Alexander Borodavka, Roman Tuma, Peter Stockley
University of Leeds, Astbury Centre for Structural Molecular Biology, Leeds, UK
Genome packaging is essential step in virus replication and potential drug
target. RNA viruses have been thought to encapsidate their genomes by
gradual co-assembly with capsid subunits. We have developed a single
molecule fluorescence assay to monitor RNA conformation and virus
assembly in real time, and applied it to a model virus, the bacteriophage
MS2. The results demonstrate that packaging is a two step process. In
the first step RNA undergoes a rapid and dramatic (~20-30%) collapse
upon addition of coat protein. The collapse is followed by a gradual
increase in size, consistent with the recruitment of additional coat protein
subunits to a growing capsid. The collapsed state is slightly smaller than
the final capsid and is similar for viral RNA fragments with different
lengths. This is consistent with formation of roughly spherical structure in
the RNA that serves as a scaffold for correct placement of the incoming
coat protein subunits. The collapse is specific to viral RNA fragments
while the equivalently sized non-viral RNAs do not show this behaviour
and yield many aberrant structures. Thus, the initial collapse is a distinct
feature of the viral RNA that evolved to facilitate assembly.
Virology workshop:
Pathogenesis
MA14
MA14 – Wed 1348
Offered paper The impact of GBV-B sequence variation on infection
outcome
Ori Bowen1, Robert Goldin2, Peter Karayiannis3, Nicola J Rose1
1
Division of Virology, NIBSC, Blanche Lane, Potters Bar, Hertfordshire, EN6
3QG, UK, 2Department of Pathology, Division of Medicine, Imperial College
London, W2 1PG, UK, 3Department of Medicine, Hepatology Section,
Division of Medicine, Imperial College London, W2 1PG, UK
GBV-B is a virus in the flaviviridae family distinct from but phylogenetically
closely related to Hepatitis C virus (HCV). GBV-B has a similar genome
length and organisation to HCV, with considerable functional homology
in defined regions and approximately 30% sequence homology over
the open reading frame. There is no licensed HCV vaccine and standard
treatment has limited efficacy. Using the surrogate GBV-B/tamarin model
of HCV to study acute infection we find that some animals display
a typical acute viraemia and others have prolonged or intermittent
infections. Understanding the impact of viral variation on the viral
dynamics -particularly clearance -could inform antiviral design. Wholegenome deep sequencing of our inoculum virus and virus recovered
from an animal with a prolonged infection revealed conservation in the
structural proteins and residue changes primarily in the C-terminus of the
NS5A protein. Since the analogous region of HCV is diverse in function
and a determinant of sensitivity to interferon therapy, these data may
indicate a region of GBV-B associated with immune selection. Continuing
investigations are aimed at analysis of samples taken throughout infection
to better understand the role of sequence variation on success of
clearance of virus which may inform immunotherapeutic development.
MA14 – Wed 1400
Offered paper First ORF of G gene in non-pathogenic strain of
pneumonia virus of mice (PVM) affects the pathogenicity of the virus
Yashar M Sadigh1, Oliver Dibben2, Andrew Dibben3
1
Pirbright Institute, Compton, Berkshire, UK, 2Department of Microbiology,
Mount Sinai School of Medicine, New York, NY, USA, 3School of Life
Sciences,The University of Warwick, Coventry, UK
We used a reverse genetics approach to study the effect on
pathogenicity of the first ORF of G gene of pneumonia virus of mice
(PVM) . We constructed recombinant viruses derived from the genome
of the non-pathogenic strain 15(Warwick). The viruses contained a G
gene derived from either pathogenic strain J3666 which contains a short
upstream ORF and an extended main ORF or the non-pathogenic
strain which contains a longer upstream ORF and a truncated main
ORF. These were used to infect mice. The viruses carrying the strain
50
J3666 G gene generated clear clinical signs, whereas the recombinant
viruses carrying the G gene from strain 15(Warwick) did not. Deletion
of the first ORF in the strain 15(Warwick) G gene resulted in an
increased pathogenicity. We used a minigenome reporter system to
study expression levels in the mutant G genes. The data showed that
in the strain 15(Warwick) G gene removal of the first ORF resulted
in a significant increase in the level of expression from the G protein
ORF. Taken together the data indicate that alteration of the level of G
protein expression by mutation of the upstream ORF directly affects the
pathogenicity of the virus in vivo.
MA14 – Wed 1412
Offered paper Persistent replication of murine norovirus in vivo selects
for an adaptive change in VP2
Armando Arias1,2, Dalan Bailey2,3, Constantina Christodoulou1,2,
Yasmin Chaudhry1,2, Ian Goodfellow1,2
1
University of Cambridge, Cambridge, UK, 2Imperial College London, London,
UK, 3Pirbright Laboratory (IAH), Woking (Surrey), UK
We have recently developed a mouse model system for the
establishment of persistent infections with murine norovirus-3 (MNV-3),
previous recovery by reverse genetics (Arias et al 2012, JGV: 93, 1432).
The sequence of full viral genomes isolated from 6 different animals has
identified several mutations that were repeated, being one change in the
minor capsid protein VP2 (T4A) found in all the animals analysed. We
have now expanded the analysis to samples obtained from 11 additional
different animals and we have found the partial or total selection of
T4A in most of them (16 out of 17 animals), suggesting that this change
has an important role in the maintenance of virus persistence in vivo.
Interestingly, when recovered by reverse genetics, higher virus titres
have been obtained for mutant T4A than for wild-type virus (WT).
The kinetics of both viruses after infection at high MOI is very similar
but again mutant T4A has shown higher viral titres at later times post
infection. Both viruses show similar thermal inactivation at 37C and 42C,
suggesting that the differences observed are not due to a functional
alteration of the viral particle. The relevance of VP2 for persistent
replication in vivo will be discussed.
MA14 – Wed 1424
Offered paper Development of a mouse model for human
norovirus
Stefan Taube2, Abimbola O. Kolawole1, Marina Hoehne4,
Ramesh Ramesh3, Christiane E Wobus1
1
University of Michigan, Ann Arbor, MI, USA, 2University of Luebeck, Luebeck,
Germany, 3Colorado State University, Fort Collins, USA, 4Robert Koch Institute,
Berlin, Germany
Human noroviruses (HuNoV) are the leading cause of viral
gastroenteritis worldwide. However, no antiviral or vaccines are available
to prevent or treat norovirus infections. Progress towards anti-noroviral
therapies has been hampered by the absence of a small animal model.
Immunodeficient mice engrafted with human CD34+ hematopoietic
stem cells to reconstitute a functional human immune system are a
powerful tool to investigate human-tropic diseases such as HIV. To
determine whether these mice were susceptible to HuNoV infection,
humanised mice, non-engrafted siblings, and immunocompetent wild
type controls were challenged with pooled stool samples from patients
positive for HuNoV. Surprisingly, both non-humanised and humanised
mice became infected as evidence by viral loads greater than that of
input. Viral genome was present in intestinal and extra-intestinal sites.
However, none of the wild-type controls became infected. These data
demonstrate that the immune status of the murine host, but not the
presence of human immune cells, play a critical role in the susceptibility
of mice to HuNoV. The development of the first mouse model for
HuNoV overcomes a major barrier in the field and will provide a critical
tool for studying HuNoV biology and accelerate the development of
urgently needed HuNoV therapeutics.
Session abstracts
MA14 – Wed 1436
Offered paper The bluetongue virus (BTV) fourth non-structural
protein (NS4) favours viral replication in primary endothelial cells and
in sheep
MAxime Ratinier1, Gerald Barry1, Luigina DiGialleonardo2,
Claudio Murgia1, Marco Caporale1,2, Massimo Palmarini1
1
MRC-University of Glasgow Centre for Virus Research, Glasgow, UK, 2Istituto
G. Caporale, Teramo, Italy
Bluetongue virus (BTV) is the causative agent of a major disease of livestock
(bluetongue). Recently, we have shown that BTV expresses a non-structural
protein (NS4) encoded by an open reading frame in segment 9 overlapping
those expressing VP6. Using reverse genetics, we successfully rescued a
BTV NS4 deletion mutant (BTV8 ΔNS4). We established that NS4 does
not affect BTV virulence in murine models of bluetongue infection, but
confers a replication advantage to BTV8 in cells pre-treated by interferon.
In experimentally inoculated sheep, BTV8 rescued by reverse genetics
(BTV8 wt) induced sustained viremia, fever and depression. In contrast,
sheep infected with BTV8 ΔNS4 did not show fever, whereas viremia was
mostly transitory and at a reduced level compared to that observed with
BTV8 wt. We also show that NS4 favours BTV8 replication in primary
sheep and bovine endothelial cells. We are currently investigating the major
roles of NS4 in viral replication. Collectively, this study suggests that NS4
plays a key role in virus-host interactions. Furthermore, the distinct nucleolar
localisation of NS4, expressed by a virus replicating in the cytoplasm, offers
new avenues to investigate the multiple roles played by the nucleolus in the
biology of the cell.
MA14 – Wed 1448
Offered paper The role of the liver stroma in hepatitis C virus
infection
Sukhdeep K Galsinh1, Ian A Rowe1,2, Luke Meredith1,
Gillian Muirhead2, Elizabeth Humphreys2, David Adams2, Chris Buckley3,
Peter Balfe1, Jane McKeating1
1
Hepatitis C Research Group, The University of Birmingham, Birmingham, UK,
2
Centre for Liver Research, NIHR Biomedical Research Unit, The University
of Birmingham, Birmingham, UK, 3Rheumatology Research Group, The
University of Birmingham, Birmingham, UK
Hepatitis C virus (HCV) infection is a major cause of global morbidity
and mortality. It is estimated that 170 million individuals are infected
worldwide and a significant proportion will develop cirrhosis and
hepatocellular carcinoma. Hepatocytes are the major site of viral
replication, however, the liver contains multiple non-parenchymal cell
types and our current understanding of the role these liver cells play
in HCV infection is limited. Stromal cells create the microenvironment
of the liver and given their close proximity to hepatocytes in vivo, we
established co-culture systems to study their role in HCV infection.
Liver myofibroblasts do not support HCV entry or genome replication,
however, they negatively regulate infection of adjacent hepatocytes in
a cell-contact dependent manner. Studying diverse steps in the viral
lifecycle demonstrates that fibroblasts limit particle entry. Importantly,
this observation is not restricted to HCV and lentiviral pseudotypes
expressing vesicular stomatitis and murine leukemia virus glycoproteins
show limited infection of fibroblast-hepatocyte co-cultures, suggesting a
global perturbation of membrane protein dynamics. These observations
highlight a novel mechanism by which stromal cells limit viral infection
and raise questions about commonly used in vitro methods to study
virus replication that generally use single cell types.
MA14 – Wed 1515
Offered paper An ex-vivo dog tracheal organ culture system for the
study of canine influenza emergence
Gaelle Gonzalez1, John F Marshall2, Joseph Hughes1,
John McCauley3, David Robb4, Pablo Murcia1
1
MRC-University of Glasgow Centre for Virus Research , Institute of Infection,
Immunity and Inflammation, College of Medical, Veterinary and Life Sciences,
51
Session abstracts
Glasgow, UK, 2Weipers Centre Equine Hospital, School of Veterinary
Medicine, University of Glasgow, Glasgow, UK, 3The National Institute for
Medical Research, The Ridgeway, Mill Hill, London, UK, 4Charles River
Laboratories, Preclinical Services, Tranent, Edinburgh, UK
Influenza viruses represent a significant risk to human and animal health.
Determining the mechanisms underpinning cross-species transmission
is critical in understanding viral emergence. Equine influenza virus (EIV)
jumped the species barrier in the early 2000’s and emerged as a novel
respiratory virus of the dog, canine influenza virus (CIV). The EIV-CIV
system provides a unique opportunity to study the mechanisms that
govern influenza emergence. We aimed to empirically determine the
evolutionary dynamics of EIV adaptation to the dog respiratory tract.
To this end we developed an air interface ex-vivo organ culture (EVOC)
system of dog tracheas. Dog tracheal explants were maintained in the
EVOC system for seven days and infected with CIV and a variety of EIVs
that circulated in horses in previous years. We defined a phenotype of
infection in explants that comprised histological lesions, detection of viral
antigen by immunohistochemistry and quantification of virus. Explants
exhibited susceptibility to CIV and EIV infection, displaying histological
changes consistent with those observed in vivo. Different EIVs exhibited
distinct infection phenotypes, with ‘young’ EIVs infecting dog tracheas
more efficiently than ‘old’ EIVs. Our results suggest that CIV emergence
was the result of adaptive mutations acquired along the evolutionary
history of EIV.
MA14 – Wed 1527
Offered paper Use of exome sequencing and RNAi to understand the
influence of host factors involved in influenza infections
Rachael S Wash1, Deepti Gurdasani1, Stephanie Franz1,
Carmen Diaz Soria1, Manj Sandhu1,2, Paul Kellam1,3
1
Wellcome Trust Sanger Institute, Cambridge, UK, 2University of Cambridge,
Cambridge, UK, 3University College London, London, UK
Whole virus genome sequencing during the Influenza A H1N1/09
pandemic showed the extent of virus variation. Preliminary data suggests
that whilst genome variation occurs, and can subtly alter replication
properties of the virus, it is not sufficient to account for severe or fatal
influenza infection. Alterations in human genes, such as IFITM3, that
interact with the virus are however associated with severe influenza
pathogenesis. To understand the influence of host genetics on virus
infection we have analysed in detail the whole human exome sequence
of patients who were severely infected with influenza during the
pandemic. Through this approach we have identified potential SNP
variants that may affect susceptibility to influenza infection. We have used
RNAi screening in A549 cells to knockdown genes where SNP variants
have been identified, followed by infection with influenza to validate this
data in vitro.To gain more insight into the effect of these variants we have
also compared influenza infection of human lymphoblastoid cell lines,
which have either the ‘reference/wild type’ genotype or the SNP variant
identified in our exome analysis associated with severe influenza. The
results of this powerful strategy to identify host factors that affect the
outcome of influenza infections will be discussed.
MA14 – Wed 1539
Offered paper HPV1 E1Ê4 protein regulates serine/arginine-specific
protein kinase 1 phosphorylation of SR proteins including the viral SR
protein E2
Claire L Brimacombe, Emma L Prescott, Margaret Hartley,
Sally Roberts
High expression levels of human papillomavirus (HPV) E1Ê4 protein
in the later stages of the HPV life cycle is associated with the productive
phase of infection. A specific role for E1Ê4 during these later stages
has yet to be determined. We have previously shown that HPV1 E1Ê4
interacts with SRPK1, a member of the serine-arginine kinase family,
and found the kinase sequestered to E1Ê4-containing inclusions in
the upper layers of HPV1-infected warts (Bell et al., 2007). Here we
demonstrate that E1Ê4 inhibits SRPK1 phosphorylation of serinearginine-rich (SR) proteins involved in controlling mRNA maturation
and translation, namely SRSF1, SRSF4, SRSF3 and SRSF7. Interestingly,
SRPK1 is known to phosphorylate E2 proteins and interactions between
E2 and SR proteins have been linked to E2 function. We demonstrate
that serine residues in serine-arginine/arginine-serine dipeptide motifs
in the hinge region of HPV1 E2 are phosphorylated by SRPK1 in vitro.
Furthermore, E2 phosphorylation is inhibited in the presence of HPV1
E1Ê4 protein. Mutation of the phosphor-acceptor serine residues in
HPV1 E2 affects the subcellular localisation of E2 in keratinocytes. Based
on our novel findings, we hypothesise that in the productive phase of
the infectious life cycle E4 modulates E2 function by inhibiting the cellular
kinase SRPK1.
MA14 – Wed 1551
Offered paper The human papillomavirus 18 E5 protein is necessary
for deregulated keratinocyte differentiation and unscheduled host
DNA synthesis during the viral life cycle
Christopher W Wasson1, Rebecca Ross1, Marietta Muller1,
Emma Prescott1, George E Blair1, Nicola Stonehouse1, Sally Roberts2,
Andrew Macdonald1
1
School of Molecular and Cellular Biology, University of Leeds, Leeds, UK,
2
School of Cancer Sciences, The University of Birmingham, Birmingham, UK
HPV E5 is a small hydrophobic oncoprotein that is able to transform cells
by hyperactivating EGFR signalling. The role of E5 in the virus lifecycle
is poorly understood and E5 knockout viruses from HPV 16 and 31
display different phenotypes in organotypic raft cultures. Whilst HPV 16
E5 is important in maintaining DNA synthesis in the upper layers of rafts,
HPV 31 E5 is important for viral DNA amplification and late viral gene
expression.
To reconcile these differences we chose to study the third major highrisk HPV type, HPV18. We developed HFKs harbouring wildtype and E5knockout genomes. Keratinocytes lacking E5 contained similar numbers
of episomes and displayed no change in proliferative ability compared to
wildtype in undifferentiated keratinocytes. In contrast upon differentiation
E5 knockout cells showed a significant reduction in host unscheduled
DNA synthesis compared to wildtype cells. This also correlated with
changes in expression levels of host differentiation markers, which were
increased in the knockout cells.
Our study demonstrates that the phenotype generated by the loss of E5
in the context of a HPV18 lifecycle is intermediate between the HPV16
and HPV31 high-risk types, suggesting differences in the role of E5
proteins between different HPV types.
MA14 – Wed 1603
Offered paper Human cytomegalovirus down-regulates the T cell
co-stimulatory molecule 4-1BBL which is critical for the activation of
HCMV-specific revertant memory CD8+ T cells
Ryan Roberts1, Richard Stanton2, Matthew Reeves1,
Gavin Wilkinson2, Mark Wills1
1
University of Cambridge, Department of Medicine, Cambridge, UK,
2
University of Cardiff, School of Medicine, Cardiff, UK
HCMV has evolved numerous strategies to circumvent CD8+ T-cell
recognition such as subversion of antigen presentation and co-stimulation
provision by the infected cell. Our most recent studies have shown
that the proliferative capacity of HCMV-specific CD8+ T cells requires
4-1BBL co-stimulation. Here we report that moDC infection promotes
4-1BBL down-regulation rendering induction refractory to cytokine
stimulation. Stable 4-1BBL-overexpressing fibroblast lines exibit reduced
surface expression of 4-1BBL following wild-type Merlin, TB40/e and
AD169 infection which we have observed is due to a viral product with
immediate early/early kinetics which targets 4-1BBL for degradation in a
52
proteasomal dependent manner. We have utilised a libraries of Merlin
deletion mutant viruses and recombinant Adenovirus vectors encoding
individual HCMV ORFs to identify the gene(s) involved. These data
illustrate another aspect of the concerted immune-evasive response
encoded by HCMV to prevent the recognition of infected cells.
MA14 – Wed 1615
Offered paper A novel viral desumoylase activity, important for
efficient viral reactivation, is a therapeutic target for latent human
cytomegalovirus infection
Matthew Reeves, Emma Poole, John Sinclair
Episodic reactivation of the lifelong latent infection established by human
cytomegalovirus (HCMV) in healthy individuals is a major cause of
disease in immune-compromised/suppressed patient populations. Latent
infection of haematopoietic progenitor cells is historically described as
a quiescent infection characterised by a lack of lytic gene expression
and infectious virus production. However, recent studies suggest
that the latent genome expresses a subset of viral genes that actively
modify the cellular environment to promote long-term persistence.
Here we show that a function for LUNA, a latency-associated gene
product impacting on HCMV latency and reactivation in myeloid cells,
promotes the disruption of cellular ND10 bodies during latent infection.
Furthermore, mutation and inhibitor studies show that ND10 disruption
depends on a novel de-sumoylase activity encoded by LUNA. Despite
little overall homology to cellular de-sumoylases LUNA expresses a
conserved cysteine residue in its C-terminal domain. Crucially, inhibition
of de-sumoylase activity profoundly inhibits HCMV reactivation
from haematopoietic cells isolated from naturally latent seropositive
individuals. These data illustrate that latent infection with HCMV results
in modifications of the cellular environment, which in this instance is
imparted via the expression of a novel viral de-sumoylase activity and
that these latency-associated changes can be targeted therapeutically to
prevent virus reactivation.
MA15Virology workshop:
Gene expression and replication
MA15 – Wed 1348
Offered paper Norovirus disruption and hijacking of host translation
Edward Emmott, Sarah Caddy, Ian Goodfellow
Division of Virology, Department of Pathology, University of Cambridge,
Cambridge, UK
As cytoplasmic-replicating viruses, norovirus replication is separated
from the host cell capping machinery and instead uses a viral protein
called VPg, which functionally fulfills the role of a 5’ cap. However
the VPg protein only requires a subset of eIF proteins for its function.
We used a novel quantitative proteomics approach -Stable Isotope
Labelling of Amino acids in Cell culture (SILAC) allowing us to compare
the abundance of host translation factors, at various times following
infection with murine norovirus (MNV) in whole cell lysates, as well as
investigating their levels in translation initiation complexes as determined
by m7G-pulldowns. This has allowed the accurate determination of the
relative abundance of large numbers of individual subunit components
within the context of viral infection. By comparing these data we show
at late time points-post infection, a defect in 43S-mRNA complex
formation and, whilst stopping short of a complete shutoff of host
translation, the cellular abundance and recruitment of large numbers
of eIF proteins is altered at various timepoints post-infection. Together
with our previous data on VPg-eIF interactions these data us to perceive
changes to the translational apparatus from both the perspective of the
virus and its host.
Session abstracts
MA15 – Wed 1400
Offered paper Regulation of hepatitis C virus by eukaryotic initiation
factor 4A2
Catherine L Jopling1, Rachel Doidge1, Martin Bushell2
1
The University of Nottingham, Nottingham, UK, 2MRC Toxicology Unit,
Leicester, UK
Hepatitis C virus (HCV) replication is promoted by various host
cofactors, including several RNA helicases and the liver-specific
microRNA-122 (miR-122). miR-122 is one of a large family of 21-23
nucleotide noncoding RNA molecules that usually function to repress
mRNA translation by interacting with the 3’ untranslated region (UTR).
miR-122 shows an unusual function in regulation of HCV, interacting
with 5’UTR sites and promoting viral replication by a mechanism that is
poorly understood.
We have identified eukaryotic initiation factor 4A2 (eIF4A2) as a novel
host factor for HCV. We find that eIF4A2 depletion strongly decreases
HCV replication, while overexpression increases it. The closely related
protein eIF4A1 has less effect. eIF4A1 and 2 are DEAD-box RNA
helicases that function in translation initiation to unwind structured
regions of the 5’UTR. However, eIF4A activity is not required for HCV
translation, which is driven by an internal ribosome entry site (IRES) that
directly recruits the 40S ribosomal subunit.
A novel function for eIF4A2 was recently revealed when it was identified
as a protein with an important role in microRNA-mediated translation
repression (Meijer et al, under review). We find that eIF4A2 regulates
HCV, at least in part, by modulating the activity of miR-122.
MA15 – Wed 1412
Offered paper A twist in the tale: structural rearrangements of
long-range RNA–RNA interactions modulating HCV translation and
replication
Andrew Tuplin1, Madeleine Struthers1, Jonathan Cook1,
Peter Simmonds2, David Evans1
1
The University of Warwick, Coventry, UK, 2Roslin Institute, The University
of Edinburgh, Edinburgh, UK
The NS5B region of the HCV genome encodes a phylogenetically
conserved RNA stem-loop designated SL9266, which is a cis-replicating
element (CRE) required for genome replication. SL9266 forms a
pseudoknot (SL9266/PK) involving a long distance ‘kissing-loop’
interaction between its terminal loop and that of SL9571 (previously SLII)
within the X-tail and a further side-loop interaction to an unstructured
portion of the genome ~150 nt. upstream.
We analysed the contribution of each base to the SL9266/PK structure
using SHAPE and replication phenotype analysis of wild-type and mutant
genomes (Tuplin et. al., 2012). Marked differences were observed in
the long-range interactions of SL9266 when genotypes 2a JFH-1 and
1b Con1b were compared. Results implied that SL9266 forms the
core of an extended pseudoknot which is able to undergo structural
rearrangement. The ‘kissing-loop’ interaction was only detectable in
JFH-1-based genomes and inhibited the formation of SL9571 in the
3’-X-tail, which was released by substitutions prevented the ‘kissing-loop’
formation, in which case SL9571 formed.
We have demonstrated that blocking SL9266/PK dramatically inhibits
virus replication and translation. We propose that SL9266/PK functions
as a temporal switch, modulating mutually incompatible translation and
replication events and are investigating its potential as a target for antiviral
therapy.
MA15 – Wed 1424
Offered paper The 2009 pandemic influenza virus nucleoprotein; does
size matter?
Helen M. Wise1, Alana Livesey2, Amanda Stuart2, JiHui Ping3,
Earl Brown3, Paul Digard1
53
Session abstracts
The Roslin Institute, The University of Edinburgh, Edinburgh, Midlothian,
UK, 2Division of Virology, Department of Pathology, University of Cambridge,
Cambridge, Cambridgeshire, UK, 3Faculty of Medicine, University of Ottawa,
Ottawa, Ontario, Canada
Influenza A virus exhibits wide strain-dependent differences in virulence
that are only partly understood at the molecular level. The 2009
pandemic virus (pH1N1) is a case in point, because its low pathogenicity
in animal models cannot be explained by reference to previously
identified influenza virulence determinants such as PB2 627 or PB1-F2.
One notable feature of the pH1N1 virus is the presence of an additional
upstream start codon (AUG) in segment 5 mRNA, suggesting the
possibility that the pH1N1 nucleoprotein (NP) has a 6 amino acid
N-terminal extension relative to other strains. Given the essential role of
NP in viral RNA synthesis, this could have functional consequences.
We show that both the upstream and the normal AUG codon are
used in vitro and in infected cells, with the long NP forming a minority
population. Mutation of the upstream AUG did not affect virus
transcription or growth in cell culture but attenuated infection in mice.
However, the long form of NP could not functionally replace the normal
form of NP for virus growth. Therefore we have identified an additional
NP related polypeptide synthesised in pH1N1 infected cells that may
have accessory functions during infection.
1
MA15 – Wed 1436
Offered paper Elucidating the role of the nucleoprotein in influenza A
virus replication and transcription
Lauren Turrell, Ervin Fodor, Frank T Vreede
The eight vRNA gene segments of influenza A are bound by viral
RNA polymerase at the 5’ and 3’ ends and the remaining viral RNA
is associated with oligomeric nucleoprotein (NP) to form a viral
ribonucleoprotein (vRNP) complex. These vRNP complexes carry out
both viral transcription and replication within the nucleus of the host cell.
The role of NP in viral transcription and replication, although essential,
is not well understood. Therefore, in order to further elucidate the role
of NP in the RNP complex, we examined the effect of mutations in
the oligomerisation and RNA binding domains of NP on the level of
viral transcription and replication. We found that the presence of these
NP mutants had a differential effect on transcription and replication in
in vivo vRNP reconstitution. Specifically, the presence of RNA-binding
or oligomerisation mutant NP resulted in significantly increased or
decreased accumulation levels of mRNA respectively, but had no effect
on the accumulation levels of vRNA. Overall, our results suggest that NP
is not merely a structural protein within the RNP complex, but that it
also plays a role in the regulation of RNP complex function.
MA15 – Wed 1448
Offered paper Mapping the post-translational modifications of
influenza viruses
Edward Hutchinson, Eleanor Denham, Benjamin Thomas,
Ashley York, Lauren Turrell, Duncan Paterson, Frank Vreede, Ervin Fodor
The proteins of influenza viruses are subject to extensive posttranslational modification, but until recently the position of most
modifications was unknown. We analysed the proteomes of influenza
A and B viruses using mass spectrometry, and found numerous sites of
phosphorylation, ubiquitination, SUMOylation, N-terminal acetylation
and methionine excision -the first time such a comprehensive approach
has been applied to a virus. In particular, we found a large number of
previously unmapped phosphorylation sites. The strong conservation
of these sites, even between different viral genera, indicates that
phosphorylation plays a fundamental role in influenza biology. The
structural contexts of the sites suggest that phosphorylation regulates
a large number of viral protein functions, particularly in viral entry and
exit, nuclear localisation, and protein multimerisation. The importance
of these regulatory functions was demonstrated experimentally using
recombinant viruses and by biochemical studies of protein function. For
the nucleoprotein (NP), individually mutating most phosphorylation
sites inhibited viral growth, confirming the sites’ importance for viral
fitness. Mutating one site prevented viral growth entirely, and we
identified a mechanism in which phosphorylation at this position (S165
in influenza A viruses, S223 in influenza B viruses), prevents uncontrolled
oligomerisation of NP, thereby regulating viral transcription and
replication.
MA15 – Wed 1515
Offered paper The adenovirus type 5 L4 promoter is activated by p53
Jordan M Wright, Keith N Leppard
The University of Warwick, Coventry, UK
During infection, adenovirus must switch the emphasis of its gene
expression from early genes to late genes. Two gene products, L422K and L4-33K, contribute to this switch by activating the Major Late
Transcription Unit (MLTU), which encodes most of the virus structural
proteins. L4-22K and L4-33K expression is driven by a novel promoter
(L4P) embedded within the MLTU, though regulatory requirements
for this promoter are not known. P53 is a global regulator of stress
responses, and its activation can lead to cell cycle arrest and apoptosis.
We have found that p53 is able to activate transcription from L4P in
293 cells. Chromatin immunoprecipitation (ChIP) studies confirm p53
association with the L4P on both plasmids and the Ad5 genome during
infection. This association peaks at 12h.p.i., coinciding with the phase of
the infectious cycle when the L4P is active, and is rapidly lost as MLP
activation commences. Furthermore, we demonstrate that L4-22K and
L4-33K negatively regulate L4P and, in the case of L4-22K, inhibits the
association of p53 with the L4P, thus establishing a negative feedback
loop. Altogether, we demonstrate a novel interaction between p53 and
adenovirus and provide evidence that p53 contributes to the timely
expression of adenovirus late genes.
MA15 – Wed 1527
Offered paper Interplay between EBV EBNA 2 and EBNA 3 proteins
in the regulation of RUNX and RGC-32 expression
C David Wood1, Andrea Gunnell1, Lina Chen1,
Michael J McClellan1, Richard D Palermo1, Aditi S Kanhere4,
Marie L Hertle2, Bettina Kempkes2, Robert E White3, Martin J Allday3,
Richard G Jenner4, Michelle J West1
1
University of Sussex, Brighton, UK, 2German Research Center for
Environmental Health, Munich, Germany, 3Imperial College School of
Medicine, London, UK, 4University College London, London, UK
We previously demonstrated that the CDK1 activator RGC-32 is
highly expressed in EBV-immortalised cells and disrupts G2 cell-cycle
arrest, implicating RGC-32 in the EBV transformation process. We also
identified the RUNX1 transcription factor as a key regulator of RGC-32
expression in B-cells.
We now demonstrate that the EBV transcription factors EBNA 2, 3A, 3B
and 3C regulate RGC-32 expression in EBV-immortalised cells through
transcriptional control of the RUNX1, RUNX3 and RGC-32 genes via
distal enhancer elements. Analysis of lymphoblastoid cell-lines generated
from EBNA 3A or 3B knock-out viruses revealed reduced RUNX1 and
RGC-32 transcription and expression of EBNA 3C upregulated RGC-32
mRNA expression in B-cell lines. These data therefore implicate EBNA
3 proteins as positive regulators of RUNX1 and RGC-32 transcription.
EBNA 2 is known to function as a negative regulator of RUNX1
expression in EBV-infected cells by upregulating transcription of the
RUNX1 repressor, RUNX3. The mechanism of EBNA 2 activation
of RUNX3 however, has not been defined. We present a functional
characterisation of the RUNX1, RUNX3 and RGC-32 enhancer
elements. Taken together our data implicate interplay between EBV
transcription factors in the direct and indirect control of the key B-cell
proliferation regulators RUNX1, RUNX3 and RGC-32.
54
Session abstracts
MA15 – Wed 1539
Offered paper ATP-dependent interaction of KSHV ORF57 with the
human transcription and export (hTREX) complex
Sophie Schumann, Adrian Whitehouse
Institute of Molecular and Cellular Biology, University of Leeds, Leeds, UK
Kaposi’s sarcoma-associated herpesvirus (KSHV) is an oncogenic virus
responsible for multiple malignancies, including Kaposi’s sarcoma. KSHV
replicates in the nucleus of the host cell and requires cellular export
factors to export viral mRNAs in order to allow efficient translation
of viral genes in the cytoplasm. However, while mammalian mRNA
export is linked to splicing, the majority of KSHV mRNAs are intronless,
prompting the virus to circumvent this step. KSHV therefore encodes
ORF57, a protein which interacts with the human transcription/export
(hTREX) complex to form an export competent ribonucleoprotein
particle, which facilitates nuclear export of viral mRNA. In this study we
show that formation of the ORF57-mediated ribonucleoprotein particle
is ATP-dependent, which presents a novel antiviral target. Our results
suggest ATP-cycle dependent remodelling of the hTREX complex,
which affects the ability of ORF57 to recruit the endogenous complex.
Following these findings we present a mechanism for specific disruption
of the ORF57/hTREX interaction, which could be used to inhibit virus
lytic replication while allowing endogenous protein complex formation.
strikingly, the majority contain in-frame duplications of up to ~400 nt at
the recombination junction.
We have demonstrated, by using different serotypes of poliovirus, that
intratypic recombination is significantly more frequent than intertypic,
indicating that sequence identity may contribute to the process.
Furthermore, in preliminary studies we have shown that the use of sublethal levels of the antiviral ribavirin enhances recombination frequency.
This both provides insights into the characteristics of the polymerase that
are important for recombination and implies that antiviral therapy may, in
addition to selecting for drug resistance, influences viral recombination in
co-infected patients.
MA15 – Wed 1551
Offered paper Analysis of mutations in the alpha helix region of the
ICP0 RING finger domain
Kathleen Pheasant, Roger Everett
MRC-University of Glasgow Centre for Viurs Research, Glasgow, UK
Herpes simplex virus type 1 (HSV-1) establishes latency in sensory
neurones after primary infection. The IE viral protein ICP0 is important
for regulating the balance between lytic and latent infections. ICP0
contains a RING finger domain which acts as an E3 ubiquitin ligase,
inducing the degradation of PML, the major component of ND10, and
other cellular proteins. ND10 play a role in restricting viral infection.
Previous work using an ICP0-inducible cell line found that RING finger
mutant N151D complements an ICP0-null mutant virus efficiently, but
was defective in reactivation of quiescent HSV-1, suggesting that the
mechanisms controlling lytic infection and reactivation may be separable.
To investigate this in more detail, we studied virus expressing ICP0 RING
finger mutants K144E and N151D. Both viruses had defects in plaque
forming ability and replication efficiency at low MOI, and degraded PML
less efficiently. They were also defective in inhibiting the recruitment of
ND10 proteins to viral genomes. However, the mutants reactivated
quiescent HSV-1 poorly even at multiplicities at which they replicate
efficiently. We conclude that there may be a greater requirement for
ICP0 activity during reactivation of chromatininsed quiescent genomes
than for stimulating gene expression from unchromatinised genomes at
the beginning of infection.
MA15 – Wed 1603
Offered paper Genetic recombination in enteroviruses
Andrew Woodman
The University of Warwick, Coventry, UK
Members of the Picornaviridae have error prone polymerases, short
replication cycles, and high yields that contribute to significant genetic
diversity. However, far greater genetic variation is achieved by
recombination than is possible in a single round of genome replication.
Genetic recombination is believed to be due to a ‘copy-choice’
mechanism, and at present, it is suggested that the RNA dependent
RNA polymerase pauses or terminates at certain RNA sequence motifs,
promoting template switching.
We have developed a system that exploits two partially replication
competent parental genomes to select the early recombination products
from a dually transfected cell. All viable progeny are recombinants and,
MA15 – Wed 1615
Offered paper Use of a reverse genetics system to identify the
functional domains of NS2 during BTV replication
Victoria A K Easton, Polly Roy
London School of Hygiene and Tropical Medicine, London, UK
Bluetongue Virus (BTV) of the Orbivirus genus (Reoviridae) has a genome
of 10 dsRNA segments which encode for 7 structural (VP1-7) and 4
non-structural proteins (NS1-4).
A characteristic of BTV infected cells is viral inclusion bodies (VIBs),
consisting mainly of NS2. The VIBs are the sites of immature virus
assembly where the structural proteins VP3 and VP7, the transcription
complex proteins (VP1, VP4, and VP6) and the virus ssRNAs are thought
to be recruited by NS2 prior to assembly. However, how NS2 recruits
each of these viral components is not clearly understood. In this study
we have identified the domain/residues responsible for BTV ssRNA
binding during virus replication. We used a combination of targeted
mutations in the viral genome together with a reverse genetics system
that allowed us to examine the effect of each mutation in VIB formation
and virus replication in vivo. In addition, to substantiate the in vivo data,
recombinant mutant NS2 proteins were synthesised using baculovirus
expression system, each mutant protein purified and characterised.
The data obtained from in vivo and in vitro studies demonstrated the
importance of NS2 in BTV replication and identified the NS2 residues
that are responsible for binding of BTV RNA segments.
Virology workshop:
Clinical virology
MA16
MA16 – Wed 1400
Offered paper Cluster of influenza A cases in a vaccinated population
of adults in a virology laboratory in Glasgow, December 2012
Amanda Bradley Stewart, Alasdair MacLean, Rhona S Miller,
Susan Bennett, Celia Aitken, Rory N Gunson
West of Scotland Specialist Virology Centre, Glasgow, UK
We report an influenza A (H3) outbreak that occurred following a
lunchtime Christmas quiz in the virology laboratory in Glasgow. Most
of the attendees had been immunised against influenza at least 6 weeks
earlier. Samples were initially screened for influenza and other respiratory
infections, and influenza positive samples were subtyped using real-time
PCR. Samples that tested positive for Influenza A were later sequenced
and a phylogenetic tree was produced.
Ten cases of influenza A, and five probable cases of influenza were
reported, these all occurred within 3-5 days of the quiz. It is speculated
that transmission occurred during the quiz which lasted over 1 hour at a
lunchtime staff meeting. The possible index was a member of staff who
later tested positive for influenza A and reported feeling unwell during
the quiz.
Nine of the ten influenza A positives patients and one influenza
B positive patient had been vaccinated. The occupational health
department were contacted to determine the vaccine batches used
to vaccinate staff (those who presented with influenza and those who
remained well) and whether this represented a vaccine batch failure. To
55
Session abstracts
date (2012/2013 influenza season) we have subtyped 33 influenza B
positive samples (14 were Victoria and 17 were Yamagata). Therefore
approximately 50% of influenza B in circulation is not covered by the
vaccine.
MA16 – Wed 1412
Offered paper Influenza pseudotypes as tools to measure
heterosubtypic neutralising antibody responses against representatives
of all Group 2 influenza A viruses
Francesca Ferrara1, Eleonora Molesti1, Eva BöttcherFrieberthäuser2, Giovanni Cattoli3, Davide Corti4, Simon Scott1,
Nigel Temperton1
1
Viral Pseudotype Unit, University of Kent, Chatham Maritime, UK, 2Philipps
University, Marburg, Germany, 3Istituto Zooprofilattico Sperimentale delle
Venezie, Legnaro, Italy, 4Institute for Research in Biomedicine, Bellinzona,
Switzerland
The evaluation of existing population immunity against influenza
viruses is an important aspect in pandemic preparedness. Serology
methods, such as haemagglutination inhibition and microneutralisation,
are widely used for this purpose and are also employed in vaccine
efficacy studies. Influenza pseudotypes represent safe tools to study the
immune response since they are replication-defective viruses and they
harbour on their envelope only the haemagglutinin that is the major
target of the antibody response. We have generated a panel of Group
2 influenza A pseudotypes and we have employed them as surrogate
antigens in neutralisation assays to study sera generated against H3N8,
H4N8, H7N1, H7N2, H7N3, H7N7, H10N1, H14N5 and H15N9
Influenza A viruses. Neutralising antibody responses are detectable
in the sera not only when they are tested against a homosubtypic
pseudotype (e.g. anti-H4N8 sera vs H4 pseudotype), but also when
the sera are tested against pseudotypes harbouring evolutionary related
haemagglutinin subtypes (e.g. anti-H14N5 sera vs H4 pseudotype). This
shows that the pseudotype neutralisation assay detects homosubtypic
and heterosubtypic neutralising antibody responses and can be used in
vaccine efficacy studies and in the evaluation of population immunity.
MA16 – Wed 1424
Offered paper Detection of a novel hantavirus (Tatenale virus) in the
United Kingdom
Kieran C Pounder1, Michael Begon1, Tarja Sironen2, Phill Watts1,
Liina Voutilainen2,3, Olli Vapalahti3, Boris Klempa5, Anthony R Fooks5,6,
Heikki Henttonen2, Lorraine M McElhinney5,6
1
University of Liverpool, Liverpool, UK, 2Finnish Forest Research Institute,
Vantaa, Finland, 3Haartman Institute, Helsinki, Finland, 4Slovak Academy of
Sciences, Bratislava, Slovakia, 5Animal Health and Veterinary Laboratories
Agency, Surrey, UK, 6National Consortium for Zoonosis Research, South
Wirral, UK
Serological studies and sporadic human cases have previously suggested
the presence of hantavirus in the UK. However, until now the species
of hantavirus present in UK wildlife has never been confirmed. Between
September 2009 and November 2011, wild rodents consisting of brown
rats (Rattus norvegicus) (n = 133), wood mice (Apodemus sylvaticus) (n =
269), house mice (Mus musculus) (n = 50), bank voles (Myodes glareolus)
(n = 35) and field voles (Microtus agrestis) (n = 8) were live caught
across north-west England (Cheshire, Liverpool and Wirral). With the
exception of a single field vole, the lungs from all rodents sampled were
negative for hantaviral RNA using a pan-hantavirus RT-PCR. However,
partial sequences for small (S) and large (L) genome segments were
recovered from the lung of the field vole and confirmed the presence of
a novel hantavirus (Tatenale Virus) in the United Kingdom. Coincidently
in 2012, HPA investigations following a case of haemorrhagic fever with
renal syndrome in Northern England, led to the subsequent isolation of
Seoul hantavirus from a wild brown rat. The prevalence and public health
impact of the two hantavirus species in the UK are not yet known.
MA16 – Wed 1436
Offered paper Vomiting Larry: a simulated vomiting system
Catherine Makison Booth
Health & Safety Laboratory, Buxton, UK
Infectious diseases such as norovirus can induce emesis (vomiting), which
can be of a projectile nature. Whilst studies have been carried out on
transmission, prevalence and decontamination of such microorganisms
within various environments, little is known about the extent to which
the surrounding environment is contaminated when an individual vomits.
This is an important, yet overlooked, consideration for infection control
purposes. The aim of this study was to develop a novel simulated
vomiting system (Vomiting Larry), which would be used to establish the
true extent to which projected fluid can spread and contaminate the
environment.
Vomiting Larry was set up within a Controlled Air Chamber (CAC)
facility at the Health and Safety Laboratory (HSL). Simulated vomiting
was undertaken using water as a vomitus substitute to which a
fluorescent marker was added so as to locate the distribution post
emesis. A fluorescent marker was used so that small splashes, ordinarily
missed under standard light, would be visible with the aid of UV lighting.
Experiments revealed that splashes and droplets travelled further than
anticipated (>3 m forward spread and 2.6 m lateral spread), highlighting
the difficulty in identifying and therefore cleaning all contaminated
surfaces after an episode of vomiting.
MA16 – Wed 1448
Offered paper Development of a real-time quantitative PCR assay for
detecting and quantifying CMV UL146 genotypes in clinical samples
MArylouisa Holton1, Tina Ganzenmueller2, Thomas F Schulz2,
Maurizio Zavattoni3, Andrew Davison1
1
Medical Research Council-University of Glasgow Centre for Virus Research,
Glasgow, UK, 2Institute of Virology, Hannover Medical School, Hannover,
Germany, 3Molecular Virology Unit, Virology and Microbiology Department,
Fondazione IRCCS, Policlinico, San Matteo, Pavia, Italy
Infections with multiple strains of CMV occur frequently in
immunocompromised patients. Organ transplant patients with these
types of infections are more likely to have higher initial viral loads and
take longer to treat. The different CMV strains found in these patients
exist in a dynamic state, with the relative abundance of major and minor
strains changing over time. Evidence suggests that multiple CMV strains
in congenital infections may be associated with more severe sequelae
and foetal death. We have developed a novel, quantitative real-time PCR
assay that can differentiate among the 14 genotypes of the CMV UL146
CXCL chemokine gene. This assay is relatively inexpensive, unbiased and
fast compared to more commonly used methods, such as PCR, cloning
and sequencing. We have validated the assay using DNA extracted
from low-passage isolates of clinical samples from immunocompromised
patients infected with multiple strains of CMV, and are now directly
analysing the original clinical samples. The preliminary data confirm that
immunocompromised patients can harbour several UL146 genotypes in
a wide range of relative amount. The assay will be used to monitor strain
dynamics and determine the frequency of multiple CMV infections in
various patient sample sets.
MA16 – Wed 1515
Offered paper Audit of reference VZV IgG serology for pregnant
women in contact with chickenpox and the potential for reducing
VZIG administration
Peter A C Maple1, Gayatri Amirthalingam2, Kevin E Brown1
1
Microbiology Services - Colindale, Health Protection Agency, Virus Reference
Department, London, UK, 2Health Protection Services -Colindale, Health
Protection Agency, Immunisation, Hepatitis and Blood Safety Department,
London, UK
56
Session abstracts
prospectively the outcome of treatment in this cohort. Compared
directly to the outcome in patients with undetectable HCV PCR, a 20%
higher relapse rate with a corresponding 20% lower sustained virological
response (SVR) was noted in patients with detectable HCV RNA below
the LOQ at week 4 and week 12. This study shows a clear correlation
between a higher incidence of an adverse outcome in those patients
with a detectable <30 IU/ml HCV RNA at week 4 and a similar trend
at week 12, with a corresponding improved response in those with an
undetectable HCV viral load during therapy
Healthy, pregnant women exposed to chickenpox should receive
prompt medical advice to determine if they are protected. In the
absence of a history of past chickenpox or shingles their immune status
should be determined, urgently. Laboratory diagnosis of past infection is
performed by measuring Varicella Zoster virus (VZV) IgG and a number
of commercial assays are available to do this. Local laboratory testing
is recommended, in the first instance, and if time permits reference
laboratory testing should be considered if results are equivocal or
negative. An audit of the utility of this approach was undertaken for
samples submitted to the Virus Reference Department (VRD), HPA
Microbiology Services, Colindale, London during 2012. Over 12 months,
71 samples which tested negative (n = 10) or equivocal (n = 61) in
local laboratories were submitted to VRD. Following reference VZV
IgG serology, 65 samples tested VZV IgG positive, 3 were equivocal
and 3 were negative. It is recommended that pregnant women, testing
VZV IgG negative, by reference serology, should receive passive VZIG
immunisation which costs £1200 per course. Submitting samples for
reference serology led to a significant reduction in VZIG administration,
saving a resource in short supply together with cost savings of £81,600.
MA16 – Wed 1527
Offered paper Detecting norovirus using the BDMAX: a fast, simple
and sensitive approach to help reduce the burden it exerts on
healthcare settings
Juliet E M Kenicer, Julie White, Peter McCulloch, Kate E Templeton
NHS Lothian, Edinburgh, UK
The primary objective of the study was to assess performance of a
multiplexed NoV GI, GII PCR with EAV internal control on the BDMAX
extraction/PCR platform (Becton Dickinson). The secondary objective
was to assess BDMAX as a suitable platform for non-professionally
registered staff in routine diagnosis.
During January and February 2012, 312 faecal and 25 vomit specimens
were collected from patients with either: vomiting, diarrhoea or both.
Samples were made into suspensions containing EAV and extracted
on BDMAX and NucliSENS easyMAG (Biomerieux.) The PCR was
executed and analysed on BDMAX and ABi 7500 (Applied Biosystems)
respectively. The NoV assay was incorporated into routine diagnosis and
turn around times (TAT) were compared to the established EasyMAG/
ABi method.
There was 2% inhibition on BDMAX and 10.6% on easyMAG/ABi.
BDMAX sensitivity was 94% (96/102) and specificity was 99% (233/235).
1414 samples were run on either the BDMAX or the easyMAG/ABi as
part of routine NoV testing. 361 samples tested on the BDMAX had a
mean TAT of 30 hours and 1053 samples tested on the easyMAG/ABi
had a mean TAT of 31 hours.
The BDMAX NoV assay performed satisfactorily and proffered
considerable practical benefits including, minimal hands on time, walkaway function and ease-of-use.
MA16 – Wed 1539
Offered paper Clinical significance of detectable but unquantifiable
HCV viral load by Abbott m2000
William L Irving2, Fouzia Jabeen1
1
Birmingham Heartlands Hospital, Birmingham, UK, 2Nottingham University
Hospitals, Notingham, UK
Accurate measurement of HCV RNA is key to the implementation
of response guided therapy in the management of HCV. With more
sensitive and specific real-time PCR techniques replacing classical PCRbased methods, the lower limit of detection (LOD) and lower limit
of quantitation (LOQ) have reduced much lower than those defining
response to treatment in previous studies. A discrepancy in the LOD
and LOQ gives low level HCV RNA results, the significance of which is
uncertain. In this study the clinical significance of detectable HCV RNA
below the LOQ with the Abbott m2000 was assessed, by evaluating
MA16 – Wed 1551
Offered paper Seroepidemiology of molluscum contagiosum virus in
representative German and UK populations
Subuhi Sherwani1, Nidhi Agarwal3, Laura Farleigh1, Sam Loveless1,
Neil Robertson1, Paul Schnitzler2, Joachim Bugert1
1
Cardiff University, Cardiff, UK, 2Universität Heidelberg, Heidelberg, Germany,
3
School of Dermatology, Skin Institute, New Delhi, India
Molluscum contagiosum virus (MCV) is a significant human skin pathogen
for children and adults. In the absence of reliable clinical data the true
prevalence of molluscum contagiosum is unknown. As MCV cannot
be grown in culture, virions can only be obtained from limited patient
material. To address this problem, we have developed a recombinant
ELISA using the molluscum surface protein MC084 as antigen. The
ELISA was found to be sensitive (100%, n=12) and specific, with low
inter-and intraassay variability. MCV seroprevalence in a representative
German population was determined. We report here the results of our
preliminary study which included 435 sera collected from patients and
healthy individuals, 0-73 years of age. These were subdivided into groups
I-V on the basis of age. The overall seropositivity rate, n= 435, against
MC084 was found to be 51% at a cut off of A450 0.2. Age related
seropositivity rates were determined as follows: 0-5 years, n=69, 37.6%;
6-10 years, n=47, 42.4%; 11-20 years, n= 72, 34.7%; 21-40 years, n=47,
53.2% and ≥40 years, n=200, 64%, respectively. In a immunesuppressed
UK population 21->40 years of age (n=99) a similar overall MC084
seroprevalence of 45.5% was found.
MA16 – Wed 1603
Offered paper HIV-1 RNA detection in patient plasma during longterm suppressive antiretroviral therapy (ART)
Stacey C King1, Paola Vitiello2, Alessandro Cozzi-Lepri3,
Andrew Phillips3, Anna Maria Geretti1, ERAS Study Group
1
University of Liverpool, Liverpool, Merseyside, UK, 2Unit of Infectious
Diseases, Busto Arsizio Hospital, Legnano, Italy, Italy, 3University College
London, London, UK
Background Whether HIV continues to replicate during apparently
successful ART remains controversial. The study aims were to validate
an ultrasensitive HIV-1 RNA load (VL) assay and evaluate the impact of
long-term ART on HIV-1 RNA detection in plasma.
Methods The Abbott RealTime assay was modified to increase
sensitivity and validated with serial dilutions of the WHO 2nd HIV-1
RNA Standard. Eligible patients were receiving first-line NNRTI-based
ART with VL consistently <50 copies/ml (no blips); they were recruited
into 10 groups based on ART duration (1-10 years).
Results The assay lower limit of detection was 1-3 HIV-1 RNA copies/
ml. The study recruited 104 adults with median age 47 years, pre-ART
VL 4.9 log10 copies/ml, nadir CD4 count 201 cells/mm3; 78% were
male, 57% white, 51% MSM. Patients started ART in 1997-2011, most
commonly (84%) with efavirenz. Considering all groups, HIV-1 RNA was
detected in 52/104 (50%) patients at median 4 copies/ml (range 1-35;
IQR 2-7). Over 10 years there was a mean VL decrease of -0.62 log10
copies/ml (95% CI: -1.37, 0.12; p=0.10).
Conclusions HIV-1 RNA remains detectable in a large number of
patients receiving apparently successful ART. The source and significance
of residual HIV-1 RNA detection warrant further studies.
57
Session abstracts
MA16 – Wed 1615
Offered paper Next-generation sequencing for HIV resistance testing
– a practical approach
Leo Chanqueo1, Valentina De Sario1, Anna Smielewska1, Kim Brugger2,
Howard Martin3, Anthony Rogers2, Hongyi Zhang1, Jane Greatorex1
1
Health Protection Agency, Cambridge, UK, 2East Anglian Sequencing and
Information Hub, Cambridge, UK, 3Dept Molecular Genetics Addenbrookes
Hospital, Cambridge, UK
There is an expectation that next generation sequencing will improve
the quality and clinical relevance of antiviral resistance testing, exposing
underlying minority resistance variants and helping to determine patient
compliance. However, the technology involved can appear complex and
expensive, and there is a lack of agreement over utility in certain clinical
scenarios.
We have carried out a proof of concept study to determine whether
existing amplicons generated in the laboratory for Sanger sequencing
could be utilised for deep sequencing. The rationale was to minimise
changes to existing laboratory procedures, reduce analysis time for staff
and improve data quality.
A simple script was developed which identified the variants, calculated
the percentages present and formatted the mutations such that they
could be pasted directly into the Stanford database. We are now
carrying out a prospective, parallel comparison with the existing Sanger
sequencing assay. Results of data analysis to determine the clinical
relevance and utility will be presented.
Replacing Sanger sequencing with next generation sequencing for
anti-viral resistance testing offers real reductions in hands on time and
should improve the overall quality of laboratory output. The relevance
of minority mutations observed in different patients sets remains to be
elucidated.
MA16 – Thu 1132
Offered paper Acute hepatitis – should the current screening strategy
be modified?
Heli Harvala, Georgina McAllister, Vincent Wong,
Ingolfur Johannessen, Sandeep Ramalingam
Specialist Virology Centre, Royal Infirmary of Edinburgh, Edinburgh, UK
Data on acute hepatitis screening were reviewed to evaluate the
effectiveness of current diagnostic testing. A Cognos search was
performed to identify individuals tested for acute hepatitis between June
2010 and December 2012, and their APEX and TRAK records were
reviewed.
All samples (total 3560) submitted for acute hepatitis investigations
were tested for HAV, HBV and HCV; a subset of these was also tested
for hepatitis E virus (HEV), EBV and CMV based on clinical criteria.
Screening identified 4 HAV, 22 HBV, 1 HCV, 25 HEV, 63 EBV and 31
CMV infections. Most subjects presenting with HAV, HBV or HEV were
jaundiced, and all subjects with acute HAV or HEV had high ALT (mean
2470U/l and 2350U/l, respectively). Individuals with EBV and CMV
infections had lower ALT (mean 443U/l and 287U/l, respectively) and
were infrequently jaundiced (7% EBV, 0% CMV). Individuals with HAV
infections were the youngest (mean 22 years) and HEV the oldest (48
years).
EBV and CMV were the most common causes of acute hepatitis. EBV
and CMV testing could focus on those with mildly abnormal ALT levels.
The high frequency of HEV detection (6.2%) suggests that HEV testing
should be included in the routine acute hepatitis screening.
MA16 – Thu 1120
Offered paper Hepatitis E infection and men who have sex with men
Brendan AI Payne1,3, Samreen Ijaz4, Ulrich Schwab1, Emma J Savage4,
O Noel Gill4, Richard Tedder4, Samuel Moses2,1, Manoj Valappil2,1
1
Newcastle-upon-Tyne Hospitals NHS Foundation Trust, Newcastle-uponTyne, UK, 2Health Protection Agency, Newcastle-upon-Tyne, UK, 3Newcastle
University, Newcastle-upon-Tyne, UK, 4Health Protection Agency, Colindale,
London, UK
Aim To establish the contribution of HIV infection and men who have
sex with men (MSM) to HEV seroprevalence.
Methods We used archived sera from the UK HPA unlinked
anonymised seroprevalence survey from genitourinary medicine clinic
attenders. Anti-HEV IgG was measured by ELISA (Fortress Diagnostics,
Antrim, UK).
Results 422 serum samples from the year 2008 were analysed (146
HIV+/MSM, 135 HIV-/MSM, 141 HIV-/hets). Anti-HEV prevalence
was: HIV+/MSM 7.5%, HIV-/MSM 10.4%, HIV-/hets 3.5% (p=0.025
for comparison of HIV-MSM and hets). MSM (p=0.044) and age band
(20-34 years vs. 35-44 years, p=0.026) were independently associated
with anti-HEV prevalence. There was no association with presence of
acute STI. To explore recent temporal trends in HEV seroprevalence
among MSM, we analysed sera from 977 MSM over a three year period,
showing a significant increasing trend (2006 2.1%, 2007 5.2%, 2008 9.3%,
p=0.003).
Conclusions We show that MSM may be a risk factor for HEV
acquisition, but HIV infection per se is not. The pathological mechanism
may plausibly include oro-anal sexual practices. The increasing
seroprevalence in MSM comes at a time of increased HEV activity in the
general UK population. MSM will therefore be at increased risk of HEV
exposure and onwards transmission.
58
Poster abstracts
Posters
mTOR is a highly conserved protein kinase that mediates stress signals
via the phosphoinositide 3-kinase (PI3K)-AKT pathway and, given its
central role in controlling cell growth, proliferation and survival, mTOR
signalling is frequently deregulated in cancer cells. The mTOR pathway
is considered an important target for anticancer drug development
and drugs that act on components of the pathway, especially mTOR,
have been approved or are in late-stage investigations as anti-cancer
therapeutics.
We have investigated the effects of mTOR inhibitors, (rapamycin,
everolimus, temsirolimus and AZD8055), combined with the oncolytic
herpesvirus HSV1716 on cell death in a panel of 10 different human
cancer cell lines. HSV1716 and mTOR inhibitors frequently combined to
enhance cancer cell killing in vitro. Surprisingly this effect was observed
at concentrations of mTOR inhibitors that prevented cell growth and
concomitantly reduced viral replication and spread. Both viral infection
and mTOR inhibition can activate the cellular autophagy response
which potentially contributes towards cell death under certain contexts
in human cancer. Further investigations of this interesting interaction
between HSV1716 and mTOR inhibition are ongoing.
Viruses and human cancer:
causes to cures
MA01
MA01/01
Novel cell-based models to investigate the role of human
papillomavirus infection in oropharyngeal carcinogenesis
Peter C Rae1, Elizabeth K Marsh1, Max Robinson2, Hisham Mehanna1,
Sally Roberts1
1
The University of Birmingham, Birmingham, UK, 2University of Newcastle,
Newcastle, UK
Over the last three decades the incidence of HPV-positive
oropharyngeal cancer (HPV-OPC), that of the tonsil or base of tongue,
has increased by 225%. Unlike other head and neck cancers associated
with alcohol and tobacco, HPV-OPC responds well to DNA damageinducing therapies: however, such treatments affect long-term quality
of life in younger patients. Therefore, understanding the mechanisms
underlying HPV infection and persistence in the oropharynx is important
for management of HPV-OPC. We have developed cell-based models
that recapitulate the full spectrum of carcinogenesis from HPV infection
to oropharyngeal carcinoma. First , we transfected tonsil keratinocytes
with high-risk HPV16 or HPV18 which are typically present in OPC. In
doing so, we establsihed stable cell lines that maintain episomal HPV
genomes throughout long-term culture; integration of the genome into
host DNA, a known risk factor for malignant cervical transformation,
also occurred in some lines. Secondly, we estabished cell lines from
oropharyngeal biopises. Each cell line was tested for HPV and, if positive,
its viral genome form assessed to build a valuable collection of HPVpositive and HPV-negative OPC cell lines. Together, our cell-based
models are a versatile resouce for head and neck cancer research.
MA01/02
Combination studies with oncolytic HSV1716 and chemotherapeutic
and targeted therapeutic anti-cancer drugs
Kirsty Learmonth, Lynne Braidwood, Joe Conner, Steven Powell
Virttu Biologics Ltd, Glasgow, UK
Oncolytic HSV1716, lacking the neurovirulence factor ICP34.5, has exquisite
replication competence for cancer cells and has been used in safety trials
in glioma, melanoma, H&NSCC and paediatric non-CNS solid tumours.
In total 77 patients have received HSV1716 to date with no evidence of
toxicity, no spread to surrounding normal tissue, no shedding in patients
and the selectivity of HSV1716 for replication only in tumour cells has
been demonstrated. As oncolytic viruses gain acceptance as therapeutics,
their interactions with other anti-cancer drugs will need to be investigated.
We have established an in vitro assay system that uses established human
cancer cell lines from various clinically relevant indications to investigate the
interactions between HSV1716 and diverse anti-cancer drugs. The assay
system monitors the effect of the drug on HSV1716 and their combined
effects on cell death. We have screened a repertoire of chemotherapeutic
and targeted therapeutic drugs and identified synergistically enhanced cell
killing interactions between HSV1716 and certain anti-cancer drugs including
doxorubicin and inhibitors of mTOR and JAK. Our initial results have
demonstrated the huge potential and also the challenges of such studies
and we have established an open innovation platform (VirttuReplicate) to
optimise HSV1716 combinatorial analyses.
MA01/03
Inhibitors of mTOR combine with oncolytic HSV1716 to enhance
cancer cell death
Joe Conner1, Lynne Braidwood1, Leigh McGibbon1,
Kirsty Learmonth1, Ed Chan2
1
Virttu Biologics Ltd, Glasgow, UK, 2SIPBS, Glasgow, UK
MA01/04
Identifying T cell responses to Merkel cell polyomavirus tumour
antigen in healthy donors
Lalit Pallan, Neil Steven, David Blackbourn, Andrew Hislop
Merkel cell Polyomavirus (MCV) is a recently discovered causative
factor in the development of Merkel cell carcinoma (MCC). This rare
but aggressive form of skin cancer is associated with the integration of
MCV into the cellular genome, leading to the expression of mutated
Large T antigen which drives proliferation of the tumour cell. The cellular
immune response appears important in control of this virus and the
pathology it causes as disease is more frequently seen in individuals with
underlying immunosuppression. However, little is known about the T cell
response to the virus or the T antigens in diseased or healthy infected
donors. Here we quantified the T cell response to the Large and Small
T proteins using interferon-gamma ELISpot assays, in which PBMCs
from healthy donors are stimulated by overlapping pools of peptides
corresponding to these two viral antigens. Stimulation of PBMCs with
Large and Small T peptides invoked weak responses in seropositive
individuals, comparable in size to other polyomavirus responses. We are
currently generating T cell clones specific to these proteins to identify
epitopes, which will be used in ELISpot studies comparing responses in
healthy versus MCC patients to identify a role for T cell control of this
malignancy.
MA01/05
The Epstein–Barr virus structural proteins encoded by BcLF1 and
BPLF1 are frequent targets of T cells in healthy individuals
James E Turner, Teja Bajt, Jill M Brooks, Alan B Rickinson,
Graham S Taylor
School of Cancer Sciences, The University of Birmingham, Birmingham, West
Midlands, UK
Background Epstein–Barr Virus (EBV) infects 90% of people worldwide.
Some of these infected individuals will subsequently develop one of
the EBV-associated cancers. To identify viral antigens that could be
included in a prophylactic vaccine, T-cell responses against two large and
abundant EBV structural proteins were investigated: the major capsid
protein (MCP) encoded by BcLF1, and the large tegument protein (LTP)
encoded by BPLF1.
Methods Results from three prediction algorithms were combined to
identify forty-one 20mer peptides from MCP or LTP each possessing
a predicted HLA-A*02 or HLA-B*07 restricted T-cell epitope. Eleven
peptide pools, containing three to five peptides, were used in interferongamma ELIspot assays to screen T-cells from nine healthy EBVseropositive donors directly ex-vivo and after 7 days of culture.
59
Poster abstracts
Results When tested ex-vivo, T-cell responses to MCP and LTP were
in most cases undetectable or small (typically <40 spots/million cells).
However, after in-vitro culture, T-cell responses were readily detected in
all donors (range 130-260 spots/million cells). T-cell clones established
from representative donors allowed the individual stimulating peptide in
the pool to be identified.
Conclusion All donors possessed low but detectable T cell responses
to MCP and LTP. These viral antigens may warrant inclusion in future
vaccine strategies.
MA01/06
Human genetic variants contribute to Epstein–Barr virus load in
lymphoblastoid cell lines and are involved in EBV lytic reactivation
Charlotte J Houldcroft1, Astrid Gall1, Anne L Palser1,
Jimmy Z Liu1, Daniel J Gaffney1, Carl A Anderson1, Paul Kellam1,2
1
Wellcome Trust Sanger Institute, Cambridge, UK, 2Division of Infection and
Immunity, UCL, London, UK
Epstein–Barr virus, a ubiquitous gammaherpesvirus with oncogenic
properties, has been implicated in gastric carcinoma, PTLD, B, T and NK
lymphomas, as well as causing the more benign infectious mononucleosis
of young adults. Studies of candidate genes in infectious mononucleosis,
and genome-wide association studies (GWAS) of EBV-related cancers,
provide evidence for a host genetic component to EBV infection and
disease. In this study, we measured EBV load in 922 HapMap and
1000 Genomes lymphoblastoid cell lines (a model of persistent latent
infection). Using 80 family trios, we estimated heritability of EBV load to
be ~15%. EBV lytic transcript expression suggested that virus reactivation
is the cause of high EBV loads in a subset of these cell lines. We
conducted a GWAS, including 715 of the samples phenotyped for EBV
load, to discover human genetic variants controlling virus load, and to
determine if these variants influence switching from latency. A number of
SNPs showed P values with genome-wide significance, including markers
located near zinc-finger proteins and molecules involved in cellular
signalling. 158 samples were grouped by EBV load and several human
genes showed differential expression in microarray data. Future work will
functionally validate these loci and look for interactions with EBV-related
disease.
MA01/07
Autophagy as a control mechanism in HPV infection
C Charsou1, K Cuschieri2, A R Williams3, S E Howie1
1
MRC Centre for Inflammation Research, Univeristy of Edinburgh, Edinburgh,
UK, 2Specialist Virology Centre Royal Infirmary of Edinburgh, Edinburgh, UK,
3
Department of Pathology, The University of Edinburgh, Edinburgh, UK
Human papilloma virus (HPV) is one of the commonest viruses
infecting keratinocytes in mucosal tissues and skin. To date there have
been identified up to 120 low and high-risk HPV subtypes, infecting
humans. High-risk subtypes 16 and 18 are responsible for almost 70%
of the cervical cancers recorded, with HPV 16 being associated with a
significant number of malignancies in the oropharynx.
Autophagy, maintains the balance between cell survival and cell death,
holding a critical role in control of intracellular infection immunity and
in the pathogenesis of a number of cancers. There is relatively little
known about the role of autophagy in HPV induced cancer. There
is contradictory evidence on autophagy marker regulation in HPV16
related cervical squamous cell carcinoma [1] while downregulation of
autophagy may be related to poor prognosis in HPV16 related cervical
cancer [2].
We are assessing whether identification and regulation of autophagy
markers in HPV related cervical and oropharyngeal cancers using
immunohistochemistry and immunofluorescence will provide effective
markers of disease progression for patients with HPV related cancer.
References 1. Zxu et al, Tumour Biol. 2012 Oct;33(5):1653-9; 2. Cheng
et al (2012, Eur. J. Gynaecol. Oncol.33, 15-20
MA01/08
Cellular splicing factor SRSF3 controls the human papillomavirus
infectious life cycle
Tetyana Klymenko, Raquel Siqueira, Sheila Graham
MRC-University of Glasgow Centre for Virus Research, Institute of Infection
Immunity and Inflammation, University of Glasgow, Glasgow, UK
Human papillomavirus type 16 (HPV16) is the most common sexually
transmitted viral pathogen and persistent infection can lead to cervical
cancer. Viral proteins are able to alter the differentiation program of the
infected epithelial cell to enable virus propagation. We previously showed
that HPV16 infection upregulates the cellular splicing factors SRSF1-3 and
this up regulation is controlled by the HPV E2 transcription factor. In this
study we investigated the consequences of this SR protein up-regulation
for the virus life cycle. In order to study virus replication in a physiologicallyrelevant system we cultured two different HPV-infected epithelial models,
W12 and NIKS/HPV16 cells. Consistent with this, siRNA depletion of
SRSF3, but not other SR proteins, in differentiated epithelial cells resulted
in reduced levels of expression of L1, the major virus capsid protein. qRTPCR analysis revealed significant changes in abundance of early mRNAs
containing the E4 ORF, while no change in levels of late mRNAs were
detected. These data indicate that cellular SRSF3 has gene regulatory
roles in both early and late phases of the virus life cycle. This is the first
demonstration of a cellular protein controlling HPV replication.
Metabolic interactions at the
host–pathogen interface
MA02
MA02/01
Metabolic reconstruction of the Dickeya genus of bacterial plant
pathogenic bacteria: insights into host range
Leighton Pritchard, Ian K Toth, Sonia Humphris
James Hutton Institute, Dundee, UK
Phytopathogenic bacteria from the genus Dickeya have differing host
ranges, cause disease on a range of crop and ornamental plants, and
pose a severe emerging risk to European potato production. We
have sequenced 25 novel isolates of this genus, and reconstructed
the predicted metabolic capacity of 29 Dickeya isolates. These
reconstructions reveal distinct metabolic capabilities that correlate with
Dickeya species and host preference.
MA02/02
Transcriptomic analysis of enterohaemorrhagic Escherichia coli
O157:H7 in planta
Louise E. Birse1, Robert Jackson1,2, Carol Wagstaff1,2, Ian Toth1,
Simon Andrews1,2, Nicola Holden1
1
The James Hutton Institute, Dundee, UK, 2University of Reading, Reading, UK
Enterohaemorrhagic Escherichia coli (EHEC) are a group of food and
contact-borne pathogens responsible for haemorrhagic colitis. The
bacteria can be transmitted not only through contaminated meat but
also by plants, which can be used as an alternative host. Four different
plant species, commonly associated with EHEC outbreaks, were infected
with EHEC O157:H7 isolates Sakai and TUV 93-0 over ten days to
assess the colonisation potential of the bacteria in both the phyllosphere
and rhizosphere of plants. The rhizosphere was found to sustain a
higher population level of bacteria over time in comparison to the
phyllosphere, yet both strains were unable to grow in response to root
exudates. Further to this, gene expression changes of Sakai in response
to plant extracts from spinach cultivar Amazon were analysed by whole
transcriptome analysis. 17% of genes showed significant change in their
expression when the bacteria were exposed to leaf lysates, whereas 7%
changed in response to root exudates. 383 genes were identified that
had differing expression levels to both leaf lysates and root exudates,
many of which were metabolic genes.
Groups of differentially regulated genes were selected for further study
60
Poster abstracts
via qPCR for confirmation of expression levels and mutagenesis to assess
their functionality.
MA02/03
Leucobacter strains are diverse natural pathogens of Caenorhabditis
Laura C Clark1, Marie-Anne Félix2, Maria J Gravato-Nobré1,
Jonathan Hodgkin1
1
University of Oxford, Oxford, UK, 2IBENS, Paris, France
The genus Leucobacter was established in 1996 and currently comprises
13 recognised species of Gram-positive coryneform. Several Leucobacter
species (L. chromiireducens subsp. solipictus; L. iarius; and the related
Microbacterium nematophilum) have been found in association with
nematodes and cause varying degrees of morbidity and mortality in the
host. Leucobacter strains Verde1 and Verde2 were found as a double
infection of a free-living nematode living on rotting banana trunks in Cape
Verde and this double infection was subsequently transferred to the
laboratory strain Caenorhabditis elegans N2. Strain Verde1 adheres densely
to the nematode cuticle giving a ‘fur coat’ appearance, and is non-lethal.
Verde2 elicits a strong but futile tail-swelling response and causes rapid
death in infected nematodes. However, nematodes carrying mutations that
render them resistant to killing with Verde2 are often lethally susceptible
to Verde1 infection, likely as a result of changes to the cuticle. We report
progress made on the detailed characterisation of Leucobacter strains
Verde1 and Verde2 and the dissection of their interaction with C. elegans.
MA02/04
Adaptation of the intracellular metabolism of Salmonella Typhimurium
to the host cell environment
Amanda C Hopper1, Steven D Bowden2, David J Kelly3,
Arthur Thompson1
1
Gut Health and Food Safety, Institute of Food Research, Norwich
Research Park, Colney, Norwich, NR4 7UA, UK, 2Department of Biology
& Biochemistry, University of Bath, Claverton Down, Bath, BA2 7AY, UK,
3
Department of Molecular Biology and Biotechnology, University of Sheffield,
Sheffield, S10 2TN, UK
Bacteria generate ATP either by substrate-level phosphorylation (SLP)
via glycolysis, or oxidative phosphorylation (OXPHOS) using ATP
synthase. Previous work suggested that intracellular Salmonella uses
SLP and OXPHOS to differing extents depending on its localisation
within epithelial cells or macrophages. To investigate this further,
different mammalian cell lines were challenged with metabolic mutants
of Salmonella Typhimurium 4/74. The transimmortalised murine small
intestinal (mIC-c12) and human cervical (HeLa) epithelial cell lines were
tested, as well as a macrophage-like murine cell line (RAW264.7) and
an adherent version of the human macrophage-like Thp-1 (Thp-1A).
Phosphofructokinase, encoded by pfkA and pfkB, is essential for glycolysis.
An S. Typhimurium pfkAB mutant was highly attenuated within mIC-c12,
Thp-1A and RAW264.7 cells, implying that SLP is an important source
of ATP for S. Typhimurium in these cell lines. A mutant lacking the
atpIBEFHAGDC operon (encoding ATP synthase) was slightly attenuated
in mIC-c12 cells, but highly attenuated in Thp-1A cells. Intriguingly these
results suggest that the ATP synthase of S. Typhimurium fulfils different
requirements depending on whether Salmonella resides within epithelial
cells or macrophages. These requirements and their implications for the
metabolism of S. Typhimurium within host cells are discussed.
MA02/05
Linking metabolism with acid survival: the GABA shunt in Listeria
monocytogenes
Conor Feehily1, Kimon-Andreas Karatzas2, Conor P O’Byrne1
1
Bacterial Stress Response Group, Microbiology, School of Natural Science,
National University of Ireland, Galway, Galway, Ireland, 2School of Food
Biosciences, University of Reading, Reading, UK
The food-borne pathogen Listeria monocytogenes utilises the glutamate
decarboxylase (GAD) system to help protect itself from a decrease
in pH. The GAD system results in the formation of γ-aminobutyric
acid (GABA). This can either be removed from the cell or accumulate
inside. The GABA shunt pathway can convert GABA to succinate and
although it has been studied in other bacteria such as Escherichia coli it
has not been described in L. monocytogenes. Using protein extracts, we
have identified the enzyme activities for the steps of the GABA shunt
in L. monocytogenes. Furthermore we have identified the genes likely
to be involved in the pathway, argD and lmo0913. Deletion of either
of these genes affects the ability of L. monocytogenes to survive at low
pH. Deletion of lmo0913 also increases the levels of GABA produced
in response to acid treatment, consistent with its role in metabolising
intracellular GABA. The deletion of lmo0913 however does not
affect the bacteria’s ability to survive within the acidic environment of
human THP-1 macrophages. Taken together with work carried out
on the upstream GAD system it appears that in L. monocytogenes the
metabolism of intracellular GABA plays a important role in acid survival.
MA02/06
Regulation of the Rid system in Campylobacter jejuni, a novel
mechanism of defence against antimicrobial peptides
Michael A White, Halah Al-Haideri, Andrew Hitchcock, Edward
Guccione, Melanie Stapleton, John B Rafferty, David J Kelly
The University of Sheffield, Sheffield, UK
The Resistance to host Innate Defences (Rid) system in the food-borne
human pathogen Campylobacter jejuni protects this bacterium against
killing by a variety of antimicrobial peptides which may be produced
by the host (see poster by Al-Haideri et al). A small helix-turn-helix
DNA-binding protein, RidR, is hypothesised to control expression of
the rid structural genes; a ridR null mutant is hypersensitive to killing by
antimicrobial peptides. We have carried out a microarray comparison of
wild-type and ridR null mutant strains and thus identified the components
of the RidR regulon. RidR has been overproduced and purified from E.
coli and used to confirm binding to the promoter regions of regulated
genes indicated by the microarray analysis. The RidR binding site is an
inverted repeat sequence that has been identified by footprinting and
site-directed mutagenesis. An additional gene, ridS, encodes a potential
sensor protein that may interact with RidR. A model has been developed
to explain how RidSR might mediate increased resistance when C. jejuni
is challenged with potentially lethal host-derived antimicrobial peptides.
MA02/07
The Rid system: a novel type of defence mechanism against host
derived antimicrobial peptides in Campylobacter jejuni
Halah Al Haideri1, Andrew Hitchcock1, Edward Guccione1,
Matthew Cliff1, Mark P Stevens2, John B Rafferty1, David J Kelly1
1
The University of Sheffield, Sheffield, UK, 2The Roslin Institute, Edinburgh, UK
Bacterial pathogens must resist a range of host innate immune defences
if they are to establish a successful infection. A key part of their strategy
is resistance to host derived cationic antimicrobial peptides (CAMPs),
which bind to bacterial membranes to cause ion-leakage and cell death.
We have discovered a previously unknown type of defence system
involved in Resistance to host Innate Defences (the ‘Rid’ phenotype) in
the food-borne human pathogen Campylobacter jejuni. The Rid system
consists of three structural proteins, RidM (an inner membrane protein),
RidL (an outer membrane protein) and RidP (a periplasmic protein).
Knockout mutants in these genes show varying degrees of sensitivity
to a range of model, human and chicken CAMPs. We have solved the
structure of the key outer membrane protein in this system (RidL) and
shown it to consist of a highly unusual beta-sheet scaffold that binds
CAMPs. Overproduction and targeting of RidL to the outer membrane
of E. coli promotes resistance to CAMPs. RidL homologues are encoded
in the genomes of a number of unrelated bacterial pathogens, suggesting
at least this component of the system may represent a more widespread
but hitherto unrecognised mechanism of CAMP resistance.
61
Poster abstracts
MA02/08
Campylobacter phase variation and its impact on immunity and vaccine
development
Lea Lango-Scholey1, Michael A Jones1, Christopher D Bayliss2
1
School of Veterinary Medicine and Science, University of Nottingham, Sutton
Bonington, UK, 2Department of Genetics, University of Leicester, Leicester, UK
Campylobacter is the most common bacterial cause of infectious
gastroenteritis, with costs to the UK economy in the region of £500-600
million per annum. The main source of disease is contaminated poultry,
and Campylobacter asymptomatically colonise the gastrointestinal tract
of chickens to very high levels. One aspect of Campylobacter biology
that has received limited attention is phase variation (PV). The genome
of C. jejuni strain 11168 contains multiple loci that are subject to PV due
to long polyG repeat tracts and are involved in modification of capsule,
lipo-oligosaccharide and flagella. These surface structures are often
targeted by host adaptive immunity and hence PV may mediate evasion
of immune responses. The aim of this project is to investigate whether
PV plays a role in immune avoidance, host colonisation, and the variability
of vaccine responses. We show how small population bottlenecks may
influence the combinations of expression states of the phase variable
genes, by measuring changes in repeat tracts of all 27 polyG tracts using
GeneScan (fragment length) analysis. The utility of this methodology is
discussed as a technique for analysing phase variable changes of C. jejuni
to host interfaces and how non-selective bottlenecks may influence this
adaptive process.
MA02/09
Campylobacter jejuni at the host–pathogen interface: the role of
periplasmic chaperones in the biogenesis of outer membrane proteins
Shadi A Zakai1, Francis Mulholland2, David J Kelly1
1
The University of Sheffield, Sheffield, South Yorkshire, UK, 2Institute of Food
Research, Norwich, UK
Campylobacters are the leading cause of human bacterial gastroenteritis
worldwide. The outer membrane (OM) is the most important factor in
their interaction with the host. The mechanism by which OM proteins
are transported and correctly assembled in the OM remains unclear.
Here, we investigated the role of periplasmic chaperones in C. jejuni
NCTC 11168, focusing on Cj0694, a PpiD homologue which may
have a chaperone role for periplasmic proteins or the translocation of
OMPs across the periplasm. We examined the biochemical functions
of this protein, and the phenotypic characterisation of a null mutant
in the cognate gene. Cj0694 was successfully overexpressed in E. coli
as a his-tagged protein and purified by Ni-NTA and ion-exchange
chromatography. Chaperone domain activity was clearly detected in
aggregation assays using the model protein rhodanese, and its peptidylprolyl cis/trans isomerase (PPIase) domain activity was demonstrated by
an enhanced refolding rate of denatured ribonuclease T1 in fluorescence
recovery assays. Evidence that Cj0694 plays a role as a periplasmic
chaperone in C. jejuni was suggested by an altered accumulation of
proteins in the periplasm of the cj0694 null mutant. Proteomic analysis of
OM and periplasmic preparations using 2D-gels and mass spectrometry
were used to identify client proteins.
MA02/10
Degradation of benzo[a]pyren by bacterial isolates from human skin
Juliane Sowada, Tewes Tralau, Andreas Luch
Federal Institute for Risk Assessment, Department for Product Safety, MaxDohrnstr. 8-10, D-10589 Berlin, Germany
The human skin is an extensive host-microbe interface and as such
it is a breeding ground to a most diverse and dense population of
microbial commensals. However, at the same time it is the organ
most exposed to potentially toxic polycyclic aromatic hydrocarbons
(PAHs) such as benzo[a]pyrene (BaP). In eukaryotes cytochrome P450mediated activation of the latter is a model for metabolism-mediated
carcinogenesis. Meanwhile, the oxidative degradation of BaP by microbes
is less well studied. The respective intermediates are often unknown let
alone characterised toxicologically. This study now reports the isolation
of BaP-degrading microbes from several habitats on human skin.
Degradation of BaP proceeded via different oxygenative pathways and
was complete in 4 out of 10 isolates. Substrate metabolism involved
several transcripts, two of them encoding enzymes with sequence
similarities to biphenyldioxygenase (bphA). Analysis of the 16s-DNA
sequences further showed that BaP-degrading isolates comprised Gram+
as well as Gram-bacteria, with Micrococci being predominant. This is the
first time that BaP-degrading microbes are identified on the human skin,
their presence being seemingly unrelated to any pre-exposure with BaP.
Further studies shall now clarify the extent of carcinogenic metabolite
formation.
MA02/11
Analysis of activation of pro-MMP-activating sequences in vitro by
gingipains from Porphyromonas gingivalis in the context of virulence in
aspiration pneumonia
Karolina Plaza1, Tomasz Kantyka1, Jan Potempa1,2
1
Department of Microbiology, Faculty of Biochemistry, Biophysics and
Biotechnology, Jagiellonian University, 30-387 Krakow, Poland, 2University of
Louisville Dental School, Department of Periodontics, Endodontics and Dental
Hygiene, Louisville, KY 40202, USA
Gingipains are responsible for over 80% of extracellular proteolytic
activity of Porphyromonas gingivalis, the main etiological agent of
periodontitis, also implicated in the development of rheumatoid
artritis and aspiration pneumonia. These pathological conditions
are characterised by massive destruction of tissues, which could
be associated with increased and uncontrolled activity of matrix
metalloproteinases. Indeed, due to the intensification of the inflammatory
response and direct tissue injury, excessive activity of MMPs seems
to be an attractive target for a pathogen that requires nutrients for
growth, additionally favoring dissemination by the connective tissue
damage. Since the mechanism of aspiration pneumonia development
remains unknown it can be speculated that in this case the gingipainrelated upregulation of MMPs activity occurs, owing to the fact that
such phenomenon is observed during lung infections with S. aureus
and P. aeruginosa. Presented study is aimed at initial analysis of ability of
gingipains to activate zymogens of MMPs employing novel method based
on soluble, proteolytically-resistant carrier protein with exposed amino
acid sequence representing investigated cleavage site. This approach will
enable better understanding of gingipains role in aspiration pneumonia,
moreover broaden our knowledge on virulence of P. gingivalis.
MA02/12
Bile aspiration: an emerging underlying cause of chronic respiratory
infection in cystic fibrosis
F. Jerry Reen, David Woods, Marlies Mooij, Claire Adams,
Fergal O’Gara
University College Cork, Cork, Ireland
Chronic respiratory infections are a major cause of morbidity and
mortality, most particularly in Cystic Fibrosis (CF) patients. The recent
finding that gastro-esophageal reflux (GER) frequently occurs in CF
patients led us to investigate the impact of bile on the behaviour of
Pseudomonas aeruginosa and other CF-associated respiratory pathogens.
Bile increased biofilm formation, Type Six Secretion, and quorum sensing
in P. aeruginosa, all of which are associated with the switch from acute to
persistent infection. Bile also modulated biofilm formation in a range of
other CF-associated respiratory pathogens, including Burkholderia cepacia
and Staphylococcus aureus. Furthermore, bile negatively influenced Type
Three Secretion and swarming motility in P. aeruginosa, phenotypes
associated with acute infection. Taken together, these findings suggest
that GER-aspired bile may have a major influence on behaviour and
biodiversity in the respiratory tract and lungs of CF and other patients.
Therefore, innovative and more effective strategies will be required
62
Poster abstracts
and also clinically latent TB infections which can persist for the lifetime
of the human host. As a pathogen which lacks conventional virulence
factors metabolic adaptation is a key determinant of this pathogenic
strategy. Phosphoenolpyruvate carboxykinase (PEPCK) alongside
pyruvate carboxylase (PCA) and malic enzyme (MEZ) control the
metabolic link between gluconeogenesis and the TCA cycle represented
by the PEP-pyruvate-oxaloacetate anapleurotic node. We hypothesise
that the combined activities of PEPCK, PYC and MEZ, their regulation
and specific contribution to growth is critical to the survival of M.
tuberculosis in the host. As PCA was reported to be an essential gene
we generated tetracyline-regulatable promoter-based conditional
mutants of PCA in both wild type and a PEPCK deleted strain of M.
tuberculosis. Gene expression was controlled using anhydrotetracycline.
Our initial results demonstrated that PCA is dispensable for growth
in vitro in media containing glucose and glycerol as the carbon
source. These mutants provide a mechanism to control and study the
PEP-pyruvate-oxaloacetate anapleurotic node of metabolism of M.
tuberculosis in vitro and in vivo.
to treat what may be a major underlying cause of chronic respiratory
disease.
MA02/13
Therapeutic doses of thiamine compounds affect the antioxidative
potential and the carbohydrate metabolism of human pathogen
Candida albicans
Natalia Wolak, Maria Rapala-Kozik
Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University,
Krakow, Poland
Vitamin B1 (thiamine) is an essential molecule for all living organisms,
mainly owing to the cofactor function of thiamine diphosphate (TDP) in
carbohydrate metabolism. Recent findings, however, have also suggested
another important role of thiamine in alleviating stress responses. In
particular, the antioxidative properties of thiamine have been studied in
Saccharomyces cerevisiae but no comparable data are available regarding
pathogenic yeasts. Such an analysis is especially important in the context
of routine application of high doses of thiamine and its derivatives in the
treatment of various neurological diseases.
This study aimed primarily at investigating the stress response of human
pathogen Candida albicans. After exposure to hydrogen peroxide,
significant changes in the carbohydrate metabolism and the antioxidative
potential of fungal cells have been observed. We confirmed an
increased demand for TDP-dependent enzymes, correlated with a
higher expression of thiamine pyrophosphokinase -the TDP-producing
enzyme. We showed also that thiamine compounds can affect the stress
response through a decrease of the expression of antioxidant enzymes,
as well as enzymes involved in DNA repair.
Taken together, our results suggest that therapeutic doses of thiamine
compounds significantly affect the metabolism of pathogenic yeast
C. albicans although the mechanism of this effect require further
investigation.
MA02/14
Serum amyloid A as early biomarker in infectious bovine subclinical
mastitis
Geetanjali Singh, Bishrutee Bhardwaaj
College of Veterinary & Animal Sciences CSK Himachal Pradesh Agriculture
University, Palampur, India
Subclinical bovine mastitis leads to heavy losses due to reduced milk yield
and quality. This is caused by a number of pathogenic bacteria such as
Staphylococcus, Escherichia coli, Streptococcus, Corynebacterium and
Proteus to name a few. Early diagnosis, treatment and prevention are
crucial for reducing the economic losses. However, unlike clinical mastitis,
subclinical mastitis is difficult to diagnose due to absence of clinical
indicators and lack of suitable diagnostic test. The classical methods of
diagnosis such as somatic cell count in milk and microbial culturing are
cumbersome and time consuming and result in delays in diagnosis. In
this study, potential biomarker proteins appearing early and specifically
in subclinical mastitis due to udder infection were assessed. It was found
that appearance of serum amyloid A (SAA) transcript in the milk was
associated with the presence of microbes & increased SCC. Further
the SAA was demonstrated in the milk of cows affected by subclinical
mastitis using 2-D gel electrophoresis followed by MALDI-MS and
ELISA. The SAA transcript and protein were not found the milk of cows
suffering from a number of systemic inflammatory diseases, thus making
SAA as intramammary-specific biomarker in subclinical bovine mastitis.
MA02/15
The role of pyruvate carboxylate in Mycobacterium tuberculosis growth
Irene Gobe, Ricardo Balhana, Dany J V Beste
University of Surrey, Guildford, UK
Mycobacterium tuberculosis is an unusual bacterial pathogen which has
the remarkable ability to cause both acute life threatening tuberculosis
MA02/16
Identification and characterisation of sugar binding adhesins in
the human gut metagenome using bioinformatics and functional
metagenomics
Conor O’Byrne, Aoife Boyde, Lokesh Joshi, Christy Agbavwe
NUI, Galway, Galway, Ireland
In the last decade, an extensive effort has been made to describe the
adhesion of bacteria to components of the human intestinal mucosa.
However, information on the surface molecules mediating this
adhesion and their corresponding receptors is less advanced. In this
project, bioinformatics and functional metagenomic approaches were
undertaken to construct metagenomic libraries used for screening of
novel proteoglycan binding elements encoded in the gut microbial
metagenome. Two types of libraries: small-insert (less than 8kb) libraries
in plasmid vectors and large-insert (up to 40kb) libraries in fosmid
vectors. Restriction mapping and DNA sequencing was performed on
multiple clones from both types of library to determine library species
diversity. Functional screens are currently underway to detect clones
that have novel adhesion properties. In this study, in silico analysis of the
human gut metagenome targeting 55 bacterial species was performed,
resulting in the identification of five putative glycan binding adhesins.
Strategies are currently in place to clone and functionally characterise
these adhesins. Preliminary findings of the study will be presented.
MA02/17
DsbA and DsbB as suppressors of biofilm formation in Salmonella
enterica serovar Typhimurium
Naeem Anwar1, Ute Römling1, Mikael Rhen1
1
Department of Microbiology, Tumour and Cell biology, Karolinska Institutet
Box-280, SE-17 177 Stockholm, Sweden
Salmonella enterica serovar Typhimurium can make biofilms, complex
communities of microorganisms in which they attach to and grow on
either biotic or abiotic surfaces making them resistant to antibiotic
treatment. Many genes involved in oxidative stress tolerance show
increased expression in a biofilm. Here, we report the role of a
periplasmic protein disulfide isomerase, DsbA and cytoplasmic
membrane disulfide oxidoreductase DsbB in biofilm formation in S.
Typhimurium. Deletion mutants of dsbA and dsbB resulted in increased
biofilm formation on Congo red plates but failed to make pellicle on the
air-liquid interface. The increased biofilm formation is associated with an
enhanced expression of the biofilm master regulator CsgD and CsgD
regulated curli fimbrial subunit CsgA. We also checked for the effect
of exogenous reductive and oxidative stresses on biofilm formation.
The dsbA and dsbB mutants showed higher production of extracellular
mucilaginous matrix under reductive stress whereas oxidative stress
completely abrogated biofilm formation in a dose dependent manner
63
Poster abstracts
without affecting the viability of bacteria. From these results, we conclude
that DsbA and DsbB act as suppressors of biofilm formation by affecting
the expression of CsgD and CsgA in S. Typhimurium.
MA02/18
Metabolic flux analysis of Pseudomonas syringae pv. tomato in virulence
gene inducing and non-inducing conditions
Sarah L McCraw, Shyam K Masakapalli, Nicholas J Kruger,
R George Ratcliffe, Gail M Preston
Department of Plant Sciences, University of Oxford, Oxford, UK
The metabolic interface between plants and their pathogens is a
key determinant in the infection process. In recent years advances in
metabolomics technologies have allowed novel insights into metabolic
pathways and fluxes in both plants and pathogens, and in the last few
years attention has turned to the metabolic interactions between them.
Research in this area may provide crucial information for the development
of more effective strategies for the control of crop diseases. My research
is focused on the interaction between Pseudomonas syringae pv. tomato
DC3000 and its host, tomato, and the development of protocols for
studying the metabolic flux of P. syringae in conditions which cause the upregulation of virulence genes. We will present work comparing metabolic
flux through central carbon metabolism in the plant pathogen P. syringae
with the non-pathogenic pseudomonad Pseudomonas fluorescens SBW25,
and the changes in metabolic flux associated with growth in virulence
gene inducing and non-inducing environments.
MA02/19
A novel host-responsive sensor mediates virulence and type III
secretion in Pseudomonas aeruginosa via interplay between the Anr/
Nar and Gac-Rsm pathways under low oxygen conditions
Julie O’Callaghan, F Jerry Reen, Claire Adams, Pat G, Casey,
Cormac G M Gahan, Fergal O’Gara
Sensitive sensory mechanisms are instrumental in affording Pseudomonas
aeruginosa the capacity to establish diverse yet severe human infections,
which can manifest in long-term untreatable disease, most especially in
cystic fibrosis (CF) patients. Although a number of factors are recognised
as playing a role in early infection, very little is known regarding the
sensors involved in this process. A steep oxygen gradient within the
mucus of the CF lung combined with the biofilm mode of bacterial
growth forces respiratory pathogens to adapt to varying oxygen
availability. This study presents the novel finding that the Pseudomonas
aeruginosa response to limiting oxygen stress includes induction of
its type III secretion system (T3SS), which subsequently contributes
towards host cell cytotoxicity. We identified P. aeruginosa PA3191
as a novel host-responsive sensor that plays a key role during P.
aeruginosa-host interactions and is required for modulation of the type
III secretion system (T3SS) in response to host cells and limiting oxygen
in vitro. PA3191 (designated GtrS) acted in concert with the response
regulator GltR to regulate the OprB transport system and subsequently
carbon metabolism including pyruvate dehydrogenase. Induction of the
T3SS is dependent on Anr, and this is mediated through direct NarL
transcriptional repression of the sRNAs rsmY and rsmZ, allowing free
RsmA protein to positively regulate the T3SS. This study reveals a
novel interplay between the Anr-NarL and RsmAYZ regulatory circuits,
and introduces RsmA as an important regulator during P. aeruginosa
adaptation to a low-oxygen environment.
Co-infections and co-colonisation
MA03
MA03/01
Micrococcus as skin probiotic
M A Alqumber1, J R Tagg2
1
Albaha Unversity, Albaha, Saudi Arabia, Saudi Arabia, 2University of Otago,
Otago, New Zealand
Micrococcus luteus strain Q24 was isolated from the skin of a
healthy 22 year old male. The strain was found to be a promising
potential skin probiotic due to its strong in vitro inhibitory activity
against anti-methicillin resistant Staphylococcus aureus and another
major skin pathogens, including many species of the Staphylococcus,
Propionibacterium, Corynebacterium, Streptococcus, Micrococcus, Prevotella,
Porphyromonas, Lactococcus and diphtheroids genera. The strain was also
found to persist in the skin of its host for 8 years. Strain Q24 inhibitor
was purified by HPLC and further analysed with MALDI-TOF. To study
its efficacy, a saline suspension of M. lutes Q24 with a concentration of
at least million CFU per dose was inoculated by swabbing one axilla of
each of 17 subjects. The inoculated strain has been shown to persist for
at least 24 hr and to reduce body malodor and corynebacteria count.
Applying the potential probiotic strain to patients with athlete’s foot
caused the itching to disappear within 10 minutes and a complete cure
within three days. Now with patent protection in place, strain Q24 is
under consideration for more applications.
MA03/02
Dynamic response to social cheats in Pseudomonas aeruginosa
Freya Harrison
University of Nottingham, Nottingham, UK
The social lives of bacteria have received increasing scrutiny in recent
years. We know that interactions between cells of the same and
different species can determine bacterial functional ecology and
pathogenicity, and we have identified aspects of population structure
that lead to evolutionary changes in social behaviour. However, there
hase been little explicit exploration of how individual cells respond
physiologically to the social strategies employed by their neighbours,
and the implications this may have for social evolution and for the
concomitant evolution of virulence in multi-species or multi-genotype
infections. I used non-growing populations of the opportunistic pathogen
P. aeruginosa to explore how cells of this species respond to the
presence of social ‘cheats’ that do not produce siderophores – a wellstudied bacterial ‘public good’. I show that cells partially compensate for
the presence of cheats by increasing siderophore production, using a
response rule previously shown to be critical for maintaining cooperation
in a radically different social context (parental cooperation in animals).
Co-culture with cheats is necessary to maintain this response rule over
evolutionary time. These results demonstrate the remarkable flexibility of
bacterial social behaviour and open a new avenue for research into the
evolution of social response rules in microbes.
MA03/03
Microbial communities and interactions in chronic lung infection in
cystic fibrosis patients
Jo L Fothergill1, Lucy Martin1, Xuan Liu2, Kevin W Southern3,
Craig Winstanley1
1
Clinical Infection, Microbiology and Immunology, Institute of Infection and
Global Health, University of Liverpool, Liverpool, UK, 2Centre for Genomic
64
Poster abstracts
Research, Institute of Integrative Biology, University of Liverpool., Liverpool,
UK, 3Department of Women and Children’s Health, Institute for Translational
Medicine, University of Liverpool., Liverpool, UK
Bacteria in the natural environment form complex, interacting
communities. This is also true for bacterial infections of animals and
humans, where much of the diversity that we see may be driven by
microbes interacting with each other as well as with the host. The
Cystic Fibrosis (CF) lung facilitates the cohabitation of diverse microbial
organisms and we are only just beginning to understand the extent of
this diversity. In order to characterise the CF lung microbiome, matched
sputum and bronchoalveolar lavage (BAL) samples from children were
sequenced using next generation sequencing of 16S rRNA sequences.
This enabled the microbial community in each sample to be identified
and compared to determine how representative sputum samples are
of the lower lung. Furthermore, BAL samples from the left and right
lung were also compared. Considerable differences in the microbial
population could be found between samples. These data have been
used to develop co-culture and multispecies biofilm models in which
microbial interactions can be investigated. Pathogens such as Pseudomoas
aeruginosa, Staphylococcus aureus, Burkholderia sp and Candida albicans
have been studied in this system. Understanding these complex
interactions may uncover novel therapeutic targets and ultimately lead to
altered CF patient management.
MA03/04
The ecological implications of synergistic quorum sensing and the
implications of cross-species interactions
James. R Gurney, Steve. P Diggle
The University of Nottingham, Nottingham, UK
Research into quorum sensing (QS) has traditionally focused on
understanding mechanism, with less attention paid to evolutionary
considerations of QS. Many bacterial species produce more than one
QS signal and a key question that receives little attention is why make
multiple QS signal molecules? Do molecules have overlapping roles
in gene regulation in a non-additive manner? Here we test this using
the opportunistic pathogen Pseudomonas aeruginosa, which produces
two major N-acylhomoserine lactone (AHL) signals. We show that
interactions of two signal molecules at the level of gene transcription
can lead to synergistic interactions, resulting in (1) drastic up-regulation
of QS-controlled genes and (2) distinct QS regulons. The ability to
produce a synergistic response is not limited to the cognate AHL signals.
Certain QS receptors show affinity for non-cognate AHLs and this,
in combination with synergistic interactions, may lead to cross-species
interactions. Our findings should be viewed as an experimental first step
in understanding why it may be beneficial to produce and respond to
multiple signal molecules, and suggests that ecological considerations of
QS will help further our understanding of the function and importance of
QS in natural environments and infections.
MA03/05
The role of bacteriophages as drivers of bacterial phenotypic evolution
Emily V Jones1, Chloe E James2, Aras Kadioglu1, Michael A Brockhurst3,
Craig Winstanley1
1
University of Liverpool, Liverpool, UK, 2University of Salford, Manchester, UK,
3
University of York, York, UK
Pseudomonas aeruginosa is an opportunistic pathogen that causes chronic
lung infections in individuals with cystic fibrosis (CF), where it grows in
biofilms. Over the course of chronic infection, P. aeruginosa populations
accumulate mutations, becoming increasingly diverse and exhibiting
phenotypes such as mucoidy, auxotrophy and antibiotic resistance;
such phenotypes are associated with altered virulence. The Liverpool
Epidemic Strain (LES) was the first transmissible P. aeruginosa strain
identified, and is associated with a worsened prognosis. Strain LESB58
displays phenotypic traits such as biofilm hyperproduction and reduced
antimicrobial susceptibility. Genome sequencing revealed the presence
of multiple prophages, and signature-tagged mutagenesis revealed three
of these to be vital to the in vivo competitiveness of LESB58. Three LES
phages have been isolated through their ability to infect strain PA01. We
constructed a model of chronic infection using artificial sputum, in which
PA01 forms biofilms, as in the CF lung. PA01 biofilms were maintained
for 240 bacterial generations in the presence or absence of the LES
phages. End-point populations were assessed for a variety of phenotypic
traits, including auxotrophy, hypermutability and loss of twitching motility,
and phages were found to play a role in diversification of bacteria by
driving development of some of these phenotypic traits.
MA03/06
TiO2-Mo photocatalytic surfaces for use in the brewing industry: the
effect of brewery soil on antimicrobial activity
Leanne Fisher, Soheyla Ostovarpour, Peter Kelly, Joanna Verran
Process hygiene plays a major role in ensuring the quality of beer.
Photocatalytic surfaces can reduce fouling and by doping TiO2 with
transition metals like molybdenum (Mo), photoactivity can be improved
and surfaces become active under fluorescent light rather than UV
light. In this study, the ability of TiO2-Mo photocatalytic surfaces to
reduce numbers of brewery organisms in the absence and presence
of a brewery soil was investigated. TiO2-Mo thin films were prepared
on stainless steel using magnetron sputtering. Brewery soil was dried
onto surfaces and organisms isolated from the brewery environment
were applied. Surfaces were irradiated under fluorescent light and viable
bacteria remaining on the surface were enumerated. Without soiling,
Pseudomonas fluorescens and Serratia marcescens counts reduced to 0
and <15cfu/cm2 respectively after 24h irradiation. Wickerhamomyces
anomalus (yeast) counts at 72h were reduced by 2 logs to >103cfu/
cm2. The addition of a brewery soil resulted in higher numbers of all
organisms remaining on the surface post-irradiation. The TiO2-Mo
surfaces were also active in the dark and could therefore have a dual
function, being photoactive and antimicrobial. The study highlights the
importance of regular cleaning in addition to exploitation of functional
surfaces that may reduce fouling.
MA03/07
The effect of Bacillus subtilis on Campylobacter in the caeca of broiler
chickens
Adrian Horton1, Dave Leemans1, Szymon Calus1, Sarah Gaunt2,
Rebecca Allen2, Justin Pachebat1, Michael Lee1
1
Aberystwyth University, Ceredigion, UK, 2Eminate, Leicestershire, UK
Campylobacter coli and C. jejuni are the leading cause of bacterial gastroenteritis in the industrialised world. Most poultry flocks are colonised
with Campylobacter spp. with 81.3% of poultry meat contaminated after
slaughter. Probiotics have been shown to reduce the load of pathogenic
bacteria. Bacillus subtilis strain KD1, when used as a feed supplement,
has been linked to the maintenance of a normal intestinal microflora
with an increase in lactobacilli, known for their anti-inflammatory and
anti-cancer properties, and a decrease in Escherichia coli. In this study we
investigated the effect of the GRAS registered B. subtilis as a probiotic
feed supplement on levels of thermotolerant Campylobacter spp. in
Ross 308 broiler chickens (n=200). Birds fed commercial feed (control)
65
Poster abstracts
carried greater loads of Campylobacter in the caeca than birds fed feed
supplemented with B. subtilis spores (intervention), except for day
36 where loads were similar. The results show that diet had an affect
on loads of Campylobacter present in the luminal contents of broiler
chicken caeca, with the greatest difference in Campylobacter loads seen
on days 14 and 21. This indicates that B. subtilis, when used as a food
supplement, has the potential to reduce levels of Campylobacter present
in chicken caeca.
MA03/08
Competition sensing: the social side of bacterial stress responses
Daniel Cornforth1,2, Kevin Foster2
1
Centre for Immunity, Infection, and Evolution, School of Biological Sciences,
University of Edinburgh; 2Department of Zoology, University of Oxford,
Oxford, UK
The fields of ecology and evolutionary biology have long recognised
two types of competition: exploitative competition that occurs indirectly
through resource consumption and interference competition whereby
one individual directly harms another. These two types of competition
have likely played a dominant role in the evolution of bacteria gene
regulation. We present the case that several of the major bacterial
stress responses detect this ecological competition by sensing nutrient
limitation (exploitative competition) as well as direct cell damage
(interference competition). We systematically review the bacterial stress
responses literature to evaluate the strength of the connection between
stress responses and competitive behaviour. A key prediction of our
perspective is that bacteria can benefit from counter attack when they
sense ecological competition but not when they sense abiotic stress. We
show that many bacteriocins and antibiotics are upregulated by stress
responses to nutrients limitation and cell damage but very rarely by heat
or osmotic stress responses which typically are not competition-related.
We present a model for bacterial competition sensing in which stress
responses, in combination with quorum sensing, are used to adaptively
respond to the degree of ecological competition.
New approaches to exploit
Streptomyces
MA05
MA05/01
Streptomyces coelicolor nucleoid proteins
Jane Moore, Anne Schumann, Beth Bradshaw, Michael McArthur
The genome of Streptomyces coelicolor encodes more than 20 gene
clusters encoding putative secondary metabolites. Many of these have
never been observed under laboratory culture conditions. This is a
common feature amongst Actinobacteria. It is therefore a priority to
unlock some of these cryptic pathways. It is hypothesised that some
of these silent gene clusters may be activated by altering the nucleoid
structure.
In eukaryotes the structure of chromatin is important for regulating
cellular differentiation by preventing access of transcriptional machinery
to specific chromosomal regions. This is a reversible process where
nucleoid proteins such as histones bind DNA. The DNA is wound
around histone proteins, compacting the DNA, resulting in gene
silencing. The histones are further modified by actylation and
methylation. This is a reversible process which alters the histones affinity
for DNA. As histone-like proteins and putative histone deacetylases
are encoded in the genome sequence of S. coelicolor M145 it is
hypothesised that these are involved in nucleoid structure and therefore
affect transcriptional regulation.
To gain a better understanding of the role nucleoid structure plays
in S. coelicolor the function of 2 histone-like proteins and 3 histone
deacetylases (HDACs) is being assessed, by deleting or over expressing
these genes.
MA05/02
Exploiting Streptomyces secondary metabolite biosynthesis using
computational approaches
MArnix H Medema1,2, Kai Blin3, Tilmann Weber3, Rainer Breitling2,4,
Eriko Takano1,4
1
Department of Microbial Physiology, University of Groningen, Groningen,
The Netherlands, 2Groningen Bioinformatics Centre, University of Groningen,
Groningen, The Netherlands, 3Mikrobiologie/Biotechnologie, Interfakultäres
Institut für Mikrobiologie und Infektionsmedizin, Eberhard Karls Universität
Tübingen, Tübingen, Germany, 4Faculty of Life Sciences, Manchester Institute
of Biotechnology, University of Manchester, Manchester, UK
The secondary metabolism of Streptomyces bacteria is a rich source
of bioactive compounds with potential pharmaceutical applications.
Computational methods have become more and more important for
exploiting this potential.
In order to devise effective strategies to make full use of the
accelerated rate of genome sequencing, we have constructed a
completely redesigned version 2.0 of antiSMASH (http://antismash.
secondarymetabolites.org)1. antiSMASH now offers options such as
analysing draft genomes composed of multiple contigs, identifying
subclusters associated with the biosynthesis of specific chemical moieties,
and predicting lantipeptide modifications.
We also introduce a complementary new software, MultiGeneBlast2,
for the comparative analysis of gene clusters or subclusters with either
the entire GenBank database or with any custom sequence database
designed by the user. An ‘architecture search’ option allows identification
of gene clusters with entirely novel combinations of enzymes.
These novel tools will empower experimental strategies to implement
gene clusters or subclusters in high-throughput synthetic biology
methodologies for activity screening and industry-scale production3,4.
References 1. Medema et al. (2011) Nucl. Acids. Res. 39: W339-W346; 2.
Medema et al. (2012) Mol. Biol. Evol., in revision; 3. Medema et al. (2011)
Nature Rev. Microbiol. 9: 131-137; 4. Medema et al. (2012) Nature Rev.
Microbiol. 10: 191-202.
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Poster abstracts
MA05/03
Functional study of an unusual tautomerase domain in the indigoidine
synthase from Streptomyces clavuligerus
A Ceniceros1, A Kovalchuk1, H Poddar4, M H Medema1, K Scherlach3,
G J Poelarends4, E Takano1,2
1
University of Groningen, Microbial Physiology, Groningen, The Netherlands,
2
University of Manchester, Manchester, UK, 3Hans-Knöl Institute, Jena,
Germany, 4University of Groningen, Department of Pharmaceutical Biology,
Groningen, The Netherlands
In silico analysis of Streptomyces clavuligerus genome[1] revealed a single
module NRPS indigoidine synthase (indC). Indigoidine is a blue pigment
first described in Erwinia chrysanthemi[2]. This NRPS has a unique
C-terminal tautomerase domain. Tautomerases convert molecules
into isomers that only differ in the position of one hydrogen atom.
[3]
. indC and indC-trunc (indC with the tautomerase domain truncated)
were expressed under the control the ermE* promoter in Streptomyces
coelicolor M1146[4]. Indigoidine was successfully produced in these strains
with a more intense pigmentation in the IndC-trunc strain. Indigoidine
produced by both strains was purified and NMR studies are being
performed to determine the role of the tautomerase domain in the
production of indigoidine. The tautomerase domain from indC was
also purified from E. coli and its activity was tested against all known
tautomerase substrates, but no activity was detected, except for
the unusual tautomerase substrate, (p-hydroxyphenyl)enolpyruvate.
Further analysis of this enzyme will be conducted in vitro, using purified
indigoidine.
References [1] Medema MH et al., Genome Biology and Evolution. 2010.
2:212-224; [2] Reverchon S et al., J Bacteriol. 2002. 184:654 -665; [3]
Poelarends GJ et al., Biochemistry. 2006. 45(25), 7700-7708; [4] GomezEscribano JP, Bibb MJ, Microb Biotechnol. 2011. 4(2):207-1
Netherlands, 2Faculty of Life Sciences, Manchester Institute of Biotechnology,
University of Manchester, Manchester, UK
γ-Butyrolactones play a role in the regulation of antibiotic production
(1). The first step in γ-butyrolactone (SCB) biosynthesis in Streptomyces
coelicolor A3(2) is proposed to be performed by ScbA, catalyzing
condensation of a glycerol derivative with a fatty acid derivative. The
SCB skeleton is further modified by two reductions, proposed to be
performed by ScbB (6-keto-reductase) and ScbC (reduction at C2-C3).
To characterise the SCB biosynthesis, we attempted to express, purify
and crystalise ScbA. However, we did not succeed in the overexpression
of ScbA in various hosts, therefore we will focus now on overexpression
of close homologues of scbA in E. coli. We have also overexpressed scbB
in E. coli. and aim to optimise expression and obtain a crystal structure.
Futhermore, to study a possible dual role of ScbA, both in biosynthesis
of SCBs and in regulation of its own transcription, additional scbA point
mutants from those created previously (2), in the conserved sites, will
be obtained and characterised in vivo. Preliminary results show that
the regulatory function of ScbA can be uncoupled from the enzymatic
function.
References 1) Takano, E. et al. 2001. Mol. Microbiol. 41:1015-1028; 2)
Hsaio, N.H. et al. 2007 Microbiology 153:1394-1404.
MA05/04
Biosynthesis of novel natural products from Streptomyces venezuelae
John D Sidda, Orestis Lazos, Lijiang Song, Anne-Claire Olivan,
Christophe Corre
Department of Chemistry, University of Warwick, Coventry, UK
ArpA-like transcriptional repressor proteins regulate biosynthesis of
a diverse range of Streptomyces natural products, primarily antibiotics
such as Streptomycin.1 Deletion of genes encoding for transcriptional
repressors is a powerful strategy for overproduction and discovery of
novel natural products.2
Gaburedins -a novel class of ureido-linked GABA derivatives -are
produced by a Streptomyces venezuelae strain lacking the gabR gene
encoding for a putative transcriptional repressor protein. The cryptic,
normally silent gab cluster located adjacent to gabR is proposed to be
responsible for gaburedin biosynthesis.
Metabolic profiles of the S. venezuelae wild type and a number of mutant
strains have been compared by LC-MS to identify key biosynthetic genes,
and a hypothetical biosynthetic pathway has been proposed. Precursordirected studies have been employed to overproduce gaburedins and to
generate novel gaburedins in vivo. Synthesis of authentic standards of the
gaburedins has also begun in order to confirm structural characterisation
data and to begin biological testing.
References 1 Y. Onishi, S. Kameyama, H. Onaka and S. Horinouchi, Mol.
Microbiol. 1999, 34, 102; 2 B. Aigle and C. Corre, Methods in Enzymol.
2012, 517, 343-366
MA05/05
The γ-butyrolactone (SCB) biosynthesis genes of Streptomyces
coelicolor A3(2)
Mirjan Petrusma1, Fabrizio Biuso1, Aysegul Erdem1, Ruben Peters1,
Eriko Takano1,2
1
Department of Microbiology, Groningen Biomolecular Sciences and
Biotechnology Institute (GBB), University of Groningen, Groningen, The
MA05/06
Transcriptome analysis of Streptomyces arpA-liketranscriptional
repressor mutants
Vincent Poon, Christophe Corre, Jonathan Moore
University of Warwick, Coventry, UK
Since the genome sequencing of several actinomycetes, a large
number of gene clusters proposed to be involved in the biosynthesis
of secondary metabolites have been identified through bioinformatic
analyses [1]. Secondary metabolite gene clusters are heavily regulated at
the transcriptional level and ArpA-like transcriptional repressor proteins
represent one class of transcriptional factors that control secondary
metabolism [2].
Preliminary bioinformatic analyses have identified highly conserved
palindromic sequences within signalling gene cluster and adjacent
secondary metabolite gene clusters [3]. The ArpA-like transcriptional
repressor proteins are proposed to interact with these conserved
palindromic sequences to perform their regulatory activity [4].
To date, predicting where these ArpA-like transcriptional repressor
proteins bind to is extremely challenging and by using a transcriptomic
approach, we expect to identify the broader picture of the biological role
of these transcriptional repressors.
References [1] Challis, G. L. (2008). Microbiology. 154 (6), 1555 -1569;
[2] Hopwood, D. A. (2007). New York, Oxford University Press; [3]
O’Rourke, S. et al. (2009). Mol. Microbiol. 71 (3), 763 -778; [4] Onaka, H.
et al. (1997). Mol. Microbiol. 24 (5), 991-1000
MA05/07
The role pyruvate kinase in growth and antibiotic production of
Streptomyces coelicolor
Jana K Hiltner1, Lorena Fernández-Martínez2, Iain S Hunter1,
Hrvoje Petkovic3, Paul A Hoskisson1
1
SIPBS, University of Strathclyde, Glasgow, UK, 2John Innes Centre, Norwich,
UK, 3Acies Bio Ltd, Llubljana, Slovenia
Streptomycetes are important producer of bioactive secondary
metabolites for use as antibiotics, immunosuppressants or anticancer
drugs. One group of particular interest is the polyketides, which are
biosynthesised through repeated condensation of acetyl CoA units,
through claisen-type reactions. The Phosphoenolpyruvate-PyruvateOxaloacetate (PEP-PYR-OAA) node is a major control point of carbon
flux as well as providing precursors for polyketides thus offering potential
to investigate its role in antibiotic production. A transposon-mutagensis
approach has been used to disrupt the pyruvate kinase genes (SCO2014
67
Poster abstracts
(pyk1), SCO5423 (pyk2)) in S. coelicolor M145 and a phenotype of
the mutants was investigated on several media (liquid YEME, SMM +
N-Acetylglucosamine/Pyruvate). No difference in the growth rate was
observed on complex medium, but a change in the antibiotic production
was observed. Furthermore the pyk2 mutant grows slower on minimal
medium but with increased antibiotic production. RT-PCR data indicate
a constant expression of pyk2 on MS agar, whereas pyk1 expression
increases during growth. On YEME both genes show a decrease in
expression during stationary phase. This points towards pyk2 being the
primary pyruvate kinase in S. coelicolor M145. The results support the
hypothesis that this node of primary metabolism can serve as a target to
increase yields in antibiotic production.
MA05/08
Expression of Type III PKS genes in Streptomyces coelicolor
Anyarat Thanapipatsiri1,2, Juan-Pablo Gomez-Escribano2,
Jan Claesen2, Mervyn Bibb2, Arinthip Thamchaipenet1
1
Department of Genetics, Faculty of Science, Kasetsart University, Bangkok,
Thailand, 2Department of Molecular Microbiology, John Innes Centre, Norwich
Research Park, Norwich, UK
Type III polyketide synthases (PKSs) are small homodimeric proteins
found in plants, fungi and bacteria1 that assemble Coenzyme A-linked
acetate-derived substrates into different length polyketides. To assist
in the analysis of Type III PKS of unknown function, Streptomyces
coelicolor M11522 [∆act ∆red ∆cpk ∆cda rpoB (C1298T)], a derivative
of strain M145 from which four antibiotic biosynthetic gene clusters
have been deleted and into which a mutation has been introduced that
pleiotropically enhances the level of secondary metabolite production,
was engineered by PCR targeting to remove all three native Type III PKS
genes (SCO1206, SCO7221 and SCO7671) providing a clean background
for heterologous expression.
Type III PKSs of actinobacterial origin were analysed bioinformatically and
their evolutionary relationships determined, revealing nine subgroups.
Eleven genes, from six different subgroups, have been cloned into the
expression vector pIJ86-Kan (KanR, ermE*p) and introduced into the
Type III PKS ‘superhost’. The recombinant strains will be analysed by
HPLC and LC-MS to determine the nature of the compounds produced
by the Type III PKSs of unknown function.
References 1. Moore, B. S.; Hopke, J. N. Chem. Biochem. 2001, 2, 35-38;
2. Gomez-Escribano, J.P., Bibb, M. 2011. Microbial Biotech., 4, 207-215.
MA05/09
Coelimycin, the metabolic product of the cpk gene cluster of
Streptomyces coelicolor: from discovery to structure elucidation
Juan Pablo Gomez-Escribano1, Lijiang Song2, Gregory L Challis2,
Mervyn J Bibb1
1
John Innes Centre, Norwich, UK, 2University of Warwick, Coventry, UK
Streptomyces coelicolor produces four families of secondary metabolites
derived from the actinorhodin, prodiginine, methylenomycin and calcium
dependent antibiotic gene clusters. Analysis of the S. coelicolor genome
sequence revealed the potential to synthesise many other compounds.
[1]
One of the cryptic gene clusters encodes a Type I polyketide synthase
and was named cpk (Cryptic PKS).[2] We recently determined the
structure of coelimycin, its metabolic product[3]. However, we now
believe that the compound was first observed over 30 years ago. In the
mid 1970’s Brian Rudd isolated several actinorhodin-production deficient
mutants that produced a diffusible orange pigment which appeared
to have antibiotic activity. He mapped the mutations genetically to a
location of the chromosome that correlates extremely well with the
physical location of the cpk gene cluster.[4] We have analysed two
of Rudd’s mutants and found that they produce large amounts of
coelimycin P1 providing evidence that he was the first researcher to
observe expression of the cpk gene cluster and its product.
References [1] Nature 2002, 417, 141-147; PNAS USA 2003, 100,
14555-14561. [2] J. Mol. Microbiol. Biotechnol. 2010, 19, 147-151. [3]
Chem. Sci. 2012, 3, 2716-2720. [4] B. Rudd, PhD thesis, University of
East Anglia, 1978.
MA05/10
The involvement of mtrAB-lpqB gene cluster in cell division and
antibiotic production in Streptomyces coelicolor
Felicity J Knowles, Nicolle F Som, Ryan F Seipke, Matthew I Hutchings
School of Biological Sciences, University of East Anglia, Norwich Research
Park, Norwich, UK
The mtrAB-lpqB gene cluster is conserved in nearly all sequenced
actinobacteria. The genes encode for a three component signal
transduction system and studies in C. glutamaticum and M. tuberculosis
have suggested roles for MtrAB in the regulation of osmoprotection,
cell envelope homeostasis and cell cycle progression. In-frame deletion
of mtrA and mtrB in Streptomyces coelicolor resulted in strains that form
small, whitish-grey colonies that produce very few spores relative to
wild-type. A second copy of FtsZ fused to eGFP in the wild-type and
mutant strains suggested that MtrA positively regulates FtsZ production
during sporulation in S. coelicolor due to the lack of eGFP fluorescence
in the mtrA mutant. These results suggest a possible role for MtrA in cell
division. Furthermore the disruption of the mtrAB-lpqB genes has an effect
on antibiotic production. Unlike the wild-type, in the mtrA, mtrB and lpqB
mutants actinorhodin is produced before the formation of aerial hyphae
and undecyloproprodigiosin is overproduced on LB agar. This suggests
that antibiotic production may be uncoupled from developmental
regulation in S. coelicolor mtrAB-lpqB mutants and that manipulation of
MtrA activity can be used to increase antibiotic production.
MA05/11
Induction of VanS-dependent vancomycin resistance requires binding
of the drug to D-Ala-D-Ala termini in the peptidoglycan cell wall
Min Jung Kwun1, Gabriela Novotna1, Andrew Hesketh2, Lionel Hill3,
Hee-Jeon Hong1
1
Department of Biochemistry, University of Cambridge, Cambridge, UK,
2
Cambridge Systems Biology Centre, University of Cambridge, Cambridge, UK,
3
Department of Metabolic Biology, John Innes Centre, Norwich, UK
An important question yet to be answered in vancomycin resistance
study is the nature of the specific ligand recognised by the VanS sensor
protein. Two distinct models exist: i) direct induction, in which the
VanS is activated by direct binding the antibiotic; ii) indirect induction,
in which the VanS is activated by binding a cell wall metabolite that is
either intermediate in cell wall biosynthesis or accumulates as a result of
antibiotic action. Recent observations strongly favour the direct induction
mechanism over the indirect one, but do not exclude a requirement for
binding as a vancomycin-cell wall metabolite complex. In this study we
use the van cluster in Streptomyces coelicolor as a model for the VanB
resistance system and demonstrate for the first time that vancomycin
primarily requires binding to the D-Ala-D-Ala termini of cell wall
peptidoglycan precursors to be perceived by the VanS sensor protein.
We were able to manipulate the relative amounts of D-Ala-D-Ala and
D-Ala-D-Lac containing peptidoglycan precursors by genetic engineering.
Further in vivo assays determined that the response to vancomycin in
these strains correlated with the abundance of D-Ala-D-Ala-containing
peptidoglycan precursors; strains producing a lower proportion of D-AlaD-Ala-containing peptidoglycan consistently exhibited a lower response
to vancomycin.
MA05/12
Vancomycin induces the zinc starvation response in S. coelicolor
Mohammad Ashraf Al Zarkan, Heather Macklyne,
Hee-Jeon Hong
Department of Biochemistry, University of Cambridge, Cambridge, UK
Our previous microarray data demonstrated that vancomycin induced
zinc homeostasis system regulated by Zur in Streptomyces coelicolor
68
and we have recently reassured this by the transcriptional analysis using
qRT-PCR. Zinc plays an indispensable role in cellular biochemistry as
a catalytic or structural cofactor for a wide variety of metalloproteins.
It can however be toxic if accumulated to excess, and its intracellular
concentration is controlled within safe limits by homeostatic regulatory
mechanisms. In bacteria, this is largely achieved by tight control of
import and export mechanisms through the zinc-responsive regulatory
protein Zur, and the Zur regulon in S. coelicolor has recently been fully
characterised. Further investigations have been carried out and finally
determined that this vancomycin-driven zinc depletion condition in the
S. coelicolor cells was indeed occurred by vancomycin binds zinc directly.
More interestingly, zinc seemed to enhance the antimicrobial activity of
vancomycin against vancomycin resistant strains as the susceptibility of
resistant cells to vancomycin was significantly increased in zinc enriched
medium. Here we propose a hypothesis to explain this rather intriguing
zinc effect on vancomycin activity against resistant strains and will discuss
the outcome of our recent in vivo assay results supporting the proposed
hypothesis in this study.
MA05/13
Developing tools for orthogonal control of gene expression in
Streptomyces
H J Frasch1, S Comba3, A R Ferrari1, H Gramajo3, E Takano1,2
1
University of Groningen, Groningen, The Netherlands, 2University of
Manchester, Manchester, UK, 3Universidad Nacional de Rosario, Rosario,
Argentina
The genus Streptomyces produces a large variety of secondary
metabolites. However, it is now known that most of the natural
products encoded in the genomes of these bacteria are not produced
under laboratory conditions [1]. We have recently proposed a new
approach for ‘awakening’ these untapped biosynthetic gene clusters that
focusses on refactoring the gene clusters in an optimised host [2]. One
aspect which is required for the ‘awakening’ is an orthogonal regulatory
circuit which is independent of the host regulatory elements. We are
developing orthogonal regulatory circuits based on inducible promoters
previously used in Streptomyces [3]. To test our synthetic circuits we
are establishing a new method that allows luminescence measurement
from Streptomyces coelicolor on solid medium in 96-well microtiter plates,
suitable for high-throughput characterisation of various promoters under
different conditions. Using this system, measurements shall be possible in
vivo, and with high temporal resolution, generating the information that
is necessary for the successful control of refactored biosynthetic gene
clusters.
MA05/14
Apramycin resistance mutation increases chromomycin
overproduction and growth rate of Streptomyces spp. 275
Jayashree K Pohnerkar1, Divya S Prajapati2
1
Maharaja Sayajirao University, Baroda, Gujarat, India, 2Maharaja Sayajirao
University, Baroda, Gujarat, India
Streptomyces are exploited for production of a wide range of secondary
metabolites. The organism of present study, Streptomyces spp. 275, was
found to produce the antitumour antibiotic, chromomycin (unpublished
results). We describe isolation of genetic alteration that enhances
chromomycin production by ~100 fold. The overproducer mutant
is resistant to high levels of apramycin unselected, and cross-resistant
to other aminoglycosides like kanamycin, geneticin, gentamycin, and
tobramycin but not to amikacin, and neomycin. We also find that
70% of spontaneous apramycin resistant mutants that overproduce
chromomycin implying the two phenotypes result from a single
mutation. Overproducer mutation, we believe is a ribosomal mutation
for the following reasons the aminoglycoside cross resistance profile
is different from that of the resistance profile of apramycin modifying
function. We ruled out that the apramycin resistance gene (aac (3) IV)
gene causes chromomycin overproduction, overproducer derivative
Poster abstracts
is significantly more viable, sporulates profusely and grows faster than
wild type. The latter phenotype is unconventional, in that spontaneous
antibiotic resistant mutant has compromised growth rate and/or at
competition disadvantage (Ochi et.al 2009). The overproducer mutant
indeed outcompetes wild type in the competition assay. The mutation to
apramycin resistance causing chromomycin overproduction thus qualifies
for being novel.
MA05/15
The isolation of novel marine Streptomyces isolates from mangrove
forests, Egypt
Ghada Yousif2, Michael Goodfellow1, Medhat abdel Fattah2,
Sherif Hassan2, Ahmed Reyad2, Wael Hozzein2
1
School of Biology, Newcastle University, Newcastle upon Tyne, UK, 2Faculty
of Science, Beni-Suif University, Beni-Suif, Egypt
Five novel Streptomyces isolates namely m.n1, m.n3, m.n19, m.n21
and m.n23 were isolated using heat pre-treatment method from three
mangrove sites along the Red Sea coast, Egypt. The isolates were chosen
from SM3 and M1 agar plates supplemented with seawater according to
their morphological and pigmentation pattern. The isolates were rich in
LL-A2pm; with no characteristic sugars in their cell hydrolysates, properties
consistent with their 16S rRNA gene data which assigning them to
the genus Streptomyces. Almost all of the isolates formed distinct
but heterogeneous subclades within their trees, sharing law similarity
values in the 16S rRNA gene sequences within their nearest taxonomic
Streptomyces neighbors ranging from 97 to 99.4%. The strains showed
characteristic spore chains morphology and spore surface ornamentation.
Moreover, the putative novel isolates, m.n1 and m.n3, can be easily
distinguished from their closest neighbors using a range of phenotypic
and chemotaxonomic characters. Interestingly, strains namely n.m1 and
n.m23 required seawater for growth, so can be considered as obligate
marine Streptomycetes isolates.
MA05/16
Defining the stages of chromosome segregation in Streptomyces
venezuelae and the role of TopA in this process
MAgdalena Donczew, Marcin Szafran, Jolanta ZakrzewskaCzerwinska, Dagmara Jakimowicz
University of Wroclaw, Wroclaw, Poland
Streptomyces venezuelae is able to grow in diffuse, homogenous manner
and sporulate to near completion in liquid media. These features make
S. venezuelae suitable for time-lapse analysis of sporulation. During
sporulation in sporogenic hyphae the multiple chromosomes are
condensed and segregated into prespores. In Streptomyces, as in other
bacteria, ParA and ParB are the major proteins responsible for proper
chromosome segregation. ParB forms nucleoprotein complexes by
binding to parS sites clustered around oriC while ParA polymers align
ParB complexes along the hyphae. We focus on the dynamics of the
ParA and ParB assembly during S. venezuelae sporulation using time-lapse
fluorescence microscopy. Functional fusions of EGFP to ParA and ParB
proteins were used to analyse the timing of protein complexes formation
and their disassembly. Since we have observed that lowering the level
of TopA (topoisomerase I) inhibits sporulation associated chromosome
segregation and septation, the localisations of EGFP tagged ParA and
ParB proteins in the TopA depletion strain were examined. We propose
the model of sporulation where active chromosome segregation occurs
at the time when the sporogenic hyphae stops growing.
MA05/17
Exploring the potential of Streptomyces coelicolor hydrophobic aerial
proteins to produce functional organic coatings in materials science
Harriet A Harold1,2, Jonathon Elvins2, David Penney1,
Geertje van Keulen1, Daniel John1
1
Swansea University, Swansea, UK, 2Tata Steel Europe, Swansea, UK
69
Poster abstracts
Coelicolor hydrophobic aerial proteins or chaplins are functional amyloids
that are produced by the filamentous bacteria Streptomyces coelicolor. The
chaplins are secreted by the mycelium and self-assemble at hydrophilichydrophobic interfaces to produce an amphipathic membrane. This
protein membrane has a dual purpose; lowering the surface tension
of water to allow the penetration of aerial hyphae and to coat these
emerging structures in an outwardly hydrophobic coat. . There are eight
members of the chaplin family; chaplins A-C are long proteins made up
of 210-230 amino acid residues and chaplins D-H which are shorter with
only 50-63 residues. Extracted mixed chaplins and a synthetic ChpH
were used to coat a variety of metal and non-metal surfaces changing the
wettability of the substrates. The chaplins formed a β-sheet structure on
the surface of the coated substrates, demonstrating the amyloid nature
of the proteins. These proteins show similar properties to hydrophobin
proteins produced by filamentous fungi, these are also surface active
proteins, shown to be capable of many medical and technical applications.
Therefore there is a great potential for exploitation in the application
of chaplins in materials science to produce an organic functional coating
capable of reversing the wettability of a surface.
MA05/18
Anti-meticilin resistant Staphylococcus aureus (anti-MRSA) activity,
taxonomy and preliminary determination of chemical constituents of
actinomycetes isolated from Thai deciduous forest soils
Chitti Thawai
King Mongkut’s Institute of Technology Ladkrabang, Bangkok, Thailand
One hundred and one actimonycete strains were isolated from the
deciduous forest soils in Maewong National Park, Thailand. These strains
were grouped using phenotypic, chemotypic and genotypic characteristics
into 6 groups. The 16S rRNA gene sequence and chemotaxonomic
analyses including morphological characteristics revealed that the
representative strains in each group belonged to the members of the
genera Streptomyces, Micromonospora Nocardia and Dactylosporangium.
Here, we found the strain MW4-36 showing morphological and
chemotaxonomic characteristics typical of members of the genus
Dactylosporangium but which was genotypically and phenotypically
distinguishable from all recognised Dactylosporangium species. Therefore,
strain MW4-36 was judged to represent the novel species of the
genus Dactylosporangium for which the name Dactylosporangium
siamense was proposed. Furthermore, the fermentation broths of these
representative strains were extracted with ethyl acetate and were tested
for anti-microbial activity. The results showed that more than 52% of
actinomycete strains exhibited the anti-methicilin resistant Staphylococcus
aureus (Anti-MRSA) activity. Antimicrobial assay-guided fractionation of
the ethyl acetate extract of Streptomyces sp. KP8-1 yielded the known
compound, geldanamycin. Based on these results, it could be concluded
that the actinomycetes found in Maewong National Park are very great
and should be represented an excellent source for the discovery of
bioactive compounds.
MA05/19
Use of red autofluorescence for monitoring prodiginines biosynthesis
Elodie Tenconi, Sébastien Rigali
University of Liège, Liège, Belgium
Prodigiosin-like pigments or prodiginins (PdGs) are promising drugs
owing to their reported antitumor, antibiotic, and immunosuppressive
activities. These natural compounds are produced by several bacteria,
including Streptomyces coelicolor and Serratia marcescens as most
commonly studied models. The bright red color of these tripyrrole
pigments made them excellent reporter molecules for studies aimed
at understanding the molecular mechanisms that control secondary
metabolite production in microorganisms. However, the natural red
autofluorescence of PdGs had surprisingly only been rarely exploited as
biophysical parameter to detect and quantify their biosynthesis. In this
work, we used S. coelicolor M145 and its ∆redD mutant (strain M510)
impaired in PdGs production to exemplify how intrinsic red fluorescence
could be utilised for rapid, low-cost, sensitive, and accurate semiquantitative analyses of PdGs biosynthesis. Additionally, and contrary to
the colorimetric-based approach, the fluorescence-based method also
allows in situ spatio-temporal visualisation of PdGs synthesis throughout
a culture of S. coelicolor. As PdGs biosynthesis is growth phase-dependent
and coincides with the onset of the physiological differentiation, red
autofluorescence could additionally be used as a natural marker to
indicate the developmental stage in the course of the S. coelicolor life
cycle.
MA05/20
Exploiting the biosynthetic diversity of Actinobacteria by automated
screening of picolitre culture droplets
Miguel A Tovar1, Emerson Zang1, Karin Martin1, Susanne Brandes1,
Franziska Mech1, Peter Horbert2, Thomas Henkel2, Marc Thilo Figge1,
Martin Roth1
1
Leibniz Institute for Natural Product Research and Infection Biology,
Hans-Knöll-Institute, Jena, Germany, 2Institute of Photonic Technology, Jena,
Germany
Despite the historic success of antibiotic discovery from actinomycetes,
landmark studies agree that more than 90% of the antibiotics produced
by Actinobacteria are yet to be found. As a new strategy to overcome
limitations of classic screening, we apply a droplet-based microfluidic
approach for high-throughput cultivation of Actinobacteria with
subsequent whole-cell screening for novel antimicrobial compounds.
Spores are encapsulated in 100 pL-culture media droplets, which are
dispersed in perfluorinated oil at frequencies above 500 Hz. Millions of
individual spores can be separated in microdroplets within hours and
subsequently incubated in a small container of 1 mL. Mycelial growth is
observed after incubation, indicating the feasibility of high-throughput
culture of actinomycetes in the droplets. Thereafter, fluorescent reporter
micro-organisms are dosed into each droplet in order to screen for
antimicrobial activity. Automated detection and sorting of droplet
subpopulations can then be performed to characterise and isolate microorganisms of interest. An additional highlight of this technique is the
rapid assessment of different culture media and microbial interactions;
both critical for secondary metabolite production. Our high-throughput
screening approach provides optimal means for microbial culture and
detection of produced bioactive compounds, and is thus a promising
tool to exploit the vast natural diversity of Actinobacteria.
MA05/21
Chromosome segregation in Streptomyces coelicolor
Beatrice Vetter1, Graham Falconer1, Emma Tilley1,
Agnieszka Kois-Ostrowska2, Jolanta Jolanta2, Dagmara Jakimowicz2,
Paul Herron1
1
University of Strathclyde, Glasgow, UK, 2University of Wroclaw, Wroclaw,
Poland
The reconciliation of monodirectional tip extension with bidirectional
chromosome segregation represents a key challenge to organisms that
undergo polarised growth such as Streptomyces coelicolor. In order to
understand this process, we have developed a Fluorescence Reporter
Operator System (FROS) in S. coelicolor. We used in vitro transposon
mutagenesis to deliver 120 copies of the tet operator (tetO) arrayed
in tandem to an oriC-proximal site within the S. coelicolor chromosome.
By fusing the cognate repressor protein (TetR) to mCherry and eGFP,
we have visualised binding of TetR to the tandem tetO array and, as a
result, the location of oriC during chromosome replication through tip
extension, erection of aerial hyphae and sporulation. Using this approach
we have generated a model describing chromosome segregation in
streptomycetes that permits chromosome colonisation of the extending
tips as well as apex-distal branches and provides a foundation for
understanding polarised growth in this important model organism.
70
Poster abstracts
MA05/22
Elucidating the complex regulation of the Streptomyces coelicolor
gamma-butyrolactone (SCB) signalling system
Lara Martin-Sanchez1, Robert Bunet1,2, Eriko Takano1,3
1
Microbial Physiology, GBB, University of Groningen, Groningen, The
Netherlands, 2Équipe de Biologie Moléculaire Marine -PROTEE, Université
du Sud Toulon-Var, La Garde Cedex, France, 3Faculty of Life Sciences,
Manchester Institute of Biotechnology, University of Manchester, Manchester,
UK
Antibiotic production is regulated by the γ-butyrolactone (SCB)
signalling system in Streptomyces coelicolor1. ScbR, γ-butyrolactone
receptor, binds upstream of its own promoter region (site R) impeding
its transcription. ScbR also binds to a non-conserved site in the
core promoter region of scbA (site A). ScbA is responsible for the
biosynthesis of γ-butyrolactones, which inhibit ScbR DNA-binding
activity to its promoter. The involvement of ScbR in the regulation of
scbA expression can be seen from the inhibition of ScbA expression by
the deletion of ScbR1. To understand this mechanism, 4 point mutations
were introduced in site A to modify the consensus ScbR binding
sequence. Gel retardation assays showed that ScbR cannot bind to this
mutated site in vitro2. This mutant could not produce γ-butyrolactones.
Expression analyses of scbA in the LW20 mutant by qRT-PCR showed
that the growth phase-dependent induction observed in the wild type
was lost in LW20 and ScbR protein was expressed constitutively as
confirmed by Western Blot. These results suggest that ScbR activates the
production of γ-butyrolactones by induction of scbA expression through
its binding to site A.
References 1. Takano E, et al. Mol Microbiol. 2001;41(5):1015-1028;
2. Bunet R. PhD thesis. Eberhard Karls Universität Tübingen 2006.
MA05/23
Elucidating the role of flotillin in Streptomyces coelicolor
Charles Webb, Paul Herron
The highly-conserved protein, flotillin, plays an important role in
the development of neurodegenerative diseases in humans, such as
Alzheimer’s and Parkinson’s disease. It has been identified as a detergentresistant membrane marker in both eukaryotes and prokaryotes.
Many studies have identified potential flotillin-interacting partners, but
in bacteria, it is always found with an NfeD protein. In Bacillus subtilis,
flotillin plays a role in sporulation. We have begun to elucidate the
nature of co-occurrence of NfeD and flotillin in Streptomyces coelicolor
using bacterial two-hybrid assays. We have also used a genetic approach
and fluorescence microscopy to identify the cellular location of these
proteins and their membrane localisation during the streptomycete life
cycle. In this way we hope to reveal information about flotillin-binding
mechanisms, which may be extrapolated to eukaryotes and disease.
MA05/24
The evolutionary history of Dps in Streptomyces genomes includes
horizontal gene transfer, duplication and neofunctionalisation
P D Facey, M D Hitchings, J S Williams, D O F Skibinski, P J Dyson,
R Del Sol
Swansea University, Swansea, UK
Dps proteins are found almost ubiquitously in bacterial genomes and the
numbers of dps genes per bacterial genome is variable -even amongst
closely related species. Typically, Streptomycetes encode for more than
one Dps protein. We offer the explanation that variation in the number
of dps per genome can be explained by gene duplication and/or lateral
acquisition. We show that the genome of S. coelicolor encodes for three
Dps proteins including a tailless Dps; which is unable to oligomerise and
thus represents the first reported Dps that appears not to readily selfassemble. Expression studies indicate that in several Streptomyces species
at least one Dps is significantly over-expressed during osmotic shock,
but the identity of the ortholog varies. In silico analysis of dps promoter
regions of duplicated dps genes suggests subsequent neofunctionalisation
of cis-acting elements to evolve an osmotically inducible dps. Lastly,
we identify a rare novel clade of Dps and show that a representative
of these proteins in S. coelicolor possesses a dodecameric quaternary
structure of high stability.
MA05/25
A systems biology approach to understanding the glyoxylate shunt
Richard A Reumerman, Paul R Herron, Paul A Hoskisson
Members of the genus Streptomyces produce many interesting
secondary metabolites, the production of which relies on central
carbon metabolism for precursors. When an increased flux through the
biosynthetic pathways is desired, an increase in precursor supply will
eventually become necessary. The glyoxylate shunt is an anapleurotic
pathway capable of replenishing TCA cycle intermediates and as such is
thought to play an important part in biosynthesis.
The precise function of the glyoxylate shunt in Streptomyces coelicolor
will be elucidated using mutants lacking or overexpressing genes
encoding the pathway’s enzymes. Kinetic parameters of these enzymes
(isocitrate lyase and malate synthase 1 & 2) will be determined in
in-vitro experiments. Using these parameters, we have developed a
mathematical model that enables a rational approach to increasing
carbon flux and thereby increasing the supply of precursors for
secondary metabolism.
MA05/26
A laterally acquired galactose oxidase-like gene is required for aerial
development during osmotic stress in Streptomyces coelicolor
Recep Liman2, Paul D. Facey1, Geertje van Keulen1, Paul Dyson1,
Ricardo Del Sol1
1
College of Medicine Swansea University, Swansea, Wales, UK, 21Faculty of
Science, Department of Genetics, Usak University, Usak, Turkey
Phylogenetic reconstruction revealed that most Actinobacterial orthologs
of S. coelicolor SCO2837, encoding a metal-dependent galactose oxidaselike protein, are found within Streptomyces and were probably acquired by
horizontal gene transfer from fungi. Disruption of SCO2837 (glxA) caused
a conditional bld phenotype that could not be reversed by extracellular
complementation. Studies aimed at characterising the regulation of
expression of glxA showed that it is not a target for other bld genes. We
provide evidence that glxA is required for osmotic adaptation, although
independently from the known osmotic stress response element SigB.
glxA has been predicted to be part of an operon with the transcription
unit comprising the upstream cslA gene and glxA. However, both
phenotypic and expression studies indicate that it is also expressed from
an independent promoter region internal to cslA. GlxA displays an in situ
localisation pattern similar to that one observed for CslA at hyphal tips,
but localisation of the former is independent of the latter. The functional
role of GlxA in relation to CslA is discussed.
MA05/27
KirCI, the in trans active acyltransferase providing malonyl units for
kirromycin assembly
Ewa M Musiol, Thomas Härtner, Anderas Kulik,
Wolfgang Wohlleben, Tilmann Weber
Universität Tübingen, Interfakultäres Institut für Mikrobiologie und
Infektionsmedizin, Mikrobiologie/Biotechnologie, Tübingen, Germany
Kirromycin is produced by Streptomyces collinus Tü 365. This compound
is synthesised by a large complex of type I polyketide synthases and
non-ribosomal peptide synthetases (PKS I/NRPS), encoded by the
genes kirAI-kirAVI and kirB. The PKSs KirAI-KirAV have no acyltransferase
domains integrated into the PKS modules. This type of PKS is named
trans-AT-PKS. The KirAVI belongs to the classical cis-AT-type PKS, where
71
Poster abstracts
the ATs are part of the PKS protein. In the gene cluster of kirromycin
two separate genes, kirCI and kirCII, were identified, which are similar to
acyltransferases. While the function of KirCII was recently described, the
role of KirCI remained unclear. A mutant of the kirCI gene was generated
and analysed for kirromycin production. The inactivation of kirCI (ΔkirCI)
resulted in a significant reduction of kirromycin production. These data
indicate that KirCI is involved in kirromycin biosynthesis. The specificity of
KirCI was investigated in an in vitro ACP-loading assay. The experiments
showed that KirCI is specific for malonyl-CoA and loads this extender
onto the ACPs of kirromycin trans-AT-PKSs.
References 1. Musiol, E.M et al., (2011). Supramolecular templating in
kirromycin biosynthesis -the acyltransferase KirCII loads ethylmalonylCoA extender onto a specific ACP of the trans-AT PKS. Chem Biol 18,
438-444.
MA05/28
The Streptomyces cytochrome bc1 complex and the Rieske iron–sulfur
protein
Adam P Hopkins, Tracy Palmer
The Actinobacteria include the antibiotic-producing Streptomyces
species in addition to serious human pathogens such as Mycobacterium
tuberculosis and Corynebacterium diptheriae. Actinobacteria have a classical
aerobic respiratory chain terminating with an aa3 type cytochrome
oxidase. Electrons are fed into this terminal oxidase by the cytochrome
bcc complex (a modified cytochrome bc1 complex containing a diheme cytochrome cc). This complex is heterotrimer of cytochrome b,
the Rieske iron-sulfur protein and cytochrome cc. After cytochrome
b oxidises quinol, the released electrons are transferred through the
2Fe-2S centre of the Rieske protein to the cytochrome cc and on to
the terminal oxidase, where oxygen is reduced. The actinobacterial
cytochrome bcc complex has several unique features, including additional
transmembrane helix (TMH) and an extracellular C-terminal globular
domain in cytochrome b, whilst the Rieske protein has a two TMH
N-terminal extension, containing an internal twin arginine signal sequence
after the second TMH, targeting the protein to the Tat pathway. Our
current work examines the cytochrome bcc complex in Streptomyces
coelicolor, particularly the unique features of the complex. We have
succeeded in isolating this complex and are working towards describing
its association. Our latest results on the composition and function of this
complex will be presented.
MA05/29
Regulatory RNAs that control development and antibiotic production
in Streptomyces
Jan Bobek, Klára Novotná, Dita Šetinová
Institute of Immunology and Microbiology of the First Faculty of Medicine,
Charles University in Prague and General Teaching Hospital, Prague, Czech
Republic
Complex development of differentiating bacteria is expected to be
controlled by an intricate regulatory network including a raised number
of regulatory RNAs. Genome-wide searches have already verified
expression of several small RNAs in Streptomyces. We are interested
whether the regulatory RNAs are involved in biosynthetic pathways
leading to production of secondary metabolites in Streptomyces
coelicolor. We focus on a candidate RNA (scr3559) which affects
production of secondary metabolites. Its predicted secondary structure
exhibits significant homology with a bacterial 6S RNA. The scr3559
RNA is expressed especially during later exponential phase of growth.
Overexpression of the scr3559 gene, inserted into multicopy number
plasmid, led to increased antibiotic production whereas a deletion
strain decreased antibiotic production. These results suggest a possible
effect of the transcript on the production of antibiotics actinorhodin
and undecylprodigiosin. Acknowledgment: This work was supported by
Czech Science Foundation No. P302/10/0468.
MA05/30
Scaling up the hunt for new antimicrobials
Tameera Rahman, Robert Powell, Shabhonam Caim, Lisa Crossman
The Genome Analysis Centre, Norwich, Norfolk, UK
The genus Streptomyces produce secondary metabolic compounds that
are rich in biological activity. Many of these compounds are encoded
by large gene clusters that are highly repetitive. Polyketide synthases
(PKS), non-ribosomal peptide synthases (NRPS) and hybrid clusters
both constitute large gene clusters found within the genus. Specific or
repeating domains in the predicted proteins are responsible for carrying
out reactions in a processive manner. These reactions build biologically
active secondary metabolites without the use of the ribosome. Due
to the repeats, these gene clusters are difficult to resolve using short
read next generation datasets and are poorly predicted using standard
approaches, leading to poor annotations. Web-based and commandline programs exist that can identify these gene clusters from finished
and large contig high quality draft sequences. To scale up screening for
these clusters from next generation datasets, we formulated specific
phylogenetic profiles for several PKS, NRPS and hybrid families. These
profiles were refined and used to scan large datasets and to identify
PKS and NRPS in metagenomic data. Further knowledge of the
environmental spread of these gene clusters and identification of new
sequences in potentially uncultured or unculturable strains could allow
the exploitation of new environmental niches or strains.
MA05/31
Towards the understanding of microbial dialogues within soil
ecosystems: studies of the interaction between Streptomyces
ambofaciens and Pseudomonas fluorescens
Justine Galet1,2, Aurélie Deveau2, Pierre Leblond1, Pascale Frey-Klett2,
Bertrand Aigle1
1
Université de Lorraine, INRA, UMR 1128, Vandoeuvre-lès-Nancy, France,
2
Université de Lorraine, INRA, UMR 1136, Champenoux, France
Streptomyces are common bacteria in temperate forest ecosystems in
which they interact with many bacterial genera and fungi. If the role of
their secondary metabolites as biological weapons is likely, they could
also provide signals in intercellular communication and play a major role
in structuring soil microbial communities. The question also arises how
the biotic environment modulates the biosynthetic pathways in these
bacteria and impacts their behaviour in the microbial communities. We
have initiated a study on interaction between Streptomyces ambofaciens
ATCC23877 and Pseudomonas fluorescens BBc6R8. S. ambofaciens
ATCC23877 produces at least five antimicrobials and two siderophores
and its genome encodes 16 additional gene clusters. P. fluorescens
BBc6R8 is a mycorrhiza helper bacterium isolated from fruiting bodies
of Laccaria bicolor. Co-culture experiments have revealed that each
bacteria affects the production of secondary metabolites in the other
partner. Thus, P. fluorescens inhibits the ability of S. ambofaciens to
produce kinamycin whose production is controlled via a mechanism of
quorum sensing. In fact P. fluorescens inhibits the regulatory cascade
controlling the antibiotic biosynthesis probably by a quorum quenching
mechanism. Reciprocally, on iron deficient medium, P. fluorescens uses
the desferrioxamines produced by S. ambofaciens and does no longer
produce its own siderophores.
MA05/32
New approaches to exploit Streptomyces: looking toward storage lipid
metabolism
T Dulermo1, M David1, A Deniset2, A Dazzi2, M J Virolle1
1
Group Métabolisme Energétique des Streptomyces, Institut de Génétique et
Microbiologie, UMR CNRS 8621, Université Paris-11, 91405 Orsay Cedex,
France; 2Laboratoire de Chimie Physique, Université Paris-Sud, 91405 Orsay
Cedex, France
The two closely related Streptomyces species, S. lividans TK24 and S.
coelicolor M145, both possess the functional pathway to synthesise the
72
Poster abstracts
blue pigmented polyketide antibiotic, actinorhodin (ACT). S. lividans TK24
produce little or no ACT whereas S. coelicolor M145 and the ppk mutant
of S. lividans TK24 produce ACT abundantly. Electron microscopic
observations and quantification of TriAcylGlycerol (TAG) content,
using Fourier Transformed InfraRed Spectroscopy (FTIRS), revealed
that S. coelicolor M145 and the the ppk mutant of S. lividans contain 30%
and 70% less TAG, than the wt strain of S. lividans, respectively . These
studies confirmed the strong prospective link between TAG metabolism
and the production of polyketide antibiotics in Streptomyces even if the
relations between these two biosynthetic routes remain to be clarified.
In consequence, manipulation of TAG metabolism (either impairment
of their biosynthesis or activation of their degradation) might constitute
novel strategies to enhance precursors availability and thus expression
of the numerous cryptic biosynthetic pathway s present in Streptomyces
genomes that might direct the synthesis of potentially useful bio-active
secondary metabolites.
Next-generation antimicrobials
MA06
MA06/01
Supramolecular arrangements of antibiotic lipoamino acids from the
potential anti-mastitis Probiotic Bacillus pumilus strain 33
M A Alqumber1, J R Tagg2
1
Albaha, Saudi Arabia, Albaha, Saudi Arabia, Saudi Arabia, 2University of
Otago, Otago, Dunedin, New Zealand
Bacillus pumilus strain 33, a cow udder isolate, was shown to produce
a heat stable 1145 Da inhibitory agent. The inhibitor was recovered
from HPLC-purified culture exudates and was found to depolymerise
into smaller molecules. Mass spectrometry indicated that these smaller
masses correspond to N-acylasparagines having varying aliphatic fatty acid
chains. The smallest obtained inhibitory component, when subjected to
MS and NMR analysis was found to be the N-acylasparagine (iso C11Asn). A proposed cationised mixed micelle aggregate of acylasparagines
is presented to explain the predominant 1145 Da molecule.
MA06/02
A FimH inhibitor prevents acute bladder infection and treats chronic
cystitis caused by multidrug resistant uropathogenic Escherichia coli
ST131
MAkrina Totsika1, Maria Kostakioti2, Thomas J Hannan2,
Mathew Upton3, Scott A Beatson1, James W Janetka2, Scott J Hultgren2,
Mark A Schembri1
1
Australian Infectious Diseases Research Centre, School of Chemistry and
Molecular Biosciences, University of Queensland, Brisbane QLD 4072, Australia,
2
Washington University School of Medicine, St. Louis, Missouri, USA, 3School of
Translational Medicine, University of Manchester, Manchester M13 9WL, UK
Background Escherichia coli O25b:H4-ST131 represent a predominant
clone of multidrug resistant uropathogens currently circulating worldwide
in hospitals and the community. Urinary tract infections (UTI) caused by
E. coli ST131 are typically associated with limited treatment options and
are often recurrent.
Methods Using established mouse models of acute and chronic UTI we
mapped the pathogenic trajectory of the reference E. coli ST131 UTI
isolate, strain EC958.
Results We demonstrated that E. coli EC958 can invade into bladder
epithelial cells and form intracellular bacterial communities early during
acute UTI. Moreover, E. coli EC958 persisted in the bladder and
established chronic UTI. Prophylactic antibiotic administration failed to
prevent E. coli EC958 mediated UTI. However, one oral dose of a small
molecular weight compound that inhibits FimH, the type 1 fimbriae
adhesin, significantly reduced bacterial colonisation of the bladder and
prevented acute UTI. Treatment of chronically infected mice with the
same FimH inhibitor lowered their bladder bacterial burden by more
than 1000-fold.
Conclusions In this study, we provide novel insight into the pathogenic
mechanisms employed by the globally disseminated E. coli ST131
clone during acute and chronic UTI and establish the potential of FimH
inhibitors as an alternative treatment against multidrug resistant E. coli.
MA06/03
Neem (Azadirachta indica/A. Juss) extracts: a possible antibacterial agent
against Staphylococcus aureus causing dental carries disease
Elizabeth Mitaki1, Murugan S.2, Judith Okoth1
1
Jomo Kenyatta University Of Agriculture and Technology, NAIROBI, Kenya,
2
KSR College Of Arts & Sciences, Erode, India
The Azadirachta indica /A.Juzz tree is reputed as possessing antimalarial,
anti periodontitic, antiviral, anti allergic, anti inflammatory and amoebicidal
among others. Staphylococcus aureus is a common cause of tooth
infection, hence a good representative of common bacterial pathogens.
Studies have been carried out on the medicinal value of the crude
extracts of neem tree only few have been done in a hospital situation.
In present study crude extracts from leaves of Azadirachta indica A. juss
(syn. Melia azadirachta) were obtained and their antibacterial activity
tested against Staphylococcus aureus isolated from dental carries patients.
chloroform extract showed no inhibition zone at concentration of 50
ul, diameter of 0.75cm at 100 ul and 1cm at 150 ul; water extract had
inhibition zone diameter of 0.5cm at concentration of 50 ul, 0.6cm at
100 ul and 0.65cm at 150 ul; methanol extract had a zone of inhibition
diameter of 0.25cm at concentration of 50 ul, 0.75cm at 100 ul and
1.3cm at 150 ul was observed. There was notable inhibitory activity by
all extracts. In this study we show that Azadirachta indica has valuable
medicinal value against one bacteria that contributes to dental decay.
Key Words;
Neem, Extract,Antibacterial,Dental Carries,Staphylococcus aureus
MA06/04
Ferredoxin containing bacteriocins pectocin M1 and M2 from
Pectobacterium sp. parasitise an existing iron acquisition pathway for
cell entry
Rhys Grinter, Daniel Walker, Joel Milner
University of Glasgow, Glasgow, UK
We have discovered two novel bacteriocins (antimicrobial proteins) in
the genus Pectobacterium. These bacteriocins (designated pectocin M1
and M2) are members of the well characterised colicin M family and
have been shown to kill only other competing strains of Pectobacterium.
Pectocin M1 and M2 consist of a cytotoxic domain homologous to that
of colicin M fused to a horizontally acquired plant-like ferredoxin. The
ferredoxin domain of these proteins substitutes the portion of colicin
M required for cell surface receptor binding and translocation across
the outer membrane of target cells. It fulfills this role by parasitising an
existing ferredoxin-based iron acquisition pathway. The presence of
such a pathway is demonstrated by the fact that strains susceptible to
these pectocins are also able to utilise plant-ferredoxin as an iron source
under iron limited conditions. It is likely that this system is important for
pathogenesis in Pectobacterium and it represents the first example of
iron piracy directly from a host protein by a phytopathogen. Additionally
these pectocins illustrate a novel mechanism for the evolution of new
bacteriocin specificities.
MA06/05
Ligand and inhibitor interactions with the intact FsrC quorum
membrane sensor kinase
MAry K Phillips-Jones1, Simon G Patching2, Shalini Edara2,
Jiro Nakayama3, Rohanah Hussain4, Giuliano Siligardi4
1
University of Central Lancashire, Preston, UK, 2University of Leeds, Leeds, UK,
3
Kyushu University, Fukuoka, Japan, 4Diamond Light Source Ltd, Oxfordshire, UK
Approximately 50% of currently marketed small molecule drugs target
membrane proteins. This past success in developing modulatory drugs
73
Poster abstracts
that target membrane proteins suggests that bacterial membrane proteins
will remain promising targets for new antibacterial drug developments
of the future. One promising candidate is FsrC, the quorum membrane
sensor of Enterococcus faecalis. However, in common with other
membrane proteins, detailed investigations such as quantitative
determinations of ligand and inhibitor binding have formerly been
hampered by the technical challenges associated with its hydrophobicity.
Here we use SRCD spectroscopy to overcome these challenges for
FsrC, by defining stabilising conditions for investigating ligand/inhibitor
binding to the intact protein solubilised in detergent micelles and
involving relatively small amounts of the protein. We reveal that binding
of the native pheromone ligand (GBAP) exerts tertiary structural
changes in the protein and that the calculated kd value for FsrC-GBAP
interactions is 2 µM. We confirm that FsrC is a site for binding by the
Fsr pathway inhibitor siamycin I, and that pheromone and inhibitor bind
at independent, non-overlapping sites in the FsrC protein. This approach
offers promise for identifying next-generation inhibitors of FsrC, quorum
sensing and indeed other membrane proteins more generally.
MA06/06
Copper increases tetracycline resistance in waste water treatment
microcosms
Seánín M McCluskey, Charles W Knapp, Paul R Herron
Copper has been used as an antimicrobial for thousands of years, but it
has recently been proposed as a ‘new’ treatment to deal with antibiotic
resistant bacteria, especially in agriculture. However, there are risks for
its use -there are links between heavy metal and antibiotic resistance.
To investigate the possibility of drug-resistance arising upon exposure
to copper, microcosms mimicking the secondary stage of wastewater
treatment were used. Bacterial populations exposed to copper displayed
significantly increased levels of tetracycline resistance in comparison
to control microcosms, or those exposed to tetracycline itself. This
link is likely due to co-and/or cross resistance mechanisms i.e. both
copper and tetracycline resistance being carried on the same piece of
genetic material (or one gene conferring resistance to both). What we
should take from this is that antimicrobial resistance can be increased/
maintained by substances other than the antimicrobial in question, and in
the future when predicting the onset of resistance (in particular for new
antimicrobials), significantly more variables than those linked directly to
the clinical setting should be considered.
MA06/07
Finafloxacin is a novel fluoroquinolone with in vitro activity against
Burkholderia species
Sarah V Harding1, Kay B Barnes1, Mark I Richards1, Andreas Vente2,
Helen S Atkins1, Andrew J Simpson1
1
Dstl, Porton Down, Salisbury, UK, 2MerLion Pharmaceuticals, Berlin, Germany
Finafloxacin is a novel fluoroquinolone that has good antimicrobial
activity against a range of bacterial species including Staphylococcus
aureus and Listeria monocytogenes. This activity is particularly apparent
at acidic pH, where other fluoroquinolones become significantly less
active. In addition it has been shown that finafloxacin accumulates in
cells at acidic pH. It is hypothesised that due to this enhanced activity at
low pH, finafloxacin may be effective against bacterial species that are
able to enter and proliferate within the acidic environment that exists
within organelles of a host, for example, within phagosomes. Burkholderia
pseudomallei and Burkholderia mallei are two pathogenic bacterial species
that possess machinery allowing them to invade, replicate and spread
intracellularly. The diseases caused by these organisms are difficult to
treat as they are resistant to several antibiotics, therefore, the evaluation
of novel therapeutics for Burkholderia species are warranted. Here,
we present preliminary in vitro data that suggests a potential use for
finafloxacin in the treatment of infections caused by the Burkholderia
species.
© Crown copyright 2013. Published with the permission of the Defence
Science and Technology Laboratory on behalf of the Controller of HMSO.
MA06/08
Displacing bacterial plasmids to reduce antibiotic resistance gene load
Claire E Miller, Mark A Webber, Jan Kreft, Christopher M Thomas
Reservoirs of antibiotic resistance genes in community, clinical and
veterinary contexts rise relentlessly due to widespread antibiotic use for
both prophylaxis and therapy. For example, CTX-M-type extendedspectrum-β-lactamases (ESBLs) conferring resistance to penicillins and
cephalosporins are increasingly prevalent in Enterobacteriaceae, such as
Escherichia coli and Klebsiella pneumoniae, which are ubiquitous in the
microbiota of the human gastrointestinal tract. CTX-M ESBLs are often
plasmid-borne, so displacing these plasmids could reduce the reservoirs
of resistance. One way to achieve this is to introduce a plasmid that
interferes with replication and maintenance functions of other plasmids.
The curing plasmid we have developed (pCURE) blocks replication of
the target plasmids and counters their post-segregational-killing systems.
A range of plasmids have been cured in the laboratory, but to extend
this to complex communities, a conjugative curing plasmid is being
developed. Functions known to cure F-like plasmids were integrated into
the broad host range IncP-1α plasmid RK2 which was shown to displace
an F-plasmid progressively from recipient bacteria, 84% being cured
after overnight growth. Work is underway to further increase the copy
number and test curing in complex bacterial communities with the long
term goal of targeting resistance reservoirs in humans and animals.
MA06/09
The mechanism of althiomycin biosynthesis in the insect pathogen
Serratia marcescens Db10
Amy J Gerc1, Lijiang Song1,2, Sarah Murdoch1, Gregory L Challis1,2,
Nicola R Stanley-Wall1, Sarah J Coulthurst1
1
University of Dundee, Dundee, UK, 2University of Warwick, Coventry, UK
There is a need to discover novel antibiotics in genetically amenable
micro-organisms. The enteric insect pathogen, Serratia marcescens
(Sma) Db10, produces a ribosome-inhibiting antibiotic called althiomycin.
Mapping the genetic locus required for althiomycin biosynthesis
revealed a six-gene operon named alb1-alb6. Chromatographic and
spectroscopic analyses of wild type and alb mutant strains of SmaDb10
confirmed that althiomycin was a product of the alb operon and the
likely biosynthetic pathway for althiomycin production was predicted.
A phosphopantetheinyl transferase enzyme required for althiomycin
biosynthesis was also identified. Expression of Alb1, a major facilitator
superfamily efflux pump, conferred althiomycin resistance on another,
althiomycin sensitive, strain of Sma. Deletion of alb1 abolished alb2-6
transcription by an as yet undefined mechanism. We hypothesised that
this decrease in transcription was a mode of ‘self-protection’ to avoid
the toxic effects of althiomycin. However, loss of transcription of alb2-6
was replicated upon deletion of alb1 in an althiomycin non-producing
strain. Consistent with higher order regulation, recent results show that
deletion of the RNA chaperone Hfq abolishes althiomycin biosynthesis.
Investigations are currently underway to determine how althiomycin
biosynthesis is regulated by Hfq, for example, whether it might be
controlled by a small RNA that binds within the alb1 coding region.
MA06/10
Comparative antimicrobial activities of different species of Acalypha
on selected clinical isolates in Obafemi Awolowo University Teaching
Hospital Complex, Ile-Ife
Racheal Hassan-Olajokun1, Love Modupe Awodiya2,
Olarinde Olaniran1
1
Obafemi Awolowo University, Ile-Ife, Osun State, Nigeria, 2Obafemi Awolowo
University Teaching Hospital Complex, Ile-Ife, Osun State, Nigeria
74
Antimicrobial activities of leaf extract of some species of Acalypha
were investigated for medicinal purposes against Staphylococcus aureus,
Escherichia coli, Pseudomonas aeruginosa, Proteus mirabilis, Alpha haemolytic
Streptococcus, Klebsiella pneumonia, Salmonella typhi and Candida albicans
using agar diffusion method. The extracts of A. wilkesiana showed the
greatest antimicrobial activity against Gram-positive, Gram-negative bacteria
and the fungus tested. Acalypha hispida burm showed antimicrobial activity
against Gram-positive and Gram-negative bacteria while A. ciliata was only
effective against Gram-negative bacteria. Results from this study confirmed
the broad-spectrum activities of some species of Acalypha plant which
traditional medicine practitioners use in treating various ailments including
fungi infections of the skin. The Acalypha extracts compared well with the
commercial antibiotics tested against the isolates.
MA06/11
Role of phage shock protein A during stationary phase in Burkholderia
pseudomallei
Stephanie J Southern1, Timothy Milne1, Mitali Sarkar-Tyson1,
Ali Tavassoli2, Petra Oyston1
1
Dstl, Salisbury, Wiltshire, UK, 2University of Southampton, Southampton,
Hampshire, UK
Burkholderia pseudomallei is a Gram-negative bacterium and the causative
agent of the disease melioidosis. Melioidosis can manifest in several forms,
including a chronic infection which can persist for months or years. This
makes treatment of B. pseudomallei infection extremely problematic,
complicated further by its inherent antibiotic resistance. This study
aims to evaluate the Phage-shock protein (Psp) response as a novel
therapeutic target in B. pseudomallei. The Psp system functions to maintain
the integrity of the inner membrane in response to extracytoplasmic
stress. Its importance for survival during stationary phase growth has
been demonstrated previously in Escherichia coli. B. pseudomallei ∆pspA
showed a growth defect after seven days at stationary phase. This was
accompanied by an increase in pH, reaching a maximum of 8.5 before
a decline in viability. This indicates that disrupting the Psp response in
B. pseudomallei affects its ability to maintain extracellular pH during
prolonged growth. Stationary phase B. pseudomallei ∆pspA was also less
able to survive in macrophages. A Galleria mellonella infection model is
therefore being developed to study its virulence in vivo. The development
of antimicrobials targeting proteins important for stationary phase survival,
such as PspA, may lead to more effective treatments for chronic infections.
MA06/12
Manuka honey inhibits adhesion and invasion of wound pathogens into
human keratinocytes
Sarah E Maddocks1, Rowena E Jenkins1, Richard S Rowlands1,
Kevin J Purdy2, Rose A Cooper1
1
Cardiff Metropolitan University, Cardiff, UK, 2The University of Warwick,
Coventry, UK
Wound infections present considerable challenges to the healing
process and choosing appropriate, effective treatments is paramount.
Anti-microbial agents have been applied to wounds for years, but the
emergence of resistant bacteria has driven the search for novel agents.
Manuka honey has a broad spectrum of antimicrobial activity and there
is currently no evidence of bacterial resistance to it. Its use is beginning
to re-emerge in the clinical setting but little is understood about the
antimicrobial mode of action of manuka honey. We have previously
shown that manuka honey disrupts biofilm development and prevents
adhesion of bacteria to the human tissue proteins fibronectin, fibrinogen
and collagen, suggesting that it has novel anti-adhesive properties in
addition to being bactericidal. In this investigation manuka honey was
shown to impair adhesion of laboratory and clinical isolates of S. aureus,
P. aeruginosa and S. pyogenes to human keratinocytes (HaCat) in vitro
and inhibited invasion into these cells by S. pyogenes and homogeneous
vancomycin intermediate S. aureus (VISA). Therefore the anti-adhesive
properties of manuka honey could make it a useful prophylactic
Poster abstracts
treatment, for example, post-surgery to help to reduce the likelihood of
surgical site infection rather than as a last resort treatment for chronic,
non-healing wounds.
MA06/13
Development of a novel antibacterial to block sporulation in
Clostridium difficile and prevent disease recurrence
Teresa Diaz-Calvo, Michael McArthur, Kostas Hatzixanthis
Procarta Biosystems Ltd, Norwich, UK
Clostridium difficile is an anaerobic, spore-forming bacterium that can
colonise the human gut with the resultant infection causing a wide range
of symptoms from severe diarrhoea and abdominal cramps to fever that
in severe cases can be fatal. The incidence and severity of infection are
strongly correlated with the extent and duration of antibiotic therapy
patients have previously received. This is because most antibiotics used
to treat the gut are broad-spectrum and will kill pathogenic and nonpathogenic bacteria alike. This sterilisation allows dormant spores of the
commensal C. difficile to germinate and rapidly colonise the gut producing
toxins that trigger inflammatory response leading to the symptoms
described. Spores are not-susceptible to antibiotic therapy, being shed
along with live bacteria in the faeces and greatly increasing the chance of
re-infection or spread to fellow patients. Hence preventing recurrence
of the disease is a major un-met medical need. This work describes the
development of a novel narrow-spectrum antibacterial to treat C. difficile
infections which specifically blocks sporulation so as to tackle the major
issue of recurrence. The novel antibacterial consists of an oligonucleotide
antibacterial, which blocks expression of the regulator of sporulation
(Spo0A) and is delivered to bacterial cells by a proprietary nanoparticle.
MA06/14
Evaluation of Mip as a novel antibacterial target in Burkholderia
pseudomallei
Isobel H Norville1, Darren Begley2, David Fox2, Dominic Jenner1,
Christina Juli4, Nicholas Harmer5, Robin Stacy3, Peter Myler3, Bart Staker2,
Lance Stewart2, Ulrike Holzgrabe4, Mitali Sarkar-Tyson1, Don Lorimer1
1
Dstl, Salisbury, UK, 2Emerald bio, Bainbridge Island, USA, 3Seattle Biomed,
Seattle, USA, 4Institut für Pharmazie und Lebensmittelchemie, Wurzburg,
Germany, 5University of Exeter, Exeter, UK
The bacterium Burkholderia pseudomallei is the causative agent of
melioidosis, a disease endemic in tropical regions. There is currently no
vaccine available against B. pseudomallei and the organism is resistant to a
range of antibiotics. Therefore identification of new drug targets is essential.
Macrophage infectivity potentiator (Mip) is a virulence factor encoded
by intracellular pathogens. Mips exhibit virulence-associated peptidylprolyl isomerase (PPIase) activity, inhibition of which may represent a
novel target for antimicrobial therapies.A Mip-like protein was identified
in B. pseudomallei (Bp-Mip) and shown to be required for full virulence.
Recombinant Bp-Mip protein exhibits PPIase activity and is inhibitable
by rapamycin and FK506. Novel Mip inhibitors have been identified and
shown to inhibit Bp-Mip PPIase activity at nanomolar concentrations. The
binding ability has been determined by HSQC-NMR and crystal structures
have been solved. In addition, these inhibitors reduce the cytotoxic effects
of B. pseudomallei on macrophages. Future work will focus on analysis of
X-ray crystal structures to optimise inhibitors.
MA06/15
Sensitivity to the glucosylated bacteriocin sublancin depends on the
phosphotransferase system
Emma L Denham1, Ruben A T Mars1, Kirsten Dörries2,
M Tanweer Khan1, Jörg Stülke3, Michael Lalk2, Wilfred A van der Donk4,
Jan Maarten van Dijl1
1
University Medical Center Groningen, Groningen, The Netherlands, 2ErnstMoritz-Arndt-University, Greifswald, Germany, 3University of Göttingen,
Göttingen, Germany, 4University of Illinois, Urbana, USA
75
Poster abstracts
The Gram-positive bacterium Bacillus subtilis 168 produces a potent
bacteriocin active against a number of Gram-positive pathogens including
Staphylococcus aureus. Sublancin does not target the cell wall and in
previous studies we identified the MscL channel as a key determinant
for sensitivity. We have since expanded our knowledge to other cellular
factors that affect the ability of sublancin to stop growth. Removal of
the glucose phosphotransferase system from a B. subtilis sensitive strain
confers resistance to sublancin. In the absence of PTS sugars, His15 of
HPr is phosphorylated by enzyme I, this phosphate is transferred to PTS
sugars when they are present. Sensitivity to sublancin could be rescued
by addition of PTS sugars or by mutation of His15 of HPr, suggesting
that the phosphorylated state is important for sublancin’s activity.
Metabolomics studies of cells treated with sublancin showed downregulation of many of the components of central carbon metabolism,
suggesting that this may be how the cell is prevented from growing.
Interestingly, tiling array analyses revealed increased expression of genes
involved in the Sigma-B regulon and in cysteine metabolism. Ongoing
studies are aimed at explaining the links between metabolism, cysteine
levels within the cell and sublancin sensitivity
MA06/16
LantiPRED: a software for in silico analysis and characterisation of
novel lantipeptide gene clusters
Kai Blin, Daniyal Kazempour, Wolfgang Wohlleben, Tilmann Weber
Institute for Microbiology and Infection Medicine, Universität Tübingen,
Tübingen, Germany
Lantipeptides are ribosomally synthetised secondary metabolites
that undergo post-translational modifications forming intramolecular
lanthionine and methyllanthionine residues. Many lantipeptides show
antibiotic activity even in low concentrations, and no significant novel
resistance mechanisms have been observerd. Thus, lantipeptides are
a very promising secondary metabolite family to exploit. So far, most
lantipeptides have been described in Firmicutes, most notably in
Lactococcus (nisin), Streptococcus (mutacin) and Bacillus (subtilin) species.
A lot of other bacteria also possess the genetic potential to produce
lantipeptides and might provide novel compounds and lead structures.
So far, no bioinformatics tools providing deeper insights in the biosythesis
of lantipeptides exist.
We have developed LantiPRED, the first specialised analysis software
to predict the biosynthetic class, molecular weight and putative posttranslational modifications of lantipeptide precursors. This poster will
present how LantiPRED can be used as part of a bioinformatics pipeline
when screening for novel lantibiotic compounds. Once a lantitpeptide
gene cluster has been identified in a genomic sequence using a generalpurpose secondary metabolite identification software like antiSMASH[1],
LantiPRED provides more detailed insights. LantiPRED is available as a
web service at http://lantipred.secondarymetabolites.org/. As part of our
ongoing development efforts on antiSMASH, a LantiPRED module is also
available directly integrated into antiSMASH 2.
MA06/17
Development of species specific protein antibiotics for the treatment
of Crohn’s disease
Carla L Brown, Karen Smith, Daniel Walker
Crohn’s disease (CD) is an incurable form of inflammatory bowel
disease which affects approximately 10-200/100,000 in Europe and USA.
CD is characterised by chronic, transmural and often granulomotous
inflammation and can occur at all regions of the gastrointestinal tract. The
novel E.coli pathotype, adherent invasive E.coli (AIEC) has been recently
linked to CD pathogenesis. AIEC form a thick biofilm on the ileal
mucosa of approximately 30% of CD patients making it highly resistant
to conventional antibiotics. Furthermore, AIEC adhere to and invade
intestinal epithelial cells and survive within host macrophages inducing
granuloma formation. In light of this data, it has been proposed that
CD treatment should involve the use of E.coli specific antibiotics. These
come in the form of potent, narrow-spectrum protein antibiotics, termed
colicins, which are produced by E.coli in response to nutrient stress. Our
preliminary data shows that colicins, unlike conventional antibiotics, are
highly active against AIEC when growing in the biofilm state. Additionally,
we have shown that colicin treatment of AIEC infected macrophages
causes a reduction in both intracellular growth of the bacteria and also in
release of inflammatory cytokines.
MA06/18
Investigating the biosynthesis of the antibiotic pacidamycin
Daniel Tromans1, David Lawson1, Mervyn Bibb1, Rebecca Goss2
1
John Innes Centre, Norwich, UK, 2University of St Andrews, St Andrews, UK
Pathogen resistance to antibiotics is becoming more prevalent every day
and there is an urgent need for new antibacterials with novel modes
of action. N-methyl-2,3-diaminobutyric acid (DABA) is an unusual and
key feature of several bioactive actinomycete natural products such
as the friulimicins and uridyl peptides. The uridyl peptides, such as
pacidamycin, inhibit bacterial cell wall biosynthesis through a clinically
unexploited target. DABA has been chemically synthesised, however the
biosynthesis of this diamine has yet to be elucidated. Previous studies on
the pacidamcyin gene cluster identified four candidate genes believed to
be involved in its production. We are using a multidisciplinary approach
involving x-ray crystallography, enzyme kinetics and gene disruption to
investigate the roles of these enzymes.
References 1. Winn, M. et al. Nat. Prod. Rep. (2010), 27, 279; 2. Rackham,
E.J. et al. Chem. Bio. Chem. (2010), 11, 1700; 3. Zhang, W. et al. PNAS (2010),
107, 16828; 4. Tromans, D. R. et al. Acta Cryst. (2012), F68, 971-974.
MA06/19
Structure and characterisation of pyocin L1, a novel lectin-like
bacteriocin from P. aeruginosa
Laura McCaughey, Daniel Walker
Pseudomonas aeruginosa is an opportunistic, Gram-negative bacterial
pathogen that has joined the rank of ‘superbugs’ due to its extreme
antibiotic resistance. A novel approach to tackling the multi-drug
resistance of P. aeruginosa is to utilise pyocins, bacteriocins from P.
aeruginosa, used for intraspecies competition. The crystallisation and
characterisation of a newly discovered bacteriocin, pyocinL1, has shown
that this pyocin is structurally homologous to monocot mannose-binding
lectins (MMBL’s). Unlike MMBL’s though, pyocinL1 has acquired a
species-specific killing activity that is also strain-specific, killing 31% of P.
aeruginosa strains tested and weakly killing some strains of P. Syringae.
Small angle X-ray scattering and analytical ultracentrifugation experiments
have shown that unlike MMBL’s, pyocinL1 is a monomer in solution.
There are two MMBL domains in the pyocinL1 structure that bind three
mannose molecules with milli-molar affinity. The mechanism of action
of pyocinL1 still remains to be determined but isolation of pyocinL1
tolerant mutants have indicated that this bacteriocin is translocated
across the outer membrane using the TonB system.
MA06/20
Microbial diversity in the cystic fibrosis lung – assessing the limitations
of current diagnostic microbiology and antibiotic susceptibility profiling
Sophie E Darch1, Shanika A Crusz1,2, Stephen Holden2,
Andrew Fogarty3, Stephen P Diggle1
1
School of Molecular Medical Sciences, University of Nottingham,
Nottingham, UK, 2Department of Clinical Microbiology, Nottingham University
Hospitals NHS Trust, Nottingham, UK, 3Division of Epidemiology and Public
Health, University of Nottingham, Clinical Sciences Building, City Hospital,
Nottingham, UK
The Cystic Fibrosis (CF) lung presents a complex polymicrobial ecology.
One of the most commonly associated pathogens is Pseudomonas
76
aeruginosa. The pathogen is capable of infection via the formation
of biofilms, which are often highly resistant to antibiotic therapies.
Routine antibiotic susceptibility testing relies on the testing of a single
‘morphotype’ planktonically. We tested 44 morphotypically identical
P. aeruginosa colonies taken from a single sputum sample. Phenotypic
assays were performed for growth and virulence factor production.
The standardised BSAC method was employed to test all strains
both individually and within mixed populations. Phenotypic analysis
demonstrates large variances in growth, virulence factor production and
quorum sensing (QS) signal molecule production between colonies,
despite all being morphologically identical. Antibiotic susceptibility data
indicates variances between colonies but also a significant increase
in resistance when colonies are mixed. We demonstrate a detailed
screening of a morphologically identical population, displaying huge
phenotypic and antibiotic susceptibility differences. This suggests that
analysing several colonies of the same morphotype (populations
of bacteria), could not only provide a more accurate method for
determining the antibiotic sensitivity of P. aeruginosa in the CF lung, but
also has the potential to be applied to other chronic infections in vivo.
MA06/21
A KorSA protein homologue (SCO3932) is a potential regulator of
the coelimycin gene cluster cpk of Streptomyces coelicolor A3(2)
Mateusz Biernacki1, Mateusz Zelkowski1, Katarzyna Litwinska1,
Magdalena Kotowska1, Krzysztof Pawlik1,2
1
Institute of Immunology and Experimental Therapy, Polish Academy of
Sciences, Wroclaw, Poland, 2Department of Toxicology, Wroclaw Medical
University, Wroclaw, Poland
Streptomyces coelicolor A3(2) is a well-known producer of coloured
polyketide antibiotics: red undecylprodigiosin, blue actinorhodin
and recently reported yellow coelimycin [1]. It was shown that the
production of coelimycin is dependent on a number of factors: medium
composition -influence of glucose [2] and glutamate[3], the quorumsensing regulatory system and others.
Here we present the results of searching for direct regulators of
coelimycin producing gene cluster. Using the DNA region between
genes cpkA i cpkD in affinity chromatography we caught the protein
SCO3932. The protein is a KorSA homolog and it is also homological to
GntR protein family containing wHTH motif and UTRA effector domain.
We have found that protein SCO3932 binds the putative promoter
region of structural genes cpkABC, and of genes cpkO and cpkN coding
for SARP-like activators. Its own promoter region is also bound by
protein SCO3932. In Streptomyces ambofaciens KorSA is a is a central
transcriptional repressor for integrative element pSAM2 [4].
References 1. Gomez-Escribano J.P., et al. (2012) Chem. Sci. 3:27162720; 2. Pawlik K., et al. (2010) J. Mol. Microbiol. Biotechnol. 19:147-51;
3. Gottelt M., et al. (2010) Microbiology (Reading, Engl.) 156:2343-53; 4.
Sezonov G., et al. (2000) J. Bacteriol. 182:1243-50.
MA06/22
Antibiotic combination and the post-antibiotic effect; possible
approaches to tackle antibiotic resistance
Amir Ali Shirazian, Yanmin Hu
St Georges University of London, London, UK
Since the discovery of penicillin, bacteria have demonstrated to be highly
capable of developing resistant mechanisms to antibiotics. In the future,
with the rapid emergence of resistant bacterial strains, most, if not all
antibiotics will become redundant. This project investigates antibiotic
combination and the post-antibiotic effect (PAE), as a mean to tackle
emergence of antibiotic resistance.
In this study, the rate of resistance development of S. aureus for
amoxicillin and clarithromycin, whilst being treated with sub-MIC
concentrations of these two singly or in combination, was measured
by recording the corresponding MICs every 5 generations. The results
indicated that after 40 generations, the MIC of cultures treated with
Poster abstracts
amoxicillin and clarithromycin alone was increased by 48 and 256 folds.
Interestingly, the MIC increase in cultures treated with combination
therapies was significantly less. Furthermore, the PAE of amoxicillin and
clarithromycin were measure singly and in combination. The results
highlighted the PAE of the combination therapy to be 2.4 and 5.6 hours
longer than the respective monotherapies.
In conclusion, this study illustrates combination therapy to prolong the
PAE, and to reduce the rate of development of antibiotic resistance,
suggesting synergistic combination therapy as an effective approach
against the rapid emergence of antibiotic resistance.
MA06/23
Quorum sensing inhibitors: silver bullets?
Richard C Allen1, Sam P Brown1,2
1
Institute of Evolutionary Biology, The University of Edinburgh, Edinburgh, UK,
2
Centre for Immunity Infection and Evolution, The University of Edinburgh,
Edinburgh, UK
The emergence of antibiotic resistant bacterial strains far outstrips the
discovery of novel antibiotics. Given this problem there has been greater
emphasis on the development of anti-virulence therapeutics to combat
microbial infections. Anti-virulence therapeutics are hypothesised to
be evolutionary robust, because the absence of direct bactericidal (or
bacteriostatic) effects may reduce selection for resistant strains.
We will present predictions from a review exercise of recent antivirulence drug literature specifically focusing on inhibitors of quorum
sensing, a signalling behaviour found in many pathogens. By applying
existing ecological and evolutionary theory to recent findings, we explain
how nutritional environment and population structuring within the host
impacts selection on resistant strains. Expanding on this framework we
also present novel predictions showing that the signalling processes
associated with quorum sensing mean that the way we inhibit quorum
sensing may be critical. We predict that the specific mechanism of
quorum sensing inhibition will change the sign of selection on resistant
strains in some circumstances. These predictions have important
implications for the rational design of anti-virulence drugs, so as to
maximise their useful lifespan.
MA06/24
Exploiting Burkholderia for antibiotic discovery
Paulina K Sydor1, Lijiang Song1, Eshwar Mahenthiralingam2,
Gregory L Challis1
1
University of Warwick, Coventry, UK, 2University of Cardiff, Cardiff, UK
The Burkholderia cepacia complex (Bcc) is a group of closely related
Gram-negative bacteria. Members of this group are known for their
many beneficial agricultural applications, but also as pathogens that
cause devastating lung infections in patients with cystic fibrosis (CF).1
All Bcc strains are highly resistant to most antibiotics and they quickly
predominate over other bacteria once they infect the lungs of CF
patients, suggesting they may themselves produce antibiotics that kill
competitors.
In collaboration with Prof. Eshwar Mahenthiralingam at the University of
Cardiff, screening a large collection of Bcc strains has identified strong
activities produced by Burkholderia ambifaria against multidrug-resistant
pathogens. A novel and unusual B. ambifaria anti-Gram-negative activity
was linked to the production of the known antibiotic enacyloxin IIa and a
novel derivative iso-enacyloxin IIa.2
Recent progress in elucidating the biosynthetic pathway of enacyloxin IIa
will be presented. Efforts to produce novel analogues of these natural
products via manipulation of the biosynthetic pathways will also be
described.
References 1. Coenye, T.; Vandamme, P. Environ Microbiol, 2003, 5,
719-729; 2. Mahenthiralingam, E.; Song, L.; Sass, A.; White, J.; Wilmot, C.;
Marchbank, A.; Boaisha, O.; Paine, J.; Knight, D.; Challis, G.L. Chem Biol,
2011, 18 (5), 665-677.
77
Poster abstracts
MA06/25
Discovery of antimicrobial peptides active against Gram-negative
pathogens
Arif Felek, John Moat, Mathew Upton
Antibiotic resistant Gram negative bacteria are a leading threat to
human health and there is an urgent need for novel therapies. This study
embraces natural antimicrobial product screening and combines it with
HPLC, mass spectrophotometry and bioinformatic techniques in order
to identify and characterise bacterially derived effective novel inhibitors of
Gram negative bacteria.
During the study two lead antimicrobial peptides (AMPs) have been
identified, purified and partially characterised. Peptide NI03, isolated from
Bacillus pumilus, has been shown to inhibit both Gram positive (MRSA)
and Gram negative pathogens and anti-Gram negative agent, peptide
NI05 was isolated from Klebsiella pneumoniae. These peptides showed
significant activity towards extended spectrum beta lactamase producing
Escherichia coli, K. pneumoniae and Pseudomonas aeruginosa isolates.
Both AMPs were observed to be highly stable against a number
of digestive enzymes and heat. The mass data (NI03: 1754.622 Da
and NI05: 1796.253) obtained from MALDI-TOF MS analysis was
compared to published literature, online databases (Bactibase) and
patent catalogues and no matches were found, indicating that the AMP’s
identified are novel agents. We have recently determined the genome
sequence of the producing isolates to identify the genetic loci required
for production of these peptide antimicrobials through de novo peptide
sequencing techniques.
MA06/26
Tetronic acid biosynthesis in abyssomicin antibiotics
Laura Vieweg1, Peter F Leadlay2, Roderich D Süssmuth1
1
Department of Chemistry, Berlin Institute of Technology, Berlin, Germany,
2
Department of Biochemistry and Cambridge Centre for Molecular
Recognition, University of Cambridge, Cambridge, UK
Throughout the past years an increasing number of tetronic acidcontaining polyketides have been discovered, many of which reveal
interesting biological activities. Among these the abyssomicins are
characterised by an oxabicyclooctane system and the tetronic acid
unit.[1] The most prominent example of the abyssomicins is the atropabyssomicin C, which is a potent antibiotic against a variety of Grampositive bacteria including pathogenic and drug resistant strains like
methicillin-resistant Staphylococcus aureus (MRSA).[2] The biosynthesis
of abyssomicin-like natural products is based on the assembly of linear
precursors, followed by a postulated Diels-Alder-reaction and further
oxidation and decoration steps.[3] While the biosynthesis of the linear
atrop-abyssomicin C precursor has been unravelled, less is known about
the formation and tethering of the tetronic acid moiety. This work
focuses on the elucidation of this mechanism.
[1]
Bister et al. (2004), Angew. Chem. Int. Ed., 43: 2574-2576.
[2]
Riedlinger et al. (2004), J. Antibiot., 57: 271-279.
[3]
Gottardi et al. (2011), ChemBioChem, 12: 1401-1410.
MA06/27
Structure and function of the essential virulence protein DsbA from
Burkholderia pseudomallei
Philip M Ireland1, Róisín Mcmahon2, Laura Marshall1, Maria Halili2,
Emily Furlong2, Stephanie Tay2, Jennifer L Martin2, Mitali Sarkar-Tyson1
1
Defence Science and Technology Laboratory, Wiltshire, UK, 2The University
of Queensland, Queensland, Australia
Burkholderia pseudomallei is endemic in South-East Asia and northern
Australia. The disease caused by B. pseudomallei, melioidosis, can
be variable in humans and may involve pulmonary infection, acute
septicaemia and a chronic or latent infection. Currently, no licensed
vaccine is available and the organism’s virulence mechanisms are poorly
understood. Prognosis following melioidosis is poor due to inadequate
therapies which are often protracted and complicated due to inherent
resistance of B. pseudomallei to many antibiotics. There is also a lack
of prophylaxis, with vaccine development in the preliminary stages.The
periplasmic protein DsbA catalyses the folding of virulence proteins in
a range of bacterial species. B. pseudomallei encodes a putative dsbA
gene containing the cys-pro-his-cys active site motif identified as the
major disulphide forming protein in Escherichia coli. This study evaluated
the redox biochemistry and determined activity consistent with that
of a protein disulphide oxidoreductase. Construction of an in-frame
deletion mutant of B. pseudomallei demonstrated that dsbA is essential
for virulence in a Balb/C mouse model of infection and for the secretion
of virulence factors. In addition, elucidation of the DsbA crystal structure
revealed similarities to Pseudomonas aeruginosa that will aid future
structure based drug design targeting this key virulence protein.
MA06/28
β-Lactam resistance; not as social as we thought?
Frances A Medaney1, Ben Raymond1, Richard J Ellis2
1
Royal Holloway, University of London, Egham, UK, 2Animal Health and
Veterinary Laboratories Agency, Addlestone, UK
Microbial sociality is of particular interest because many virulenceassociated traits such as quorum sensing, siderophore production
and toxin secretion are cooperative. Antibiotic resistance conferred
by enzymatic breakdown of drugs can be considered a cooperative
trait, as it detoxifies the environment for all cells. Previous studies have
demonstrated the survival of antibiotic sensitive E. coli and Salmonella in
the presence of a resistant strain at high concentrations of antibiotic. The
production of β-lactamases, which cleave and deactivate penicillins, is
often cited as a social trait in bacteria. In this study we use the naturally
occurring extended spectrum β-lactamase (ESBL) resistance plasmid,
pCT, to confer resistance to β-lactam antibiotics. Otherwise isogenic
plasmid-free E. coli were competed with the pCT-carrying strain under
a variety of conditions. The resistant strain shows protective clearance
of β-lactams, facilitating the growth of susceptible E. coli, only under
very specific conditions, suggesting that cooperative antibiotic resistance
occurs only in limited circumstances.
MA06/29
Zirconium nitride silver nanocomposite coatings to combat external
fixation pin infection
David Wickens, Glen West, Joanna Verran, Peter Kelly,
Kathryn Whitehead
External bone fixation is a frequently applied medical procedure for
rehabilitating severe fractures. The pins used require strict cleaning
regimes at the entry site. However, the pin sites are still putative
ports for conceivable pathogenic microbial infection. The study aim
was to potentially combat and reduce pin tract infections (PTI) using
zirconium nitride silver coatings; investigate the coating characteristics,
determine the antimicrobial properties against Staphylococcus aureus
and Staphylococcus epidermidis using LiveDead™ and CTC staining,
and demonstrate the coatings biocompatibility against human cells (U937 monocyte inflammatory cell line). Zirconium Nitride (ZrN) is a
hard wearing compound possessing corrosion resistance, thus giving it
biomaterial potential. Combining with silver gives the coating potential
for a multifunctional surface with antimicrobial characteristics. Magnetron
sputtering was used to reactively co-sputter zirconium and silver, to
produce nanocomposite thin films with a range of silver concentrations.
Addition of silver to the coatings increased antimicrobial efficacy towards
the Staphylococci. No antimicrobial leaching of silver was observed from
any of the surfaces, however S. aureus was retained in higher numbers
than S. epidermidis. The bacteria were reduced by 1-log when added to
the U-937 cells. These coatings have demonstrated potential to be used
on external fixation pins to reduce PTIs.
78
Poster abstracts
MA06/30
Development of epidermicin, a potent novel anti-staphylococcal
bacteriocin
Samantha Halliwell, Tarek Gibreel, Jeremy Derrick, Peter Warn,
Mathew Upton
Resistance to antibiotic therapy is a major threat to human health.
Epidermicin is a novel bacteriocin with rapid potent bactericidal activity
against a wide range of Gram-positive pathogens. The peptide was
identified during our ongoing natural product screening programmes,
which are supported by draft genome sequence determination of
inhibitor producing bacteria. Epidermicin perturbs the bacterial cell
membrane resulting in rapid depolarisation and cell death. In a multidrug
resistant, Staphylococcus epidermidis biofilm, epidermicin caused rapid,
concentration dependent membrane depolarisation, leading to increased
membrane permeability and leakage of cellular ATP and potassium
ions. In silico predictions, using the I-TASSER online server indicate, with
high confidence, that epidermicin forms a three helix bundle-type fold.
Epidermicin’s closest homolog, lacticin Q, forms large torroidal pores in
membranes of target bacteria and it is probable that epidermicin has a
similar mode of action. Epidermicin can protect Galleria mellonella larvae
from infection with MRSA in a dose dependent fashion and we are
developing the molecule for testing in cotton rat nasal decolonisation
models. We suggest that epidermicin, and other bacteriocins, have
excellent potential for development into novel therapeutics to combat
infections caused by antibiotic resistant bacteria.
Institute of Microbiology and Infection, The University of Birmingham,
Birmingham, UK, 2School of Biosciences, University of Nottingham, Sutton
Bonington campus, Sutton Bonington, Loughborough, UK
The outer membranes of Gram-negative bacteria function as a barrier
to protect cells from toxic compounds. The inner leaflet of the outer
membrane is composed of phospholipids, whilst the outer leaflet is
predominantly lipopolysaccharide (LPS). LPS consists of lipid A to
which sugar units are added to generate the core LPS. The core LPS is
further modified by the attachment of a repeat oligosaccharide unit, the
O-antigen. All laboratory strains of Escherichia coli K-12 are phenotypically
rough, being unable to synthesise O-antigen due to mutations in the rfb
gene cluster. Here we have generated an E. coli K-12 strain, DFB1655
L9, which expresses full length O-antigen and demonstrate that it has
increased resistance to many environmental insults, e.g. low pH and
human serum exposure. Surprisingly, growth experiments and Biolog
Phenotype Microarray analysis, which compares the metabolic activity of
strains under ca. 2000 different conditions, indicated that strain DFB1655
L9 was more sensitive to the fatty acid biosynthesis inhibitor triclosan.
As triclosan resistance has been demonstrated in many bacteria, with
resistance mechanisms including mutations within fabI, derepression of
drug efflux pump expression and the production of inactivating enzymes,
the loss of O-antigen biosynthesis represents a novel mechanism for
triclosan resistance.
1
MA06/31
A putatively novel broad spectrum antibiotic from the Streptomyces
isolate DEM 30543
George Kemp1,2, Nick Allenby2, Michael Goodfellow2,3, Jeff Errington2,4
1
Biopharmaceutical and Bioprocessing Technology Centre, Newcastle
University, Newcastle upon Tyne, UK, 2Demuris Ltd, Newcastle upon Tyne,
UK, 3School of Biology, Newcastle University, Newcastle upon Tyne, UK,
4
Institute for Cell and Molecular Biosciences, The Centre for Bacterial Cell
Biology, Newcastle University, Newcastle upon Tyne, UK
Screening of the Goodfellow actinomycete collection has identified
a putatively novel antibiotic with broad spectrum activity from the
Streptomyces isolate DEM30543. The compound has been shown to
be active against a range of both Gram positive and Gram negative
bacteria, including several pathogenic strains such as Methicillin-resistance
Staphylococcus aureus (MRSA) and Pseudomonas aeruginosa.The biological
and chemical characteristics of the molecule have been investigated
further. The producing strain DEM 30543 has been fermented at both
laboratory and pilot scale (500 litres) where growth and antibiotic
production profiles have been characterised. Using whole cell bioassays it
has been indicated that the potential mechanisms of action are inhibition
of cell wall synthesis or disruption of cell wall metabolism. This has been
shown in independent bioassay systems using both Escherichia coli and
Bacillus subtilis. The Minimum Inhibitory Concentration (MIC) value of
the molecule against Bacillus subtilis and Escherichia coli is ~1µg/ml. MIC
values for pathogenic strains are yet to be determined. Stability studies
have shown the antibiotic molecule is stable at ambient temperatures as
well as elevated temperatures (37 and 55 degrees Celsius) for prolonged
periods. Preliminary 1H NMR and 13C NMR studies have been
conducted revealing the core structure of the molecule.
MA06/32
Restoration of O-antigen biosynthesis in Escherichia coli K-12 increases
sensitivity to the biocide triclosan
Douglas F Browning1, Timothy J Wells1, Fernanda L S França1,
Faye C Morris1, Yanina R Sevastsyanovich1, Jack A Bryant1, Matthew D
Johnson1, Peter A Lund1, Adam F Cunningham1, Jon L Hobman2,
Robin C May1, Mark A Webber1, Ian R Henderson1
MA06/33
Investigation of temocillin as a possible additive to orthopaedic cement
for local antibiotic delivery
Stewart Barker1, Tim Nichol1, Ian Stockley2, Robert Townsend2,
Thomas J Smith1
1
Sheffield Hallam University, Sheffield, UK, 2Sheffield Teaching Hospitals NHS
Trust, Sheffield, UK
In the UK during 2010, bone cement was used in approximately 90,000
arthroplasty (joint replacement) operations. Adding one or more
prophylactic antibiotics to bone cement reduces postoperative infection
rates. Due to problems with antibiotic-resistant bacteria, particularly
during revision surgery to replace infected prostheses, a wide range of
antibiotics is needed. However, currently not all antibiotics have been
tested in bone cement. Here, the Gram negative-specific antibiotic
temocillin was investigated to determine its elution properties from
polymethylmethacrylate (PMMA) bone cement that was also pre-loaded
with gentamicin, a commonly used antibiotic in this application. The
antibiotic-loaded PMMA was submersed in buffer and samples were
removed and analysed by HPLC-MS. Temocilin was also separated from
the eluted gentamicin by FPLC and minimal inhibitory concentrations
(MICs) were determined against a range of antibiotic-resistant Escherichia
coli. HPLC-MS data showed temocillin concentrations of ≥ 50 µg/mL,
from the first hour of elution. This far exceeds the MICs of sensitive
strains, which range from 1.9-15.3 µg/mL, showing that the eluted
temocillin retained antimicrobial activity. Likewise, the gentamicin also
eluted in high concentrations (≥240 µg/mL) from hour one. These are
encouraging data for the application of temocillin as a locally delivered
antibiotic in orthopaedic surgery.
MA06/34
Synthesis of novel antibacterial proteins in the chloroplast of the green
microalga Chlamydomonas reinhardtii
Laura Stoffels1, Bambos Charalambous2, Saul Purton1
1
Institute of Structural and Molecular Biology, University College London,
London, UK, 2Research Department of Infection, University College London,
London, UK
Endolysins are antibacterial proteins that are produced by bacteriophages
to digest the bacterial cell wall for phage progeny release at the end of
the lytic cycle. These efficient enzymes are highly specific for the cell
wall of the target bacteria without affecting other species. Development
79
Poster abstracts
of resistance against endolysins is very rare, because they evolved to
target molecules in the cell wall that are essential for bacterial viability.
Taken together, this makes them promising novel antibacterial agents.
C. reinhardtii offers already established techniques for the expression of
foreign genes in the chloroplast and is an attractive expression platform
for therapeutic proteins, due to the lack of endotoxins and potentially
infectious agents. Furthermore it can be inexpensively cultivated in full
containment and under sterile conditions in simple photobioreactors.
Two bacteriophage endolysins targeting two major human pathogens
were successfully expressed in the chloroplast of C. reinhardtii, and their
specificity and efficiency in killing the target bacteria was assayed in vitro.
MA06/35
Characterising the chaperone protein SurA, and its potential role in
Burkholderia infection
Tara R C A Macey1, Isobel Norville2, Mitali Sarkar-Tyson2,
Nicholas Harmer1
1
University of Exeter, Exeter, UK, 2Dstl, Salisbury, UK
Burkholderia pseudomallei is a facultative intracellular pathogen, and
the causative agent of the devastating tropical disease melioidosis. It
causes significant levels of disease throughout the tropics, most notably
in South-East Asia and Northern Australia. However, with significant
mortality rates, and an often latent and indistinguishable infection, B.
pseudomallei is considered amongst the most serious biological warfare
threats. Survival protein A (SurA) is a periplasmic peptidyl-prolyl
isomerase and chaperone. In E. coli, it assists in the folding and assembly
of outer membrane proteins important in infectivity and virulence. In B.
thailandensis E264, a close but less virulent relative of B. pseudomallei,
surA mutants are highly attenuated. To dissect the role of the three
domains of SurA, we used each combination of domains to complement
these mutants. We characterised these mutant-complements through
assays for motility, polymyxin B susceptibility and infection of the larvae
of the wax moth Galleria mellonella. These assays indicate the importance
of SurA in virulence, but moreover reveal the N-terminal domain
to be the most significant domain for supporting infection. A mass
spectrometry comparison of membrane proteins from wild-type, mutant,
and mutant-complement B. thailandensis, confirms the role of SurA as a
chaperone protein.
MA06/36
The use of oxacillin, a β-lactam antibiotic to target the quorum sensing
system during MRSA infections; a novel anti-virulence application for
an old antibiotic
Justine Rudkin1,2, Andrew Edwards3, Katrina Lennon1, James O’Gara2,
Nicholas Waterfield1, Ruth Massey1
1
University of Bath, Bath, UK, 2NUI Galway, Galway, Ireland, 3Imperial College
London, London, UK
Methicillin Resistant Staphylococcus aureus (MSRA) is a prolific cause
of hospital associated infections and is currently spreading beyond our
healthcare facilities into the community. Resistance to multiple classes
of antibiotics is aiding the success of these strains, adding to their
status as ‘superbugs’. Like many bacterial infections, novel methods of
treatment and control are increasingly needed. As the rate of resistance
development continues to outpace the rate at which new antibiotics
make it to market, novel approaches need to be taken. One approach
widely discussed is targeting bacterial quorum sensing systems. We
have previously shown that activation of mecA, which encodes penicillin
binding protein 2a and methicillin resistance in MRSA strains has a
negative effect on the Agr quorum sensing system, blocking toxin gene
expression (Rudkin et al, 2012. Pozzi et al, 2012). Here we report that
the β-lactam antibiotic oxacillin, to which MRSA is resistant, activates
mecA gene expression, therefore down regulating toxin production by
blocking the Agr quorum sensing system. Administration of oxacilin in
an invertebrate model of MRSA infection significantly reduced morbidity
revealing a potentially novel therapeutic application of this old drug.
MA06/37
Investigating the antibiotic productivity of Streptomyces rimosus
Alison C MacFadyen1, Florence Pethick1, Zhenyu Tang2,1,
Vartul Sangal1, Paul A Hoskisson1, Ralph Kirby3, RuAngelie Edrada-Ebel1,
Iain S Hunter1, Paul R Herron1
1
University of Strathclyde, Glasgow, UK, 2East China University of Science and
Engineering, Shanghai, China, 3National Yang-Ming University, Taipei, Taiwan
Streptomyces rimosus, the industrial strain used in the production of
the type-II polyketide antibiotic oxytetracycline (OTC), has undergone
extensive strain improvement over the past 50 years. This has resulted
in OTC levels increasing from less than 0.5g per litre in the original
soil isolate to over 70g per litre for contemporary production strains.
In order to understand this increase in antibiotic productivity, we
have sequenced the genome of the original soil isolate, S. rimosus
ATCC 10970, as well as several strains taken from the Pfizer strain
improvement program. These newly obtained genome sequences have
allowed us to discover many previously unknown secondary metabolite
biosynthetic clusters as well as to begin to understand the genetic basis
of elevated levels of antibiotic titre by production strains . We have also
been able to use this data to identify the gene cluster associated with
rimocidin production. Although previous data has demonstrated that
S. rimosus produces rimocidin, until now the identity and arrangement of
a majority of the genes within the biosynthetic cluster was unknown.
We present the complete biosynthetic cluster for the production of
rimocidin and predictions on the role of selected genes within the
cluster.
MA06/38
Expression of bacteriophage endolysins in the Chlamydomonas
chloroplast for use as novel antibacterials
Henry N. Taunt, Saul Purton, Bambos Charalambous
Bacteriophage endolysins hold great promise as antibacterials since they
can lyse a particular bacterial pathogen without affecting the body’s
natural flora, do not result in acquired resistance in the pathogen, and
are affective on mucosal surfaces. They also hold several benefits over
whole phage therapy, including ease of production and application. As an
initial study we have expressed an endolysin in a relatively new platform,
the Chlamydomonas chloroplast, which combines routine techniques for
foreign gene expression, low cost cultivation, and lack of endotoxins or
potentially infectious agents.
In vitro studies conducted using cell extracts prepared from the
Chlamydomonas transgenic line have shown selective inhibition of the
target bacterium, and highly enriched protein fractions have displayed
clear dose-dependent activity. Production of the transgenic lines has
been scaled up to a large lab-scale with potentially easy scalability to
industrial levels of production.
In an extension to this study, three chimeric constructs have been built
fusing two different endolysins together in order to give multi-pathogen
specificity while maintaining absence of activity against commensal
bacterial flora. Good protein accumulation has been observed following
initial expression in E. coli, and selective lysis of target cells detected.
Studies of Chlamydomonas transformants are in progress.
MA06/39
Isolation and purification of a novel antibiotic (DEM32253)
fromStreptomyces sp.
Hamed Mosaei Sejzi1,3, Michael Goodfellow1,2, Jeff Errington1,3,
Nick Allenby1
1
Demuris Ltd, Bioincubator Suite, William Leech Building,Medical
School,Newcastle University, Newcastle upon Tyne, UK, 2School of Biology,
Ridley Building, Newcastle University, Newcastle upon Tyne, UK, 3Institute
for Cell and Molecular Biosciences (ICaMB), Catherine Cookson, Building,
Medical School, Newcastle University, Newcastle upon Tyne, UK
80
Poster abstracts
gram-negative bacteria including ESBL producing strains was tested
using the BS ISO 22196:2011 method. A log10 reduction in viability
of >5 was obtained within 4 h for the disinfectant test strains. Activity
against the hospital isolates was slower but still gave log10 reduction
factors of >5 for ESBL producing Escherichia coli and >3 for VRE, MRSA
and Acetobacter baumannii within 24 h. The results demonstrate the
importance of testing antimicrobial materials destined for healthcare use
against isolates of current interest in hospitals as well as standard test
strains. Preliminary results of evaluation of the activity of coated ceramic
tiles in a hospital setting will be reported. The coatings have potential
applications where reduction of microbial environmental contamination
is desirable.
Despite the growing threat of bacterial resistance, new classes of
antibiotics are rarely introduced to the clinical phase.[1] Amongst all the
antibiotics produced by Actinomycetes, almost half of them are made
by genus Streptomyces.[2] The strain, DEM32253 was isolated from the
marine environment with antimicrobial activity against Gram positives.
The strain was screened in bio-assays (in vivo) to determine the activity
and mode of action in parallel with the 16s rRNA and phylogenetic
tree analysis. Media optimisation tests were followed by Purification and
Identification of the active compound using Polymeric Adsorbent beads,
SPE (Solid Phase Extraction), HPLC and FTMS (The Fourier Transform
Mass Spectroscopy). Microscopic assays indicated that the antimicrobial
is effective on bacillus cell wall. Transcription and cell wall biosynthesis
inhibition was observed in the preliminary screenings. The cell wall
inhibitor molecule was purified and novel accurate mass (~737 AMU)
was achieved. Addressing the structure of molecule and the class of
antibiotic is now being investigated.
References 1. Donadio, S., et al., Sources of novel antibiotics-aside the
common roads. Applied Microbiology and Biotechnology, 2010. 88(6): p.
1261-1267; 2. Watve, M., et al., How many antibiotics are produced by the
genus Streptomyces? Archives of Microbiology, 2001. 176(5): p. 386-390.
MA06/40
Transposon mutagenesis identifies regulation pathways involved
in curli repression of efflux mutants of Salmonella enterica serovar
Typhimurium
Stephanie Baugh, Aruna S Ekanayaka, Laura J V Piddock,
Mark A Webber
The University of Birmingham, Birmingham, West Midlands, UK
Salmonella is known to have nine multidrug resistance (MDR) efflux
pumps able to export a wide variety of substrates including antibiotics.
However these efflux systems also have roles in virulence, cell division
and biofilm formation. This study investigates the mechanism linking
MDR efflux and biofilm formation in Salmonella Typhimurium.
Transposon mutagenesis was used to identify genes involved in biofilm
formation and phenotypic characterisation used crystal violet biofilm
assays and Congo red curli staining.
Mutants lacking any of the MDR transporters or the outer membrane
export porin, tolC, were found to have a biofilm formation defect.
This defect in all mutants was due to down regulation of curli at a
transcriptional level. Transposon mutagenesis of a selection of the
efflux mutants identified colonies with normal curli production. Rescue
mutants had transposon insertions within various genes and fully restored
expression of curli and biofilm formation.
This study reveals new insights into the regulation of efflux and biofilm
formation in Salmonella and begins to unravel the regulation pathways
involved in curli repression observed. This work also has therapeutic
impact as chemical inactivation of efflux in vivo represses transcription of
curli and therefore abolishes biofilm formation, giving rise to a potential
new anti-biofilm agent.
MA06/41
Novel antimicrobial coatings for reduction of transmission of
infectious diseases via surfaces
Souad O Elfakhri1, David W Sheel1,2, Paul Sheel2, Frederic W (Eric)
Bolton1, Howard. A Foster1
1
University of Salford, Salford, Lancs,, UK, 2CVD Technologies Ltd, Salford,
Lancs, UK
There is increasing recognition that the healthcare environment acts as
a reservoir for transmission of healthcare acquired infections (HCAI).
One method of reducing environmental contamination this would be
use of antimicrobial materials. The antimicrobial activity of thin copper
containing silica films prepared by chemical vapour deposition were
evaluated against hospital related pathogens. The activity of the coatings
on glass against standard strains of bacteria used for disinfectant testing
and bacteria of current interest in HCAI including VRE, MRSA and
MA06/42
DEM30355/A a novel first in class polyketide from an Amycolatopsis sp.
Bernhard Kepplinger1,2, Michael Goodfellow2,3, Jeff Errington2,4,
Nick Allenby2
1
Biopharmaceutical Bioprocess Technology Centre, Newcastle University,
Newcastle Upon Tyne, UK, 2Demuris, Newcastle Upon Tyne, UK, 3School of
Biology, Newcastle University, Newcastle Upon Tyne, UK, 4Institute for Cell
and Molecular Biosciences (ICaMB), Newcastle University, Newcastle Upon
Tyne, UK
The genus of Amycolatopis produces various important compounds
such as vancomycin and rifamycin[1, 2]. Demuris has obtained a range
of promising hits from this genus. Following the hypothesis that novel
strains isolated from extreme habitats will produce new compounds,
the strain DEM30355 was chosen for further investigation[3]. This strain
shows twelve base pairs difference in its 16S RNA sequence to its
nearest type strain and was isolated from a dessert environment. The
active compound shows activity against a broad range of Gram-positive
pathogens including Methicillin-resistant Staphylococcus aureus (MRSA)
at a MIC between 4-16mg/L. Little toxicity was observed towards
eukaryotic yeast and the human cancer cell line Hep3G. Structural
elucidation was performed via NMR and X-ray diffraction experiments.
This first in class polyketide fulfils lipinskis rule of five and shows little
similarity with any other antibiotic.
References 1. Levine, D.P., Vancomycin: a history. Clin Infect Dis, 2006.
42 Suppl 1: p. S5-12; 2. Bala, S., et al., Reclassification of Amycolatopsis
mediterranei DSM 46095 as Amycolatopsis rifamycinica sp. nov. Int J Syst
Evol Microbiol, 2004. 54(Pt 4): p. 1145-9; 3. Goodfellow, M. and H.P.
Fiedler, A guide to successful bioprospecting: informed by actinobacterial
systematics. Antonie van Leeuwenhoek. 98(2): p. 119-42.
MA06/43
Novel strategies to enhance current antibiotic therapies
Riccardo V D’Elia, Thomas R Laws, Alun Carter, Roman A Lukaszewski,
Graeme C Clark
DSTL -Porton Down, Salisbury, UK
Francisella tularensis is a Gram-negative, intracellular pathogen that has
the ability to subvert and overcome the host’s immune response. It has
been reported in mouse models of F. tularensis subsp. tularensis Schu-S4
that infection via the inhalation route results in an initial delay in host
inflammatory responses, with no detectable levels of pro-inflammatory
cytokines detected in the first 48-72hrs of infection. This is then followed
by an excessive and potentially damaging immune response, reminiscent
of a ‘cytokine storm’. In these studies we look to determine whether or
not the typical pro-inflammatory markers seen late during F. tularensis
infection are essential to host survival or in fact are detrimental and
ultimately futile.
Understanding the exact role of the inflammatory response during
F. tularensis infection may lead to the discovery of targets for next
generation antimicrobials and/or treatment strategies that can increase
the effectiveness of current therapies, such as antibiotics, against the
potentially fatal disease tularemia.
© Crown Copyright. Dstl, 2012
81
Poster abstracts
MA06/44
Reprofiling drugs as novel treatments for Gram-negative bacterial
infection
Jessica L. Rooke, Amanda E Rossiter, Faye C Morris,
Yanina R Sevastsyanovich, Faridah Abu, Laura J Piddock, Ian R Henderson
One of the great challenges facing modern medicine today is the rise
of antibiotic resistance, especially in Gram-negative infections. Gramnegative bacteria are problematic as the outer membrane is a formidable
barrier that creates innate resistance to many antibiotics effective against
Gram-positive strains. The disruption of the outer membrane would
allow a whole range of pre-existing antimicrobials to become effective
against Gram-negative bacteria. One such antibiotic is vancomycin,
which is effective against Gram-positive bacteria, but cannot penetrate
the outer membrane of Gram-negatives. In this study we reprofile the
NINDS library of clinically approved neurological drugs to identify if
any alone, or in conjunction with vancomycin can hinder the growth of
Escherichia coli BW25113. To date, 180 of the 1040 drugs have been
tested in a liquid culture growth assay and by flow cytometry analysis.
One drug, eugenol has been identified that when used in conjunction
with vancomycin significantly reduces E. coli growth. Thus, reprofiling
clinically available drugs for use against Gram-negative bacteria could lead
to quickly available novel therapies.
MA06/45
Photodynamic antimicrobial chemotherapy for Clostridium difficile
Luisa De Sordi1,2, Mohammed A Butt2,3, Gokhan Yahioglu4,5,
Charles A Mosse2, Sinan Battah6,7, Ioanna Stamati4,5, Mahendra
Deonarain4,5, Laurence B Lovat2,3, Elaine Allan1, Peter Mullany1
1
Microbial Diseases, UCL Eastman Dental Institute, London, UK, 2UCL
National Medical Laser Centre, London, UK, 3Gastroenterology, University
College Hospital, London, UK, 4PhotoBiotics Ltd, Imperial College London,
London, UK, 5Division of Cell & Molecular Biology, Imperial College London,
London, UK, 6Organix Ltd, University of Essex, Colchester, UK, 7Department
of Biological Sciences, University of Essex, Colchester, UK
Clostridium difficile is the leading cause of antibiotic-associated diarrhoea
and pseudo membranous colitis in the developed world. This study aims
at developing Photodynamic Antimicrobial Chemotherapy (PACT) for
the treatment of C. difficile infections. PACT utilises the ability of lightactivated photosensitisers (PS) to produce lethal free radical species. We
screened 15 PS against C. difficile. In the presence of oxygen, nine killed
99.99% of the ‘hypervirulent’ C. difficile strain R20291 after exposure to
red laser light (0.2 J/cm2). Four PS were able to kill 99.9% of bacteria
in anaerobic conditions. The efficacy of PACT was confirmed in five
other C. difficile strains belonging to different ribotypes (173, 011, 249,
027 and 020). Applicability of PACT to eradicate C. difficile spores was
also shown, by inducing germination with the bile salt taurocholate,
followed by PACT. Toxicity of effective compounds on the colorectal
adenocarcinoma cell-line HT-29, showed that taurocholate and the PS
chlorin e6, S4 and talaporfin were not toxic to human cells. Therefore,
PACT shows promise as a new treatment for C. difficile colitis, killing
both vegetative cells and spores via a 2-step approach of germination
followed by bacterial targeting, with light delivered at the site of infection.
MA06/46
New therapeutic options for multidrug resistant bacterial infections:
marine-derived bioactive compounds
MArlies Mooij1, Catriona Harrington1, John O’Halloran1, Rob Phelan1,
Teresa Barbosa1, John Morrissey2, Alan Dobson2, Fergal O’Gara1,2
1
BIOMERIT Research Centre, Microbioogy department, University College
Cork, Cork, Ireland, 2Marine Biotechnology Centre, Microbiology department,
University College Cork, Cork, Ireland
Marine sponges have proved to be a rich source of bioactive compounds
with potential therapeutic applications. Many of these metabolites
are microbial-derived, as sponges host a large and diverse microbial
community. In order to identify bioactive compounds with antimicrobial
properties, bacterial isolates and metagenomic libraries derived from
marine sponges have been tested. To date, over hundred marine-derived
bacterial isolates have been shown to produce bioactive compounds
with antimicrobial properties. Several bioactive compounds are further
characterised, but two are potentially very interesting as they are active
against methicillin resistant Staphylococcus aureus and a range of multidrug
resistant Gram-negative pathogens, including extended spectrum betalactamase producing E. coli. Many bacteria use the cell density dependent
cell to cell-signalling system, quorum sensing (QS) to coordinate their
virulence. Therefore, QS is a promising target for antimicrobial drugs.
Marine-derived microorganisms and metagenomic libraries have been
analysed for the presence of quorum quenching compounds. Indeed a
large range of quorum quenching producing microorganisms have been
identified and are currently further characterised. Candidate marinederived modulators of cell-signalling could lead to the development
of new therapeutic agents, which are urgently needed due to global
emergence of antimicrobial resistance.
MA06/47
Investigation of the antimicrobial activity of essential oils of culinary
herbs and spices against selected gastrointestinal pathogens
Mike Chorlton1, Chris O Phillips2, Tim C Claypole2, Eugene Rees1,
Nidhika Berry1, Paula Row1
1
Public Health Wales Microbiology ABM, Singleton Hospital, Swansea, UK,
2
Welsh Centre for Printing and Coating, and College of Engineering, Talbot
Building, Swansea University, Singleton Park, Swansea, UK, 3College of
Medicine, c/o Grove Building Reception, Swansea University, Singleton Park,
Swansea, UK
Pathogenic gut microorganisms, and dysbiosis of the gastrointestinal
microbiota are a significant cause of mortality and morbidity
worldwide. Due to increasing resistance of gastrointestinal pathogens
to conventional antibiotics, alternative antimicrobial agents that could
target gastrointestinal infections are urgently needed. Essential oils
(concentrated mixtures of aromatic compounds usually obtained by
the distillation of plant tissues) have been shown in many cases to
have antibacterial activity. Here we have investigated the antimicrobial
activity of essential oils of a wide range of culinary and medicinal herbs
against selected gastrointestinal pathogens, namely Salmonella enterica,
Clostridium difficile, two clinical isolates of Escherichia coli and Candida
albicans by disc diffusion assays. Seven of the essential oils (aniseed,
asafoetida, cinnamon, clove, oregano, thyme and winter savory) were
effective at inhibiting the growth of all five of the organisms tested
whereas a further eight essential oils (coriander, galangal, garlic, lemon
balm, lemon grass, May Chang, peppermint and rosemary) inhibited the
growth of three or four of the organisms. Batch to batch variation was
evident in the antimicrobial activity of some of the essential oils. This
was examined further by identification of the compounds present in
some of the oils by thermal desorption gas chromatography with mass
spectrometry.
MA06/48
Actinomycetes as potential source of antibiotic against multiple drugresistant pathogens
Jenileima D Kshetrimayum1, Vibha Krishnan2, Nick Allenby1,
Jeff Errington1,3
1
Demuris Ltd. William Leech Building,Medical School,Newcastle University,
Newcastle upon tyne, UK, 2School of Biology, Ridley Building , Newcastle
university, Newcastle upon tyne, UK, 33Institute for Cell and Molecular
Biosciences (ICaMB), Newcastle University, Newcastle upon tyne, UK
Multiple drug resistance microorganisms have become a major global
concern. New antibiotics are needed to combat the spread of these
antibiotic resistant microorganisms. Actinomycetes notably Streptomyces
have been a major antibiotic producer known since 1940 (1, 2). Demuris
Ltd. has a large collection of actinomycetes such as Amycolaptosis,
82
Poster abstracts
Actinomadura, Gordonia, Streptomyces, Micromonospora, Microbispora,
Streptomyces, Nocardia and Rhodococcus isolated from different and
extreme habitats. Traditional screening method contributed about
2% of Demuris Ltd. collection inhibiting E.coli, of which 30% kill New
Delhi metallo-beta-lactamase-1 E.coli (NDM-1), 26% kill Pseudomonas
aeruginosa and 9% kill Mycobacterium terrae. 16s rDNA sequence analysis
and target -based screening has led to determination of taxonomic
diversity and discovery of broad spectrum bioactive compounds.
Re-analysing of freeze-dried mycelium extracts and purification of
compounds by reverse-phase HPLC methods showed as initial
identification and purification of bioactive secondary metabolites. Further
organic extraction and HPLC-MS data will help in narrowing down the
known the potential bioactive secondary metabolites producer (1).
References 1. Goodfellow, M & Fiedler, H.P. (2010) A guide to
successful bioprospecting. Antonie van Leeuwenhoek. 98 (2):119-42; 2.
Berdy, J (2012) Thoughts and facts about antibiotics: Where we are
now and where we are heading. The Journal of Antibiotics 65: 385-395.
MA06/49
Isolation and identification of antimicrobial compound producing novel
streptomycetes from Fire Mountain, China
Nicola Taylor1,2, Michael Goodfellow2,3, Nick Allenby2,
Jeff Errington2,4
1
Biopharmaceutical Bioprocess Technology Centre, Merz Court, Newcastle
University, Newcastle upon Tyne, UK, 2Demuris Ltd, Bioincubator Suite,
William Leech Building, Medical School, Newcastle University, Newcastle
upon Tyne, UK, 3School of Biology, Ridley Building, Newcastle University,
Newcastle upon Tyne, UK, 4Institute for Cell and Molecular Biosciences
(ICaMB), Catherine Cookson, Building, Medical School, Newcastle University,
Newcastle upon Tyne, UK
Actinomycetes remain a unique source of novel antibiotics needed to
control the spread of drug resistant microbial pathogens (1). Investigating
extreme and underexplored habitats reduces the likelihood of reisolating known species and compounds.
Hundreds of streptomycetes were isolated from a Fire Mountain soil
sample using a range of selective media supplemented with antifungal
antibiotics. 135 representative isolates were assigned to 30 colour-groups
based on aerial spore, substrate mycelial and diffusible pigment and
melanin pigments.
Fourteen representative isolates from the colour-groups were examined
for the presence of isomers of diaminopimelic acid and sugars in wholecell hydrolysates to allow preliminary classification as Streptomycetes.
Following 16S rRNA gene analysis, 19 strains were Streptomyces sp.,
ten of which form distinct phyletic line in 16S streptomycetes gene tree.
The remaining isolate was found to be putatively novel Nocardiopsis sp.
The taxonomic integrity of these strains was supported by corresponding
data.
Fourteen representatives of each colour-group were screened against
a panel of known pathogens. Four isolates showed inhibitory activity
against gram-positive and gram-negative organisms. Future work includes
target-based screening and HPLC-MS for further characterisation of the
bioactive compound.
Reference Goodfellow, M & Fiedler, H.P. (2010) A guide to successful
bioprospecting. Antonie van Leeuwenhoek. 98 (2):119-42.
Virology workshop:
Vaccines and antivirals
MA09
MA09/01
Potential of a sequence-based antigenic distance measure to indicate
equine influenza vaccine strain efficacy
Janet M Daly1, Debra M Elton2
1
University of Nottingham, Sutton Bonington, UK, 2Animal Health Trust,
Newmarket, UK
The calculation of p(epitope) values, a sequence-based measure of
antigenic distance between strains, was developed for human influenza.
The potential to apply the p(epitope) value to equine influenza vaccine
strain selection was assessed. There was a negative correlation between
p(epitope) value and vaccine efficacy for pairs of vaccine and challenge
strains used in cross-protection studies in ponies that just reached
statistical significance (p=0.046) only if one pair of viruses was excluded
from the analysis. Thus the p(epitope) value has potential to provide
additional data to consider in the decision-making process for updating
equine influenza vaccine strains. However, further work is required
to define the epitopes of the equine H3N8 haemagglutinin protein
recognised by equine antibodies, which could lead to refinement of the
p(epitope) value calculation. Furthermore, other factors such as vaccine
potency and virulence of circulating strains may also influence vaccine
efficacy.
MA09/02
Generation of a recombinant infectious bronchitis virus (IBV) to study
cellular tropism
Phoebe R Stevenson-Leggett1,2, Erica J Bickerton1, Paul Britton1
1
Pirbright Institute, Compton, Newbury, UK, 2University of Surrey, Guildford,
Surrey, UK
The gammacoronavirus, IBV, causes infectious bronchitis in chickens.
Although vaccines exist, control of the virus is difficult as there are
many different serotypes and cross-protection is poor. Vaccines must
be grown in embryonated eggs which is highly inefficient therefore a
method using cell lines would be beneficial.
IBV has four structural proteins known as the spike (S), membrane (M),
envelope (E) and nucleocapsid (N). The spike protein comprises two
subunits, S1 and S2; S1 is responsible for attachment to host cells and
S2 is responsible for fusion. Altering the S protein of an IBV strain can
change the tropism of the resulting recombinant virus. Using the reverse
genetics system the S protein from the Beaudette (Beau-R) strain of
IBV has been inserted into the background of the M41 strain. This
involved transfecting Vero cells with a plasmid containing the S gene and
using transient dominant selection to produce a recombinant vaccinia
virus containing the M41 cDNA with the Beaudette-derived S gene.
Recombinant IBVs based on M41 containing the Beaudette S gene will
be analysed for virus growth on Vero cells.
Growth characteristics of the rIBV on different cell types will be analysed
using confocal microscopy and growth curves.
MA09/03
The use of equine influenza pseudotypes for serological screening
Simon D Scott1, Eleonora Molesti1, Nigel J Temperton1,
Francesca Ferrara1, Eva Böttcher-Friebertshäuser2, Janet Daly3
1
Viral Pseudotype Unit, School of Pharmacy, University of Kent, Chatham
Maritime, UK, 2Institute of Virology, Philipps University, Marburg, Germany,
3
School of Veterinary Medicine and Science, University of Nottingham,
Nottingham, UK
Standard assays used for influenza serology present certain practical
issues, such as inter-laboratory variability, complex protocols and
necessity for handling certain virus strains in high biological containment
facilities. In an attempt to address this, avian and human influenza
HA pseudotyped retroviruses have been successfully employed in
antibody neutralisation assays. In this study we have generated equine
influenza pseudotyped lentiviruses for serological screening. This was
achieved by co-transfection of HEK293T cells with plasmids expressing
the haemagglutinin (HA) protein of different H3N8 subtype equine
influenza virus strains, HIV gag-pol and firefly luciferase reporter genes
and harvesting virus from supernatant. To produce infective pseudotype
particles it was also necessary to co-transfect a plasmid encoding the
TMPRSS2 endoprotease to cleave HA. High titre pseudotype virus
(PV) was then used in PV antibody neutralisation assays (PVNAs) to
successfully distinguish between vaccinated and non-vaccinated equines.
83
Poster abstracts
The sera were also screened by single radial haemolysis (SRH) assay.
There was a 65% statistical correlation between the results of the two
assays, with the PVNA assay appearing more slightly more sensitive.
Future work will extend the testing of the PVNA with a larger cohort
of serum samples to assess sensitivity/specificity, inter/intra-laboratory
variability and to define a protective titre.
MA09/04
Generation of a recombinant infectious bronchitis virus (IBV) with
chimaeric S gene
Erica J Bickerton, Paul Britton
Pirbright Institute, Compton Laboratory, Newbury, Berkshire, UK
Vaccines against the avian coronavirus, IBV, are currently grown on
embryonated eggs, a cumbersome and expensive process; growth on
a cell line such as Vero cells would be beneficial. The IBV Beau-R strain
replicates in both primary chick kidney (CK) cells and in Vero cells,
whereas H120, a vaccine strain, replicates in primary cells only. The IBV
spike (S) glycoprotein, comprising S1 and S2 subunits, has a vital role
in virulence in vivo and is responsible for cellular tropism in vitro. The
Beau-R S2 subunit confers ability to grow on Vero cells. A recombinant
IBV (rIBV) with a chimaeric S gene has been generated using reverse
genetics, BeauR-H120(S1). The resulting virus has the S1 subunit of
H120 within the genomic background of Beau-R. The rIBV has been
passaged on Vero cells, growth characteristics analysed by microscopy
and growth curves carried out on CK and Vero cells; replication is
observed to be comparable to Beau-R on CK and Vero cells. The most
immunogenic region of the S glycoprotein, the S1 subunit, of a vaccine
strain can be incorporated into a virus able to grow on Vero cells,
enabling IBV vaccines to be grown in cell lines rather than embryonated
eggs.
MA09/05
Identification of a panel of tricyclic compounds that inhibit HCV entry
Vanessa Cowton, Sarah Beattie, Arvind Patel
MRC-University of Glasgow Centre for Virus Research, Glasgow, UK
The entry step of HCV infection is a multi-stage process involving
attachment, receptor interaction, cellular uptake by clathrin-mediated
endocytosis and fusion of the viral and endosomal membranes. We
developed a plate based assay to monitor HCV entry using a HCV
pseudoparticle (HCVpp) system in Huh7 cells. This assay was used to
identify small-molecules that inhibit HCV entry from a library of >1000
bioactive compounds. The effect of the compounds on cell viability
was monitored in parallel to exclude false positives due to cell toxicity.
Inhibition of HCV entry was confirmed using HCV JFH-1 virus. The
screen identified a panel of tricyclic antidepressants with similar structure
as inhibitors of HCV entry. The inhibitory effect is specific for HCV as
entry of an alternative virus that also uses the clathrin route was only
inhibited at high concentrations. This also suggests that the clathrin
pathway is not the target for these compounds. Interestingly, the efficacy
of the compounds against different HCV genotypes was variable with a
strong bias towards genotype 2A which was used for the screen. This
may indicate subtle differences in the entry routes of HCV genotypes.
Further studies into the mechanism underlying the inhibitory effect of
these compounds are ongoing.
MA09/06
Development of a pseudotype neutralisation assay-based diagnostic
kit for in-field vaccine evaluation and serosurveillance for highly
pathogenic viruses
Stuart Mather1, Nigel J Temperton1, Edward Wright2,
Simon D Scott1
1
Viral Pseudotype Unit Medway, School of Pharmacy, University of Kent,
Chatham Maritime, UK, 2Viral Pseudotype Unit Fitzrovia, School of Life
Sciences, University of Westminster, London, UK
Virus neutralisation assays quantitatively detect levels of neutralising
antibody response against antigenic surface glycoproteins on many
viruses, following vaccination or natural infection. However, high biosafety
level requirements and extensive personnel training prevent these
tests from broad laboratory application, especially in resource-limited
regions. Therefore, development of methods for vaccine evaluation
and serosurveillance which can be used in these areas are urgently
required. To address these issues lentiviral pseudotype viruses (PVs)
have been utilised. PVs are chimeric, replication-deficient particles
that mimic the infective mechanisms of their wild-type counterparts.
Pseudotype neutralisation assays (PNAs) circumvent the requirement for
high biosafety precautions whilst maintaining comparable sensitivity and
specificity with existing assays. This study ascertains pseudotype stability
through subjection to environmental conditions likely to be encountered
in assembly, transport and usage of a PNA-based diagnostic kit.
Pseudotypes of clinically-important viruses (e.g. influenza and lyssaviruses)
have been used and titres monitored through cumulative freeze-thaw
cycles, lyophilisation, and varying temperatures and humidities. Results
demonstrated the ability to retain acceptable levels of virus activity
following treatments, indicating the potential of PNA-based kits for global
distribution and diagnostic application. Such flexible and durable kits
could permit accurate in-field vaccine evaluation and serosurveillance for
many viruses of endemic and pandemic concern.
MA09/07
Development of LPAI and HPAI H7 avian influenza pseudotypes
for serological applications utilising a combination of protease cotransfection and site-directed mutagenesis
Eleonora Molesti1, Giovanni Cattoli2, Francesca Ferrara1,
Eva Böttcher-Friebertshäuser3, Calogero Terragino2, Nigel Temperton1
1
2
FAO, OIE and National Reference Laboratory for Newcastle Disease and
Avian Influenza, Istituto Zooprofilattico Sperimentale delle Venezie, Legnaro,
Italy, 3Institute of Virology, Philipps University, Marburg, Germany
HPAI and LPAI H5 and H7 viruses have been disastrous for the
economies of affected countries reliant on poultry production. In a
situation similar to H5, H7 strains show adaptation to humans and
therefore pose a serious public health concern. Applying knowledge
acquired from study of H5 virus evolution and spread to the
development of sensitive serological methods will improve our ability
to understand and respond to the emergence of other HPAI and
LPAI viruses with pandemic potential. We describe the production of
pseudotypes bearing envelope glycoproteins of LPAI and HPAI H7 avian
influenza for use as tools in pseudotype-based (pp-NT) neutralisation
assays. A crucial feature of H7 pseudotype production is efficient
intracellular cleavage of haemagglutinin. We show that the LPAI strain
A/chicken/Italy/1082/1999 possesses a monobasic cleavage site and
requires TMPRSS2 to effect cleavage of the HA. The HPAI strain A/
Pakistan/34668/95 possesses a sub-optimal furin cleavage site resulting
in low yields. In order to circumvent this we have used site-directed
mutagenesis to replace the polybasic cleavage site with a monobasic
site that can subsequently be cleaved (during production) via the cotransfection of the TMPRSS2 protease. Sensitive pp-NT assays were then
carried out on post-vaccination sera using these new surrogate viruses.
MA09/08
Investigating the antibody neutralisation potential of a rabies virus
lentiviral pseudotype: an international inter-laboratory trial
Derek M Healy1, Daniel L Horton1, Ashley C Banyard1,
Edward Wright3, Trudy Goddard1, Jennifer Evans2,
Lorraine M McElhinney1,4, Anthony R Fooks1,4
1
Animal Health & Veterinary Laboratories Agency, Woodham lane,
Weybridge, Surrey, KT15 3NB, UK, 2University of Warwick, School of Life
Sciences, Coventry, CV4 7AL, UK, 3MRC/UCL, Wohl Virion Centre, Division
of Infection and Immunity, University College London, UK, 4The National
84
Consortium for Zoonosis Research, University of Liverpool, Leahurst, Chester
High Road, Neston, CH64 7TE, UK
Rabies infection causes an acute viral encephalitis that is almost invariably
fatal. Transmission most commonly occurs following the bite of an
infected dog, and annually >55,000 people die from this disease, the
majority being located in the developing world. Dog vaccination and
responsible dog ownership are key to the control and elimination of
rabies and the ability to detect virus neutralising antibodies is key to the
evaluation of vaccination status following pre-immunisation. Current
diagnostic tests are expensive, and as such are not readily performed in
resource limited settings. To overcome this, lentiviral pseudotypes have
been applied as an alternative platform for post vaccinal assessment
and serosurveillance. The assay requires a small amount of serum
in comparison to the standard fluorescent virus neutralisation assay
(FAVN). Furthermore, the ability to tailor the assay according to facilities
available through flexibility of reporter gene expression enables transfer
of this technology across areas where the virus remains endemic, i.e.
Africa and Asia. These reporter gene detection systems eliminate the
need for expensive equipment and facilities allowing the assay to be
performed in laboratories throughout the world. An inter laboratory ring
trial has shown that the pseudotype assay for rabies virus is both reliable
and reproducible.
MA09/09
Mapping the antigenic evolution of an H5N1 clade 2.2 A/chicken/
Egypt/1709-1/07 and the drift variant A/chicken/Egypt/1709-1/07 by a
sensitive pseudotyped-based assay
Eleonora Molesti1, Adelaide Milani2, Giovani Cattoli2,
Nigel Temperton1
1
2
FAO, OIE and National Reference Laboratory for Newcastle Disease and
Avian Influenza, Istituto Zooprofilattico Sperimentale delle Venezie, Legnaro,
Italy
The propensity of influenza virus to mutate and evolve into new types
with divergent antigenic determinants is an important challenge in the
control of influenza infection. In Egypt, an H5N1 avian virus belonging
to clade 2.2 was notified in February 2006 and the country has faced
a huge number of outbreaks since. Subsequently, to control infection
in poultry and prevent the risk of human exposure, an inactivated
vaccine containing an H5 virus belonging to a different lineage to the
Eurasian H5N1 (H5N2/Mexico) was employed. Despite this, the
continuous circulation of the virus and the mass vaccination programme
has resulted in progressive genetic evolution and significant antigenic
drift with multiple mutations near the major antigenic sites leading
to the emergence of a new strain grouped within clade 2.2.1. To
establish whether the administration of the H5N2/Mexico vaccine
is likely to provide cross-protective antibody responses against
different clades, lentiviral pseudotypes derived from H5N1 HPAI
viruses (A/Egypt-1709-01/2007) and the antigenic drift variant (A/
Egypt-1709-06/2008) were constructed and used in pseudotype-based
neutralisation (pp-NT) assays. Overall results indicated a significantly
lower level of antigenic cross-reactivity of post vaccine sera against the
pseudotyped-particles bearing the HA of the 1709-06 drift variant.
MA09/10
Development of immune-focused recombinant vaccine candidates for
the prevention and treatment of hepatitis C virus infection
Mohamed R Hamed1,2, Matthijs Backx1, Alexander W Tarr1,
Richard J P Brown1, Richard A Urbanowicz1, Jonathan K Ball1
1
School of Molecular Medical Sciences, and Biomedical Research Unit in
Gastroenterology, The University of Nottingham, Nottingham, UK, 2Faculty of
Medicine, Mansours University, Mansoura, Egypt
Hepatitis C virus (HCV) causes acute and chronic liver diseases in
humans. Its two envelope glycoproteins, E1 and E2, interact with host
cell receptors, CD81 and SRB1, and provide a target for neutralising
Poster abstracts
antibodies. Past vaccine studies using unmodified E2 proteins have failed
to convincingly generate broadly neutralising antibody responses. This
study sought to generate immune-focused, vaccine candidates for the
prevention and treatment of HCV.
To minimise the problem of genetic diversity between our vaccine
constructs and contemporary viruses, we generated synthetic constructs
based on both the consensus and most recent common ancestral
sequences of HCV genotype 1 viruses. These synthetic constructs
showed functionality in the HCV pseudo-particle (HCVpp) system and
were conformationally intact when examined by monoclonal antibody.
In addition we identified minimal functional vaccine constructs that
enhanced the exposure of the CD81 binding site. Monomeric protein
generated using a Drosophila expression system was used in a guinea
pig vaccine trial. The elicited antibody responses predominantly targeted
linear epitopes and had variable potency in the HCVpp system against a
panel of neutralisation-resistant, patient-isolated HCVpps.
This study demonstrates a novel vaccine approach of using minimally
diverse and minimal functional constructs generated using the Drosophila
expression system.
MA09/11
Structural and in silico studies of enterovirus EV71 inhibitors
Luigi De Colibus1, Xiangxi Wang2, John A B Spyrou1,
Jonathan Grimes1, Gerhard Puerstinger3, Elizabeth E Fry1, Zihe Rao2,
David I Stuart1
1
Division of Structural Biology, University of Oxford, Oxford, UK, 2National
Laboratory of Macromolecules, Institute of Biophysics, Beijing, China,
3
Department of Pharmaceutical Chemistry, Innsbruck University, Innsbruck,
Austria
The family Picornaviridae comprises small icosahedral RNA animal viruses,
including enteroviruses. Human enterovirus 71 (EV71) is very often
associated with outbreaks ranging from mild childhood exanthema and
hand-foot-mouth disease to severe neurologic disease including aseptic
meningitis, encephalitis, and poliomyelitis-like paralysis. This accounts for
the urgency of developing anti-EV71 agents.
As all enteroviruses, EV71 has a depression around the icosahedral
five-fold axes called the ‘canyon’ which host receptors frequently bind
to, causing the expulsion of a natural lipid, the pocket factor. This crucial
event in the infection strategy is the prelude to virus uncoating and
genome release. Therefore, antiviral compounds able to replace the
pocket factor with higher affinity are potent picornavirus inhibitors.
Several classes of molecules have been proven to displace the pocket
factor and lead to the inhibition of functions associated with viral
uncoating and host infection. We have solved four crystal structures of
EV71 in complex with pyridyl imidazolidinone derivatives, shedding light
on key residues involved in the binding with these inhibitors.
Using a structure based approach such as in silico docking we aim to
design novel and potent EV71 inhibitors.
MA09/12
Cloning malarial antigen, PfRH5, into modified vaccinia Ankara (MVA)
under control of a T7 promoter
Christopher D Cousins1,2, Katharina Lauer1, Radi Alsafi1,
Megan Whittaker1, C P Chikkanna-Gowda1, Thomas Blanchard1,3
1
Microbiology & Virology Unit, School of Translational Medicine, University of
Manchester, Manchester, UK, 2Salford Royal NHS Foundation Trust, Salford,
UK, 3Department of Infectious Diseases and Tropical Medicine, North
Manchester General Hospital, Manchester, UK
Background Malaria continues to be a major epidemic with unacceptably
high incidence and mortality rates. Effective vaccination would
undoubtedly be beneficial to the global effort in controlling the disease.
However, despite decades of work, there is still no licensed malaria
vaccine. Plasmodium falciparum reticulocyte-binding protein homologue
5 (PfRH5) is a promising blood-stage vaccine candidate. A recombinant
modified vaccinia Ankara (MVA) vaccine candidate expressing PfRH5
85
Poster abstracts
driven by a poxviral promoter has previously been described1.
Methods We have fused a PfRH5 synthetic gene with influenza
haemagglutinin and a T7 expression cassette, and transferred it into
MVA via homologous recombination. Recombinant MVA encoding the
T7-driven PfRH5/HA fusion protein has subsequently been isolated
by transient dominant colour section employing blue-white-screening
(LacZ).
Conclusions/ Discussion Recombinant MVA, which should express
membrane-bound PfRH5/HA fusion, has been isolated. The use of
transient-dominant colour selection will enable the recycling of the
marker cassette for future construction of complex multi-pathogen
vaccine candidates. The use of a T7 expression system should permit the
strong regulated expression of PfRH5/HA.
References [1] Douglas AD, Williams AR, Illingworth JJ, Kamuyu G,
Biswas S, Goodman AL et al. The blood-stage malaria antigen PfRH5 is
susceptible to vaccine-inducible cross-strain neutralising antibody. Nat
Commun. 2011;2:601.
MA09/13
Protective immunity induced by a recombinant modified vaccinia
Ankara (MVA) expressing African horse sickness virus (AHSV) VP7
Laura V Bailey, Nicola Manning, Eva Calvo-Pinilla, Peter P C Mertens,
Javier Castillo-Olivares
The Pirbright Institute, Pirbright, Woking, Surrey, UK
African horse sickness (AHS) is an arthropod-borne disease of equids.
Horses are most susceptible to AHS and mortality rates can exceed
95% in naïve horse populations. In the absence of effective treatment,
vaccination plays an important role in the control of AHS. However,
there are a number of concerns associated with the usage of live
attenuated vaccines particularly in countries free of the disease. These
concerns have stimulated the development of novel recombinant
vaccines including the use of live viral vector vaccines such as modified
vaccinia Ankara (MVA). We have carried out a vaccination and challenge
experiment to investigate the protective immunity induced by MVAAHSV-VP7, a recombinant MVA virus expressing the outer core capsid
protein (VP7) of AHSV serotype 4. For this study, interferon alpha
receptor gene knock-out (IFNAR -/ -) mice were vaccinated with MVA
VP7 on days 0 and 28 and subsequently challenged (day 38), together
with non-vaccinated control mice, with either AHSV-3, AHSV-4 or
AHSV-9. Antibody responses in mice were analysed and protective
immunity measured by evaluating clinical responses and virus levels in
blood measured by standard virus plaque assays and real time PCR (RTPCR). The results of this study will be presented and discussed.
MA09/14
Role of protein oligomerisation in the immune response to a
candidate influenza vaccine
Elena Robinson1, Marta Szajna2, Marina Botto2, Ian M Jones1
1
School of Biological Sciences, University of Reading, Reading, UK,
2
Rheumatology Section, Faculty of Medicine, Imperial College London, London,
UK
Influenza vaccine development must take account of antigenic shift and
the slower paced antigenic drift associated with the natural process of
virus evolution. Efficacy, scalability and time from new strain emergence
to antigen generation are crucial factors when evaluating new methods
of influenza vaccine production. Vaccination with purified haemagglutinin
fused to the human immunoglobulin Fc domain enables a rapid response,
easy reformulation and high immunogenicity. However, the mechanism
by which purified HA fusion proteins elicit strong immune responses
remains unclear.
We have investigated the role of HA-Fc oligomerisation in the induction
of antigen specific responses by comparing the immune response to Fc
tagged full length HA0 and truncated HA1 proteins. We show that HA0Fc forms hexameric complexes in solution, probably via the unpaired
-SH side chain interactions of cysteine residues of the Fc hinge region
while HA1-Fc forms only to a dimer. We demonstrate that this change
in the state of oligomerisation alters significantly the immunogenicity of
the candidate vaccine and furthermore that complement fixation may be
a component of the response observed.
MA09/15
Development and validation of a high throughput screening assay
targeting the HCV NS2 autoprotease
Joseph Shaw, Colin Fishwick, Mark Harris
Hepatitis C virus (HCV) infects over 180 million people worldwide,
leading to an emphasis on identifying direct acting anti-virals that can
contribute to a future combination therapy. Autoproteolysis at the HCV
NS2-NS3 junction is an event known to be essential in the virus lifecycle
to begin processing of the non-structural proteins from the polyprotein
and control the onset of viral replication, yet to date there are no antivirals targeting this activity.
We have developed a high-throughput screening assay targeting the
autoproteolytic activity of the HCV NS2 protein. NS2-containing
sub-genomic replicons have been produced which allow selection of
stable cell lines and accurate quantification of HCV replication through
a neomycin phosphotransferase-firefly luciferase fusion protein. NS2
containing stable replicons support strong levels of HCV replication, as
monitored through luciferase activity, for multiple HCV genotypes. Using
stable replicons with and without NS2 present, compounds identified
from in silico techniques are being screened for specific anti-NS2 activity.
MA09/16
Novel antiviral activity of L-dideoxy bicyclic nucleoside analogues
versus vaccinia and measles viruses in vitro
Chris McGuigan1, Karen Hinsinger1, Laura Farleigh1, Ranjith N Pathirana2,
Joachim J Bugert1
1
Cardiff University, Cardiff, UK, 2University of Ruhuna, Matara, Sri Lanka
Dideoxy bicyclic pyrimidine nucleoside analogues (ddBCNAs) with
[D] chirality have been described by us to inhibit replication of human
cytomegalovirus. We herein report that further potency against vaccinia
virus (VACV) was achieved using novel [L] analogues. A structure
activity relationship was established: Antiviral activity versus VACV
was highest with an ether side chain with an optimum of nC9H18-OnC5H11. This gave an IC50 of 190 nM, a 60 fold enhancement over
the FDA approved antiviral cidofovir in BSC-1 cells. Antiviral activity was
observed as early as 2 hours p.i. for VACV in luciferase reporter assays,
with a sustained effect in vitro over 4 days in plaque assays. L-ddBCNAs
mildly affect cell proliferation in BSC-1 cells with a selectivity index of
526 for the lead L-ddBCNAs over four days. Interestingly, L-ddBCNAs
also inhibit wild type measles virus syncytia formation in B95a cells
with a TCID50 of 7.5 μM for the lead compound. We propose that
L-ddBCNAs represent promising antiviral candidates versus measles and
poxviruses and we suggest a mechanism of action versus one or more
cellular targets that are essential for viral replication.
MA09/17
Persisting live attenuated SIV vaccination in multiple lymphoid tissues
elicits localised innate immune responses associated with protective
vaccination
Deborah Ferguson1, Giada Mattiuzzo1, Claire Ham1, Richard Stebbings1,
Nicola Rose1, Ed Mee1, Mark Page1, Martin Cranage2, Neil Almond1,
Greg Towers3, Neil Berry1
1
NIBSC, South Mimms, UK, 2St Georges, Univ London, London, UK, 3UCL,
London, UK
Vaccination of Mauritian cynomolgus macaques with the attenuated
nef-truncated C8 variant of SIVmac251(32H) induces early, potent
protection against challenge with pathogenic, heterologous SIVsmE660.
Intravenous inoculation with SIVmacC8 results in rapid, widespread virus
86
Poster abstracts
dissemination with prolific simultaneous productive infection of multiple
lymphoid organs (mesenteric lymph nodes, spleen and small intestine).
Intracellular viral RNA was detectable in lymphoid tissues by qRT-PCR 3
days post-inoculation, peaking 7-14d , resolving to low, persisting levels.
Vaccine virus was characterised by foci of persisting infected cells as
detected by in-situ hybridisation where SIVmacC8 triggered a broad antiretroviral innate response. TRIM5α, APOBEC3G and type-1 interferon
signalling genes IRF-7 and STAT-1 were upregulated in lymphoid tissues,
but did not prevent viral persistence within multiple lymphoid tissues.
Immunohistochemistry revealed widespread activation of macrophages,
B-cells and dendritic cells as early as 3 days post-inoculation; high
DC-SIGN expression in spleen and MLN persisted 3-20 weeks postSIVmacC8 vaccination. Retrospective analysis of a vaccination/challenge
study revealed persistence of vaccine-induced splenic DC-SIGN
expression was a marker of superinfection resistance to SIVsmE660.
Persisting stimulation of selected innate responses at localised lymphoid
sites may be critical for early superinfection resistance and conditioning
appropriate cognate immune responses required for persistent
protection that may be needed for an effective HIV/AIDS vaccine.
These observations are consistent with previously published biochemical
data and with new virologic data from mutants designed to test these
specific interactions. Our HIV-1 IN model has also been an
integral part of a collaborative drug design effort.2,3 A lead compound
with micromolar activity has been modified to yield a derivative with low
nanomolar activity against WT HIV-1, and activities against known drugresistant mutants are comparable to, if not better than, those of two
FDA-approved IN inhibitors.
References 1Hare et al. (2010) Nature. 464(7286):232-6; 2Johnson et
al. (2012) Antimicrob Agents Chemother. 56(1):411-9; 3Métifiot et al.
(2012) ACS Chem Biol. In press.
MA09/18
Comparative pathogenicity of cell-propagated avian adenovirus-4 and
development of cell culture based vaccine
Asma Jabeen1,2, Khalid Naeem1,2, S M Saqlan Naqvi1, Tahira Kamal1,2
1
PMAS Arid Agriculture University, Rawalpindi, Pakistan, 2National Agricultural
Research Centre, Islamabad, Pakistan
The present study was designed to develop an efficient cell culturebased vaccine for AAV-4 the causative agent of hydropericardium
syndrome of chickens. A field isolate of AAV-4 was propagated in
chicken embryo liver cell culture till 5th passage. Liver homogenate virus
(105.0TCID50/ml) caused 100% mortality via subcut route. Passage-1virus
(P-1 105.0TCID50/ml) caused 80% mortality whereas Passage-5 (P-5
103.0TCID50/ml) was non-pathogenic to birds.The liver homogenate
and cell culture passaged viruses were used for preparing formalin
inactivated water and oil based vaccines, P-5 virus was also prepared
as live vaccine. The antibody responses to all vaccines were tested
by ELISA.Groups of chicks injected s/c with 0.2 ml of inactivated liver
homogenate water and oil-base vaccines showed S/P ratios of 0.341 and
0.363 respectively, the groups given 0.2 ml of inactivated P-1 cell culture
passaged oil or water-based vaccines showed average S/P ratios of 0.989
and 1.025. However,the groups given 0.2ml 0f P-5 inactivated oil and
waterbased vaccines showed average S/P ratios of 1.032 and 0.557. Live
cell culture-based vaccine showed an S/P ratio of 1.169. All vaccinated
groups survived the challenge. (105.0TCID50/ml) and 100% mortality was
observed in PBS control group. Cell culture-passaged inactivated vaccines
qualified standards of safety, sterility and potency.
MA09/19
Modelling and inhibition of HIV-1 integrase
Barry C Johnson1, Mathieu Metifiot2, Steven J Smith1,
Xuezhi Zhao3, Stephen Hare4, Peter Cherepanov5, Terrence R Burke, Jr3,
Yves Pommier2, Stephen H Hughes1
1
National Institutes of Health, HIV Drug Resistance Program, Frederick, MD,
USA; 2National Institutes of Health, Laboratory of Molecular Pharmacology,
Bethesda, MD, USA; 3National Institutes of Health, Chemical Biology
Laboratory, Frederick, MD, USA; 4Imperial College London, Division of
Infectious Diseases, London, UK; 5Cancer Research UK London Research
Institute, Clare Hall Laboratories, London, UK
Retroviral integrase (IN) is the enzyme responsible for inserting the viral
DNA (vDNA) into the DNA of a host cell (hDNA). Recently solved
crystal structures of the prototype foamy virus (PFV) intasome, including
an IN tetramer with vDNA and hDNA mimics, have allowed for
improved modeling of the HIV-1 intasome.1,2 Our modeling efforts have
generated evidence of a novel dimer contact within the HIV-1 intasome
and identified residues that appear to be critical for hDNA binding.
MA09/20
Lymphocyte adoptive transfer in MHC-identical cynomolgus macaques
Edward T Mee1, Richard Stebbings2, Elaine Giles1, Joanna Hall1,
Neil Almond1, Nicola J Rose1
Divisions of 1Virology and 2Biotherapeutics, National Institute for Biological
Standards and Control, Health Protection Agency, South Mimms, UK
Defining immune correlates of protection against HIV remains an
important goal in the development of an effective vaccine. Macaque
studies show that potent sterilising immunity against SIV can be
induced by immunisation with live-attenuated virus. CD8+ T-cells or
whole lymphocyte depletion suggests that these populations are not
essential to protection but not that they are incapable of conferring
protection. Transfer of cells from vaccinated to naive animals prior
to challenge would allow for testing of this hypothesis. Lymphocyte
adoptive transfer has largely been limited to inbred rodent models,
however, based on their limited genetic diversity we established a
selective breeding program to produce MHC-identical Mauritianderived cynomolgus macaques. We have transferred lymphocytes from
a macaque immunised with SIV peptides into related and unrelated,
MHC-matched macaques and MHC-mismatched recipients. Cells persist
in MHC-matched animals, with higher levels in macaques related to the
donor, suggesting a role for minor histocompatibility antigens, but are
rapidly cleared from MHC-mismatched macaques. Cell function is to be
evaluated. Our next step is to determine whether lymphocytes from
a macaque immunised with a candidate vaccine can protect recipients
against challenge with SIV. This approach represents a significant advance
in the utility of the SIV/macaque model of HIV infection.
Virology workshop:
The virome and viromics
MA10
MA10/01
Presenceof fowlpox virus and evidence of FWPV-REV integration in
vaccinated laying hens in Kosovo
Afrim Hamidi1, B Behluli1, Skender Muji1, Agim Rexhepi1,
Kurtesh Sherifi1, Olivier Sparagano2, David George2,
Dorte Luschow3, Mohamed Hafez3
1
University of Prishtina, Prishtina, Kosovo, 2University of Northumbria,
Newcastle upon Tyne, UK, 3Free University Berlin, Berlin, Germany
In October 2008, two outbreaks of fowlpox virus (FWPV) were
registered at a laying hen facility NW Kosovo, despite prior vaccination
of birds at 14-15 weeks of age. Both forms of the disease (cutaneous
and diphtheritic) were visually confirmed in both outbreaks. During the
outbreak, 30 samples representing material from cutaneous nodular
lesions and fibrion-necrotic proliferative lesions (from mucosa of mouth
and pharynx mucous membrane), were randomly selected from 350
severely infected birds for laboratory analysis. Multiplex polymerase chain
reaction combined with restriction enzyme analysis for direct detection
of FWPV and reticuloendotheliosis virus (REV) were used to isolate and
confirm FWPV and determine the possible integration of REV into its
genome. Using one primer set, which framed a region within the avian
poxvirus 4b core protein gene, it was possible to detect FWPV-specific
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Poster abstracts
DNA from all three pooled samples tested. This is the first study to
demonstrate FWPV/FWPV-REV occurrence in Kosovo, with infection by
both the cutaneous and diphtheritic forms confirmed. The relevance of
these results to the poultry industry and to developing our understanding
of this disease and its future treatment per se are briefly discussed.
MA10/02
Characterising the human respiratory virome using next generation
sequencing
Fiona Thorburn1, David Murdoch2, Richard Legget3,
Mario Caccamo3, Joseph Hughes1, Rory Gunson4, Pablo Murcia1
1
MRC -University of Glasgow, Centre for Virus Research, Glasgow, UK, 2University
of Otago, Christchurch, New Zealand, 3The Genome Analysis Centre, Norwich,
UK, 4West of Scotland Specialist Virology Centre, Glasgow, UK
In each person microbes outnumber human cells approximately 10:1.
The Human Microbiome Project was established to describe the
microbial population present on and in healthy individuals since evidence
increasingly suggests links between the microbiome and disease. The
advent of Next Generation Sequencing (NGS) technologies has made
possible the study of the structure of this symbiotic population with
unprecedented precision. The common cold is the most prevalent
human infection[i] with 20-30% of cases having an unknown aetiology[ii],
possibly caused by previously uncharacterised viruses or those which
have escaped diagnosis through mutation. We aim to characterise the
viral component of the microbiome (virome) of the common cold
using NGS. Nucleic acids extracted from nasopharyngeal swabs (NPS)
collected from healthy individuals during an upper respiratory tract illness,
will be amplified and sequenced using Sequence-Independent SinglePrimer Amplification and NGS, respectively. A variety of bioinformatic
tools (including Velvet and BLAST) are used to determine the structure
of the upper respiratory tract virus population in each individual.
References [i] Ron Eccles. Understanding the symptoms of the common
cold and influenza Lancet Infect Dis 2005; 5: 718-725; [ii] Terho
Heikkinen. The Common Cold The Lancet Volume 361, Issue 9351, 4
January 2003, Pages 51-59
MA10/03
Survey of rotavirus genotypes of scouring pigs in the UK
Rebecca Chandler, Laura Hancox, Kenneth Mellits
Rotavirus is an RNA virus with a broad host range. In children it is the
leading cause of severe gastroenteritis worldwide, most have acquired
the infection by the time they are 5 years old . In pigs, rotavirus is the
most prevalent cause of diarrhoea in neonates and at weaning . Rotavirus
is endemic in pig farms in the UK; where it causes losses in production.
To date there is no clear surveillance data of porcine rotavirus genotypes
circulating in the UK. The aim of this study was to determine genotypes
causing clinical symptoms within the UK and investigate evidence of
zoonosis between species.
Faecal samples were collected from 41 clinically infected pig farms in 11
counties across the UK. VP7 and VP4 genes were genotyped by RTPCR and sequencing product of VP7 and VP4 genes. Of the sequenced
samples five G types G3, G4, G5, G9 and G11 and six P types P[6],
P[7], P[8], P[13], P[23], and P[32] have been identified. The sequenced
samples will be compared to reference sequences of porcine, bovine
and human origin to investigate zoonosis. This study has shown an
unexpected high diversity of genotypes compared to other European
studies.
MA10/04
Full-length genome sequencing of influenza A on the ion torrent
PGMTM sequencer
Karen Jones, Astrid Ferlinz, Simone Günther, Andy Felton
Life Technologies, Glasgow, UK
The evolution of influenza A viruses represents a major challenge for
both human and animal health. Influenza A viruses may cross species
barriers and transmit to humans with the potential to cause pandemics.
We describe a rapid sequencing method and standard operating
procedure using the Ion Torrent PGMTM sequencing platform for rapid
and cost effective Influenza A virus typing. This includes the analysis,
reporting and sharing of data.
We amplified the 8 genome segments of Influenza A by a multiplex RTPCR (PathAmpTM Flu A Reagents) and performed a standard sequencing
reaction on the Ion Torrent PGMTM Sequencer. Base calling, alignment
and coverage analysis were carried out with the Torrent Suite Software.
Our results show the accuracy of NGS to define the full length sequence
of Influenza A genome. Comparison of the NGS to Sanger sequencing
on a single H1N1pdm isolate showed 100% nucleotide concordance.
The high throughput obtained so far indicates that we can multiplex
more than 10 samples for a single sequencing run on the Ion PGMTM 316
chip.
Virology workshop:
Assembly and structure
MA11
MA11/01
The efficiency of producing progeny mouse hepatitis virus is not
closely related to the prevalence or size of double membrane vesicles
Hawaa M N Al-Mulla, Benjamin W Neuman
University of Reading, Reading, UK
Coronaviruses induce the formation of double membrane vesicles
(DMVs) in infected cells. Mouse hepatitis virus (MHV) is the prototype
of Betacoronaviruses and one of most extensively studied coronaviruses.
In this study, MHV-Bristol temperature sensitive 31 (ts) was studied,
which has a single mutation in nsp3 at nucleotide 4131 (Stokes et.al
2010), in an attempt to understand the function of coronavirus nsp3.
This mutation makes the virus unable to synthesise viral RNA when the
infection is initiated and maintained at the nonpermissive temperature.
However, the ts virus was able to synthesise genomic and subgenomic
RNA at the same rate as wild-type virus at 33°C. Transmission electron
microscopy analysis revealed that ts virus makes significantly smaller and
fewer DMVs at 33°C. The lack of difference in the size and number of
intracellular virus particles demonstrated that changes in apparent DMV
size and number were unlikely to be an artefact of the preparation
and imaging techniques. This suggests that the efficiency of producing
progeny virus is not closely related to the prevalence or size of DMVs
and therefore, that high levels of DMV formation not necessary for
completion of the coronavirus life-cycle.
MA11/02
Investigating novel FMDV receptors and entry
Kyle Chamberlain1,2, Veronica Fowler1, Nick Knowles1, Paul Barnett1,
Terry Jackson1
1
The Pirbright Institute, Woking, Surrey, UK, 2University of Surrey, Guildford,
Surrey, UK
Field strains of foot-and-mouth disease virus (FMDV) use integrins
as receptors while cell culture adapted variants can also use other
receptors such as heparan sulphate (HS). Recently a virus (A/Iran/87
(A-)) with a deletion at the integrin-binding site was isolated from a
vaccine stock. Also, this virus lacks the known HS contact residues and
infects HS-deficient CHO cells, suggesting that cell-entry is mediated
by a novel receptor. Sequence comparison identified only a limited
number of residue changes within the capsid of A/Iran/87 (A-) which
are surface exposed and have the potential to make direct contacts
with cellular receptors, including a SAR tri-peptide located at VP2 7880. Recombinant A/Iran/87 (A-) viruses with AAR, SAA or AAA in
place of the SAR have been generated. Viral RNA for these viruses was
transfected into BHK cells. Repeated attempts to recover infectious virus
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Poster abstracts
by subsequent infection of BHK cells was successful only for the viruses
with the wt capsid or AAR. The AAR virus was passed through BHK
cells 4 times and sequencing of the capsid coding region of the resulting
virus showed that the AAR was retained. This suggests that the R of the
SAR motif may be important for receptor binding.
MA11/03
Functional analysis of the CA-SP1 junction in HIV-1 Gag: a critical
assembly domain and molecular target of HIV-1 maturation inhibitors
Christopher T L Murgatroyd, Catherine S Adamson
University of St Andrews, Fife, UK
The CA-SP1 junction in HIV-1 Gag plays a critical role in virus assembly
and also acts as the molecular target of HIV-1 maturation inhibitors, which
specifically inhibit CA-SP1 cleavage during viral proteolytic maturation.
We previously identified two mutations, SP1-A3V and SP1-A3T that both
confer in vitro resistance to the first-in-class maturation inhibitor bevirimat
and impose a replication defect on HIV-1. Interestingly we previously
observed that the SP1-A3V replication defect is reversed by a second-site
compensatory mutation CA-G225S and partially rescued by inhibitory
concentrations of bevirimat. To further investigate these observations we
have conducted further selection experiments using SP1-A3V to identify a
panel of second-site mutations. Characterisation of these mutants in Jurkat
T cells has demonstrated that, like the CA-G225S mutation, SP1-V7I and
SP1-P10Q mutations reverse the SP1-A3V but not the more severe
SP1-A3T replication defect. However the SP1-T8I mutation compensates
for both the SP1-A3V and SP1-A3T mutations. We are investigating
the mechanism of the replication defect imposed by the SP1 residue 3
mutations and how the second-site mutations or drug binding reverse
this effect. These studies will provide further understanding of the role of
this region of Gag in HIV-1 assembly and aid the development of secondgeneration HIV-1 maturation inhibitors.
MA11/04
Uncoating of foot-and-mouth disease virus induces permeability in
model membranes
Sarah Gold, Toby Tuthill
The Pirbright Institute, Pirbright, Surrey, UK
Non-enveloped viruses must penetrate the cell membrane without using
membrane fusion and the mechanisms for this remain poorly understood.
Foot-and-mouth disease virus (FMDV) is a livestock pathogen and
member of the non-enveloped picornavirus family. The picornaviruses
poliovirus and rhinovirus release their genomes from intact particles and
well-characterised capsid alterations are proposed to form pores for
genome delivery through the membrane. FMDV uncoating is thought
to be different because the capsid dissociates during entry and no
information is available for how this virus interacts with and penetrates
the membrane. In this study, FMDV interacted with liposome model
membranes in floatation assays and induced membrane permeability
in fluorescent dye release assays. Membrane interactions were induced
by exposure of virus to either physiological temperatures (indicating
virus-membrane interactions by capsid breathing) or exposure to low
pH, the natural trigger for FMDV uncoating. Conditions that triggered
virus uncoating also increased the rate of permeability, suggesting that
uncoating and membrane interactions were linked. After exposure of
FMDV to low pH, particles were detected with altered sedimentation,
consistent with the formation of empty capsids. Together this suggests a
new model for FMDV entry, where uncoating, membrane permeability
and genome release from a transient empty particle are coordinated.
MA11/05
Isolation, characterisation and assembly of recombinant foot-andmouth disease virus capsid precursors expressed in mammalian cells
Joseph Newman1, Stephen Curry1, Terry Jackson2, Toby Tuthill1
1
The Pirbright Institute, Woking, UK, 2Imperial College London, London, UK
FMDV is a pathogen of livestock and a member of the picornavirus
family of non-enveloped viruses. The FMDV capsid precursor is a
monomeric subunit (P1) which is cleaved at two sites by a viral protease
(3Cpro) in order to assemble into pentameric intermediates and then
intact capsids.
In this study, the requirements for assembly and properties of the
pentameric assembly intermediates were studied using recombinant
P1 produced in a reticulocyte lysate cell free system or in mammalian
cells using a vaccinia virus expression system. A panel of P1 expression
constructs was generated, including wild type, myristoylation signal and
cleavage site mutants, and a mutation introducing an artificial cleavage
site at the VP4/VP2 junction.
Purified FMDV 3Cpro was expressed in E. coli, mixed with recombinant
P1 and cleavage analysed by SDS-PAGE and immunoblot or autoradiography. The sedimentation of monomers and assembly products
was analysed by centrifugation in density gradients. Cleavage of wild type
P1 resulted in detection of faster sedimenting structures, consistent with
formation of pentamers. Analysis of P1 with 3C cleavage site mutations
showed that the two sites could be cleaved independently and that
cleavage at both sites were required for the formation of pentamers.
MA11/06
Structural characterisation of a hepatitis B core construct with an
N-terminal His tag
Richard McGonigle, Rebecca Pink, David Bhella
CVR, Glasgow, UK
The core protein of Hepatitis B Virus (HBV) possesses the unusual
property that it is expressed and forms a capsid in a variety of expression
systems and in the absence of any other viral components. HBV core
assembles to form two differently sized icosahedral particles, the T=4
form that packages the viral genome in HBV infections and a T=3 form
of unknown significance.
We have performed a structural analysis of particles produced by a
modified Hepatitis B core protein with a 34-nucleotide addition at the
N terminus that encodes a polyhistidine tag. This construct has been
created to simplify production of large quantities of core antigen for
diagnostic purposes, however our analysis shows some interesting
properties of the resulting core particles. The structures of T=3 and
T=4 capsids have been solved by cryo-electron microscopy and threedimensional image reconstruction to sub-nanometre resolution. In
addition to the characteristic dimeric spike seen in wild-type capsids we
see an additional smaller trimeric spike on the capsid surface comprising
the N-terminal insertion.
Virology workshop:
Innate immunity
MA12
MA12/01
Involvement of the adenovirus E4 Orf3 target, PML-II, in the induction
of interferon response
YiXiang Chen, Keith N Leppard
University of Warwick, Coventry, UK
Promyelocytic leukaemia (PML), a multi-isoforms protein is strongly
implicated in diverse cellular activities including the innate response to
virus infection. One isoform, PML-II, is specifically targeted by adenovirus
E4 Orf3, a protein necessary for virus growth in interferon targeted cells.
We investigated the role of PML-II in the initial induction of the IFN
response. Knockdown of PML-II not only inhibits the expression of IFNβ
and many IFN-stimulated genes (ISGs) such as ISG15, ISG20, ISG54,
ISG56 and IRF7, but also affects the induction of proinflammatory
cytokines and chemokines including interleukin-6 (IL-6), IL-8, tumour
necrosis factor-alpha (TNFα) and RANTES. In mechanistic studies,
knockdown of PML-II affected transcription factors IRF3 and NFκB activities. However, it had no effect on IRF3 or NF-κB nucleus
89
Poster abstracts
translocation and IRF3 phosphorylation. Subsequently, we found PML-II
bound co-activator CBP together with IRF3 or NF-κB to form a protein
complex. Importantly, depletion of PML-II affected IRF3 and NF-κB
binding to chromatin DNA at the promoters/enhancers of genes such
as IFNβ and IL-6, and also affected the recruitment of co-activator CBP/
p300 to these promoters. This suggested a new mechanistic role for
PML-II in the activation/regulation of innate immune response genes
during the response of cells to infection stress.
MA12/02
Respiratory syncytial virus induces but antagonises innate antiviral
responses in well-differentiated paediatric primary bronchial epithelial
cells
Rémi Villenave1, Lindsay Broadbent1, Isobel Douglas2,
Jeremy D Lyons2, Peter V Coyle3, Michael N Teng4, Ralph A Tripp5,
Liam G Heaney1, Michael D Shields1,2, Ultan F Power1
1
Centre for Infection & Immunity, School of Medicine, Dentistry & Biomedical
Sciences, Queens University Belfast, Belfast, Northern Ireland, UK, 2The Royal
Belfast Hospital for Sick Children, Belfast, Northern Ireland, UK, 3The Regional
Virus Laboratory, Belfast Trust, Belfast, Northern Ireland, UK, 4Morsani College
of Medicine, University of South Florida, Tampa, Florida, USA, 5Department of
Infectious Diseases, University of Georgia, Athens, Georgia, USA
RSV is the principal cause of severe lung disease in young infants.
However, how RSV causes disease is poorly understood. We recently
reported an RSV infection model based on well-differentiated primary
paediatric bronchial epithelial cells (WD-PBECs), which reproduced
several hallmarks of RSV infection in infants (Villenave et al, PNAS
109:5040-5, 2012). We therefore exploited this model to study
components of innate immune responses to RSV. RSV infection
virtually abrogated super-infection with Sendai virus and reduced superinfection with a different RSV strain. This was mediated, in part, by
secreted factors. However, our data indicated that type I IFNs were not
implicated. Alternatively, IL-29 was detected in BALs and basolateral
medium from RSV-infected infants and WD-PBECs, respectively, and
IL-29 pre-treatment reduced SeV growth in WD-PBECs in a dosedependent manner. Neither secreted factors nor IL-29, at doses found
in basolateral medium from RSV-infected WD-PBECs, prevented RSV
replication in pre-treated WD-PBECs. Using an RSV ΔNS1/NS2 deletion
mutant, we demonstrated that RSV NS1/2 proteins are essential for RSV
replication in WD-PBECs and implicated in antagonising IL-29-induced
antiviral responses in Vero cells. Intriguingly, RSV-infected cells in WDPBECs demonstrated massive reductions in Mx1 expression. Therefore,
RSV induces but effectively antagonises antiviral innate immune
responses in WD-PBECs.
Virology workshop:
genome
MA13
MA13/01
TenpIN, a protein-RNA toxin-antitoxin complex from the insect
pathogen, Photorhabdus luminescens, provides specific bacteriophage
resistance
Feng Rao, George P C Salmond
Toxin-antitoxin (TA) systems are ubiquitous among bacteria and
archaea, and are known to play a role in stress responses, bacterial
persistence, and phage resistance. Photorhabdus luminescens, an insect
pathogen with an exotic lifestyle, has 60 of these genetic modules in its
genome. The open reading frame (ORF) plu4873 was identified by a
comprehensive search for toxins of the Type III TA family, defined by
a direct interaction between an RNA antitoxin that acts to suppress
a proteinaceous toxin. Renamed TenpN, the plu4873 product was
confirmed to behave as a component of a TA system, with the region
5’ of the ORF, encoding the RNA antitoxin, named TenpI. TenpIN
was shown to enable bacteriophage resistance, like the paradigm Type
III system ToxIN from the plant pathogen, Pectobacterium. However,
the preliminary evidence suggests that it may show some significant
differences in the primary and, possibly, quaternary structures compared
with the ToxIN paradigm. TenpIN also confers remarkable phage
specificity in engineered E. coli derivatives.
MA13/02
Calicivirus modulation of stress granule assembly
MAjid Noori Humoud AL-Sailawi, Elizabeth Royall,
Nicolas Locker, Lisa Roberts
Department of Microbial and Cellular Sciences, FHMS, University of Surrey,
Guildford, Surrey, UK
Caliciviruses are single-stranded positive RNA viruses that are responsible
for several important diseases in human and animal hosts. To date, the
replication mechanisms of human caliciviruses are poorly understood
because of a lack of a suitable cell culture system. Feline calicivirus
(FCV) or mouse norovirus (MNV) share many properties with the
human caliciviruses, and provide models to increase our understanding
of calicivirus translation and replication. Recent evidence suggests that
the dynamic nature of mRNA expression is a key coordinator of viral
pathogenesis, with different host genes expressed at different times
during infection. The expression of mRNAs can be regulated through
their storage and/or decay in subcellular compartments such as stress
granules, to stall their translation, or processing bodies (P-bodies), for
their further degradation. Moreover, proteins within P-bodies or stress
granules can enhance or limit viral infection. Viral proteins can also be
found in these compartments, suggesting an important interplay between
RNA turnover and viral life cycle. This is an exciting emerging field in
virology and we have set out to investigate how calicivirus infection
affects the formation of P-bodies and stress granules and will present
evidence that calicivirus infection modulates the formation of stress
granules.
Virology workshop: Pathogenesis
MA14
MA14/01
Schmallenberg virus pathogenesis, tropism and interaction with the
innate immune system of the host
MAriana Varela1, Esther Schnettler1, Marco Caporale2,
Claudio Murgia1, Gerald Barry1, Melanie McFarlane1, Eva McGregor1,
Ilaria Piras3, Andrew Shaw1, Catherine Lamm1, Anna Janowicz1,
Martin Beer4, Mandy Glass1, Vanessa Herder5, Kerstin Hahn5,
Wolfgang Baumgärtner5, Alain Kohl1, Massimo Palmarini1
1
Centre for Virus Research, University of Glasgow, Infection, immunity and
inflamation, Glasgow, Scotland, UK, 2Istituto G. Caporale, Teramo, Italy,
3
Dipartimento di Medicina Veterinaria, Universita’ degli Studi di Sassari,
Sassari, Italy, 4Institute of Diagnostic Virology, Friedrich-Loeffler-Institut,
Greifswald-Insel Riems, Germany, 5Department of Pathology and Center of
Systems Neuroscience, University of Veterinary Medicine, Hannover, Germany
Schmallenberg virus (SBV) is an emerging orthobunyavirus of ruminants
associated with outbreaks of congenital malformations in aborted and
stillborn animals. Here, we developed molecular and serological tools,
and an experimental in vivo model to study SBV pathogenesis, tropism
and virus-host cell interactions.
Using a synthetic biology approach, we developed a reverse genetics
system for the rescue and genetic manipulation of SBV. We developed
an experimental mouse model of infection and showed that SBV
replicates in neurons where it causes malacia and vacuolation of
the cerebral cortex. These virus-induced acute lesions are useful in
understanding the progression from vacuolation to porencephaly and
extensive tissue destruction, often observed in naturally occurring
Schmallenberg cases. Finally, we investigated the molecular determinants
90
of SBV virulence. We found a biological SBV clone that after passage
in cell culture displays increased virulence in mice. We also found that
a SBV deletion mutant of the non-structural NSs protein (SBVΔNSs) is
less virulent in mice than wild type SBV. Attenuation of SBV virulence
depends on the inability of SBVΔNSs to block IFN synthesis in virus
infected cells. In conclusion, this work provides a useful experimental
framework to study the biology and pathogenesis of SBV.
MA14/02
Changes in PDZ protein expression following establishment of HPV
episome replication in primary human keratinocytes
Claire D James, Elizabeth K Marsh, Sarah M Leonard, Sally Roberts
High-risk HPV types, which are strongly associated with ano-genital and
oropharyngeal cancers, encode a PSD95/DLG1/ZO1 (PDZ) interaction
motif at the C-terminus of the E6 oncoprotein. This motif is known
to interact with a number of cellular PDZ proteins involved in the
regulation of cell polarity, cell proliferation and cell signalling. However,
the physiological role of E6 targeting of PDZ proteins in the life cycle
of HPV it is not yet clear. Using a cell-based model of the HPV16 and
HPV18 life cycle, we analysed the gene expression levels of a subset of
PDZ substrates of E6, using SYBR green qPCR. Following establishment
of episomal HPV genomes, there is an upregulation of selected PDZ
targets of HPV16 and HPV18 E6 at a transcriptional level. Moreover,
upon maintenance of replicating viral genomes, this upregulation is
sustained in primary cells. Interestingly, this increase in gene activity does
not correspond with an increase in protein levels. These data highlight a
novel HPV effect on the expression of specific PDZ proteins and offer
new insights into early events involving the relationship between cellular
PDZ proteins and HPV replication in primary keratinocytes.
MA14/03
Characterisation of ovine nectin-4, a novel peste des petits ruminants
virus (PPRV) receptor
Jamie J Birch1, Nicholas Juleff1, Michael P Heaton2, Ted Kalbfleisch3,
James Kijas4, Dalan Bailey1
1
The Pirbright Institute, Surrey, UK, 2U.S. Meat Animal Research Center,
Clay Center, Nebraska, USA, 3School of Medicine, Louisville, Kentucky, USA,
4
CSIRO, Queensland, Australia
Small ruminants infected with peste des petits ruminants virus (PPRV)
exhibit lesions typical of epithelial infection and necrosis. However,
the only established host receptor for this virus is the immune cell
marker signalling lymphocyte activation molecule (SLAM). We have
confirmed that the ovine Nectin-4 protein, when over-expressed in
epithelial cells, permits efficient replication of PPRV. Similar results were
reported for canine distemper and measles viruses, indicating that, like
SLAM, this protein is likely a universal morbillivirus receptor. In addition,
we performed quantitative analysis of Nectin-4 and SLAM in mRNAs
taken from 49 separate tissues of a healthy sheep. We found high
Nectin-4 expression in the mouth, upper respiratory tract and stomach
tissues, while SLAM expression predominated in lymphatic tissues. Coexpression was detected most commonly in the lungs, albeit at lower
levels. This ovine Nectin-4 expression profile correlates well with the
epithelial pathogenesis observed during PPRV infection in the natural
host. Finally, the genetic variation of Nectin-4 in 75 sheep (sampled from
around the world) was also investigated in order to determine the role
of host-encoded polymorphisms in susceptibility to PPRV infection. The
identification of ovine Nectin-4 as the PPRV receptor will contribute to
our understanding of PPRV pathogenesis.
MA14/04
The PrPC C1 fragment derived from the ovine A136R154R171 PRNP
allele is highly abundant in sheep brain and inhibits disease associated
fibrillisation of full-length PrPC protein in vitro
Lauren Campbell, Andrew C Gill, Nora Hunter, Wilfred Goldmann
Poster abstracts
Roslin Institute/The University of Edinburgh, Edinburgh, UK
TSE’s (transmissible spongiform encephalopathies) or prion diseases are
a group of neurodegenerative disorders caused by an unconventional
infectious agent thought to be devoid of nucleic acid, known as
prion. Expression of the cellular prion protein (PrPC) is crucial for the
development of prion diseases. Resistance to prion diseases can result
from reduced availability of the prion protein or from amino acid changes
in the prion protein sequence. We propose that increased production of
a natural PrP α-cleavage fragment, C1, is also associated with resistance
to disease. We show, in brain tissue, that ARR homozygous sheep,
associated with resistance to disease, produced more C1 fragment
than the disease-susceptible ARQ homozygous and highly susceptible
VRQ homozygous animals. Through the use of fibrillisation assays we
have shown that recombinant C1 can form amyloid fibrils in-vitro and
assessed the toxicity of these fibrils to neuronal cells in culture. Only
the C1 fragment derived from the ARR allele inhibits in-vitro disease
associated fibrillisation of other allelic PrPC variants. We propose that
the increased α-cleavage of ovine ARR PrPC contributes to a dominant
negative effect of this polymorphism on disease susceptibility.
MA14/05
The multifunctional roles of the rotavirus non-structural protein NSP4
MArk G Boyce1, Weiming Yang2, Malcolm McCrae1
1
The Pirbright Institute, Pirbright, Surrey, UK, 2Johns Hopkins University School
of Medicine, Baltimore, MD, USA
NSP4 was initially shown to be an integral ER membrane protein
functioning in the the trans-membrane transport of double layered
particles from the cytoplasm of the infected cell into the lumen of the ER
for final maturation by addition of the virion outer shell. More recently
NSP4 has been characterised as the first viral enterotoxin, whose
proposed secretion from infected cells and spread in a paracrine fashion
to kill surrounding uninfected cells could explain a key observation of viral
pathogenesis, namely that diarrhoea precedes extensive viral replication
in gut tissue. However these two functions of NSP4 raise the question
of how could a single protein be both an integral membrane protein and
also be secreted from infected cells? The use of a combination of time
elapse confocal analysis of virus infected cells and of cells transfected
with a plasmid expressing NSP4 provided a potential resolution of this
paradox. These studies showed that NSP4 expression promotes the
transient development late in the virus replication cycle of an extensive
network of long cytoplasmic extrusions. These extrusions can make
intercellular connections to surrounding cells and transfer material from
infected/transfected cells to uninfected cells, thereby obviating the need
for extracellular secretion of NSP4.
MA14/06
The multifunctional MCMV m169 transcript is essential for activating
Ly49 (P, L and D2) recognition of MHC-I/m04 complexes by NK cells
during lytic murine cytomegalovirus infection
Anne L’Hernault1, Vanda Juranic Lisnic2, Miranda de Graaf1, Marina Babic
Cac2, Astrid Krmpotic2, Stipan Jonjic2, Lars Dölken1
1
Department of Medicine, University of Cambridge, Cambridge, UK,
2
Department of Histology and Embryology, University of Rijeka, Rijeka,
Croatia
Differences in the Ly49 receptor repertoire of natural killer cells play
a major role in the susceptibility of different mouse strains to acute
MCMV infection. During lytic MCMV infection the MCMV proteins m06
and m152 inhibit antigen presentation via MHC-I by targeting MHC-I
molecules for degradation. To avoid missing-self recognition by NKcells the viral m04 protein rescues some MHC-I complexes to the cell
surface. Interestingly, these m04/MHC-I complexes are recognised by
activating NK-cell receptors including Ly49P, L and D2 thereby triggering
NK-cell-mediated killing of infected cells.
Although expression of m04 has been shown to be required, it is not
sufficient to activate NK-cells via Ly49P. Here, we show this activation to
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Poster abstracts
be dependent on the most abundant MCMV transcript m169. Recently,
we identified this transcript to mediate degradation of two cellular
miRNAs to aid lytic virus replication. Nevertheless, activation of Ly49P/L/
D2 was neither dependent on miR-27a/b degradation nor on the 17
kD protein this transcript encodes. In contrast, deletion of or sequence
alterations in its 5’-UTR completely abolished NK-cell activation. Our
data unravel a viral transcript with multiple both non-coding and coding
RNA functions. We will present our current data on the underlying
molecular mechanisms.
MA14/07
High-throughput quantitative proteomic analysis of dengue virus –
host cell interactions
Andrew Davidson, Han-Chen Chiu, Kate Heesom, Jim Bird,
David Matthews, Holger Hanneman
University of Bristol, Bristol, UK
Dengue virus (DENV) causes the most important arthropod-borne
infection of humans. However, little is still known concerning the virushost protein interactions that are required to facilitate virus replication
and evade host defenses. We are using Stable Isotope Labelling of
Amino acids in Cell culture (SILAC) combined with high throughput
mass spectrometry (MS), to quantitate global changes in cellular protein
levels in response to DENV infection and identify interactions between
cellular and DENV proteins.
Initially, changes in the nuclear and cytoplasmic proteomes of A549 cells,
in response to DENV-2 infection, were analysed by SILAC-MS. Changes
in the amount of a selected set of proteins, found to significantly increase
or decrease in DENV infected A549 cells were then confirmed in two
human cell lines (A549 and HEK293) over a time course of DENV-2
infection by Western blotting and confocal microscopy. To complement
the holistic analysis, high throughput co-immunoprecipitation analysis
is being conducted using cell lines stably expressing specific DENV
proteins. Novel cellular proteins that bind to the DENV NS5 protein
in a serotypic manner have been identified, including proteins that are
severely decreased during DENV-2 infection. The role of these proteins
in virus replication is currently being investigated.
MA14/08
Murine norovirus (MNV) infection is associated with mitochondrial
dysfunction as shown by SILAC quantitative proteomics
Sarah L Caddy1,2, Edward Emmott2, Ian Goodfellow2
1
Imperial College London, London, UK, 2University of Cambridge, Cambridge,
UK
Noroviruses are a major cause of human gastroenteritis worldwide, for
which no vaccine or anti-viral treatment is available. In order to further
understand norovirus-host cell interactions and to identify cellular
pathways required for and modified by norovirus during replication in
cell culture, we used quantitative proteomics to study the effect of MNV
replication on cells. The proteome of MNV infected cells was elucidated
using stable isotope labelling by amino acids in cell culture (SILAC)
quantitative proteomics. This highlighted cellular pathways altered during
MNV infection, with the most significant change being the induction of
mitochondrial dysfunction. Subsequently, mitochondrial enrichment of
MNV infected stable isotope labelled cells was conducted in triplicate
and proteins analysed. A total of 93 proteins were identified as being
decreased in abundance in the mitochondrial-enriched fraction. Proteins
of interest include those involved in mitochondrial dynamics and innate
immunity. SILAC data has since been validated using western blotting
and confocal microscopy, and further investigations are planned to
ascertain the functional effect of manipulating identified proteins on viral
titres. It is predicted that these mitochondrial proteins are altered by
MNV to enhance survival and replication.
MA14/09
Investigation of the role of naturally occurring changes in equine
influenza virus haemagglutinin in cross-species adaption
Adam Rash1, Alana Kilby1, Liz Medcalf1, Emily Holliday3,
Laura Peachey3, Greg Dowd3, Janet Daly2, Debra Elton1
1
Animal Health Trust, Newmarket, Suffolk, UK, 2University of Nottingham,
Nottingham, UK, 3Royal Veterinary College, London, UK
The first barrier that influenza A viruses have to cross when infecting
a new host species is binding to a receptor on the cell surface. The
viral glycoprotein, haemagglutinin (HA), is responsible for binding to
terminal sialic acid residues on the cell surface. Influenza viruses from
different hosts have different receptor preferences, such as specificity
for a2-3 or a2-6 linkages. Equine influenza viruses, like the avian viruses,
bind receptors with a2-3 linkages and show preference for terminal
Neu5Gc rather than Neu5Ac. Specific amino acid changes within the
220 loop of HA have been shown to shift receptor preference as well
as affect binding affinity. Analysis of equine influenza HA sequences
from strains with differing replication abilities between eggs and cells
identified naturally occurring point mutations within this region. In
addition, an equine influenza virus passaged on canine tracheal explants
highlighted a change at one of these positions within the 220 loop.
Viruses containing 220 loop mutations were rescued by reverse genetics
and characterised. HA mutants with a canine-like 220 loop mutation
replicated more efficiently in a canine cell line than the wild-type equine
HA. Haemagglutination assays with erythrocytes from different species
also highlighted differences in binding abilities between the mutants.
MA14/10
Characterisation of HSV-1 latency establishment in the ROSA26R
reporter mouse model utilising thymidine kinase (TK) deletion
mutants
Michael P Nicoll, Stacey Efstathiou
During HSV-1 latency, gene expression is restricted to the latencyassociated transcripts (LATs). LATs possess anti-apoptotic functions and
encode microRNAs that down-regulate virus gene expression.
We have previously described the use of a ROSA26R reporter mouse
model of infection to assess the frequency at which wildtype or LAT
deletion mutants establish latency. In the absence of LAT expression,
HSV-1 establishes latency in a greater number of trigeminal ganglion
(TG) neurons relative to LAT+ virus. Despite the elevated establishment
observed we have been unable to determine differences in LAT+ and
LAT-replication kinetics during acute infection using standard assays for
virus infectivity. Here we report a strategy for the characterisation of
latency establishment using LAT+ and LAT-viruses inhibited for neuronal
replication following deletion of the viral thymidine kinase (TK) gene.
Following infection with TK mutants, productive neuronal infection
is curtailed and restricted to first order neurons directly innervating
peripheral tissues. TK-deleted LAT+ and LAT-HSV-1 recombinants
establish latency at an equivalent frequency, demonstrating that
enhanced replication within TG neurons is required for an increased
frequency of establishment with LAT-virus. Furthermore, these data
suggest LAT is non-essential for first order neuron survival, despite their
potential to experience early viral gene expression.
MA14/11
Characterisation of YIPF4 a novel HPV E5 binding protein
Marietta Muller1, Ramya Gundurao1,2, Christopher Wasson1,
Oliver Watherston1, Jurgan Haas2, G Eric Blair1, Nicola Stonehouse1,
Andrew Macdonald1
1
University of Leeds, Leeds, UK, 2Division of Pathway Medicine, The University
of Edinburgh, Edinburgh, UK
92
Poster abstracts
The human papillomavirus (HPV) multifunctional oncoprotein E5
is involved in host cell transformation and immune evasion. The
mechanisms by which E5 achieves these are unclear. It is plausible that E5
modifies intracellular vesicular trafficking of cell surface receptors crucial
for proliferation and immune defence. The aim of this project was to
identify proteins that interact with E5 and contribute to these processes.
A yeast-two-hybrid identified a novel cis-Golgi protein, termed YIPF4, as
an interacting partner of HPV16 E5. Deep sequencing, Western blot and
immunocytochemistry analysis confirmed expression of YIPF4 in primary
keratinocytes. Both proteins co-localise at cellular membranes
and co-immunoprecipitation revealed a conserved interaction of YIPF4
with E5 proteins from clinically important HPVs, indicating a potentially
invaluable role for this complex during the HPV life cycle or E5 mediated
pathogenesis. Ongoing functional studies will reveal how the interaction
of E5 with YIPF4 exactly contributes to transformation and immune
evasion.
MA14/12
The genetic stability and attenuation of pneumonia virus of mice
(PVM) during consecutive passages
Yashar M Sadigh1, Andrew Easton2
1
Pirbright Institute, Compton, Berkshire, UK, 2School of Life Sciences, University
of Warwick, Coventry, UK
Serial tissue culture passage of the pathogenic strain J3666 of pneumonia
virus of mice (PVM) results in significant reduction in pathogenicity.
Groups of mice were inoculated with 5000, 500 and 250pfu of PVM
from passages 5, 6 and 7. Severe clinical signs were seen in the groups
of mice inoculated with PVM passage 5. Mice inoculated with PVM
passage 6 showed less severe clinical signs, and mice inoculated with
PVM passage 7 showed only very mild clinical signs. Comparison of the
nucleotide sequences of the SH and G genes of the three passages
showed no differences. However, comparison with the sequence of
the low passage parental identified several differences. More strikingly, it
was shown that point mutations in both of the SH and G genes cause
alterations in the main ORFs. To investigate the sequence changes in
this region in more detail, DNA clones from passage 4 of PVM were
prepared and sequenced. The data indicated that the virus population
was a mixture of at least two sub-populations. The loss of pathogenicity
associated with differences in the SH and G genes is due to selection of
one of the variants from the original mixed population.
MA14/13
Human cytomegalovirus modification of the actin cytoskeleton
confers efficient protection against natural killer and CD8+ cytotoxic
T lymphocytes
Richard J Stanton1, Virginie Prod’homme1, Melanie Moore1,
Rebecca J Aicheler1, Marcus Hainzmann2, Susanne M Bailer2,
Robin Antrobus3, Michael P Weekes3, Paul J Lehner3, B Vojtesek5,
Kelly L Miners1, Marco Purbhoo6, Stephen Mann1, Andrew J Davison4,
Eddie Wang1, Peter Tomasec1, Gavin W G Wilkinson1
1
Cardiff University, Cardiff, UK, 2Max von Pettenkofer-Institut, München,
Germany, 3University of Cambridge, Cambridge, UK, 4Medical Research
Council–University of Glasgow Centre for Virus Research, Glasgow, UK,
5
Regional Centre for Applied Molecular Oncology, Brno, Czech Republic,
6
Imperial College, London, UK
Natural Killer (NK) cells are crucial to the control of human
cytomegalovirus (HCMV) infection, and as a result the virus carries
a large number of genes dedicated to modulating the NK response.
HCMV has become a paradigm for immune evasion, containing six
genes and a microRNA that have been shown to be inhibit NK cell
attack. Understanding the mechanisms by which these work has revealed
numerous details concerning the normal functioning of the immune
system. To identify novel HCMV encoded genes which modulate the
NK response, ORFs were individually screened following cloning into
adenovirus vectors. This revealed a gene that is capable of modulating
the immune response through manipulation of the actin cytoskeleton.
HCMV-directed loss of F-actin fibers, and inhibition of cytoskeletal
remodelling resulted in an extremely broad inhibition of both NK and
T cells. Conjugate formation, synapse structure, degranulation and killing
were all inhibited by inhibition of F-actin in the target. This indicates a
requirement for the infected cell to ‘co-operate’ in its own killing. In
attacking this requirement, HCMV efficiently inhibits a broad range of
effector cells.
MA14/14
The human transmembrane protease serine 2 is necessary for the
production of Group 2 influenza A virus pseudotypes
Francesca Ferrara1, Eleonora Molesti1,
Eva Böttcher-Frieberthäuser2, Davide Corti3, Simon Scott1,
Nigel Temperton1
1
2
Philipps University, Marburg, Germany, 3Institute for Research in Biomedicine,
Bellinzona, Switzerland
The monomer of influenza haemagglutinin (HA) is synthesised as a single
polypeptide precursor that during maturation is cleaved by proteases
into two active subunits. Other studies have demonstrated that the
human Transmembrane Protease Serine 2 (TMPRSS2) can cleave the
HA of human seasonal influenza viruses and therefore has an important
role in pathogenesis. As a model of this cleavage we have investigated
the use of human TMPRSS2 to produce high-titre influenza HA lentiviral
pseudotypes from Group 2 influenza viruses. Such pseudotypes
represent powerful and safe tools to study viral entry mechanisms
and immune responses. Influenza pseudotype particles are obtained
by co-transfecting human embryonic kidney HEK293T/17 cells using
plasmids coding for the influenza HA, HIV gag-pol and a retroviral vector
incorporating firefly luciferase. However, in order to successfully produce
Group 2 pseudotypes, it was necessary to co-transfect a plasmid
expressing the TMPRSS2 endoprotease to achieve the necessary HA
cleavage for infective particle generation. These lentiviral pseudotypes
were shown to transduce HEK293T/17 cells with high efficiency. This
demonstrates that TMPRSS2 can cleave, in vitro, both the HA of
human seasonal influenza and also other Group 2 HA influenza strains.
Pseudotypes represent an adjunct tool for predicting pandemic potential
of emerging influenza viruses.
MA14/15
Influenza A virus NS1 induces re-localisation of cellular ADAR1 to
nucleoli during infection
Artur A Arikainen1, Helen Wise2, Paul Digard2
1
University of Cambridge, Cambridge, UK, 2The Roslin Institute, The University
of Edinburgh, Easter Bush, Edinburgh, UK
We recently found that influenza A virus infection induces the relocalisation of adenosine deaminase acting on RNA 1 (ADAR1) to the
nucleolus. ADAR1 is an enzyme that converts adenosine residues in
RNA to inosine (A-to-I). Evidence for non-specific editing of influenza
RNAs has been reported, but the significance of this has not been
demonstrated. We previously showed that influenza A NS1 is both
necessary and sufficient to induce nucleolar re-localisation of ADAR1 in
several cell types and that mutant forms of the NS1 protein with lesions
in the RNA binding or TRIM25 interaction sites failed to induce this relocalisation. This correlates with the inability of these mutants to suppress
IRF3 activation. We also show by GFP-trap pull-down assays that
ADAR1 interacts with influenza NS1 in an RNA-independent manner.
Additionally, ADAR1 interacts with components of the viral polymerase
and over-expression of GFP-ADAR1, but not GFP-ADAR2, promoted
viral polymerase activity in a mini-replicon assay, independently of
NS1. We conclude that the influenza A virus NS1 protein induces the
nucleolar re-localisation of ADAR1 and that this interaction benefits the
virus. However, the full mechanism of this is yet unknown.
93
Poster abstracts
MA14/16
An H5N1 avian influenza virus with low pathogenic phenotype despite
a multi-basic HA cleavage motif
Jason Long1,2, Wendy Howard2, Jill Banks2, Wendy Barclay1
1
Imperial College London, London, UK, 2Animal Health and Veterinary
Laboratories Agency, Weybridge, UK
Highly pathogenic avian influenza (HPAI) viruses contain a multi-basic
HA cleavage site (MBCS), which facilitates systemic dissemination of
the virus. Yet, other factors may be necessary for a HP phenotype. A/
turkey/England/50-92/91 H5N1 (50-92) is a HPAI virus isolated from a
poultry farm outbreak in the UK 1991. Interestingly, different clones from
the original isolate displayed great variation of pathogenicity. Sequencing
revealed three differences in the HA gene between two of the clones.
Mutation A172T, a predicted glycosylation site; E205K, described to
increase propensity of α-2,6 Sialic acid binding; and G348R, a striking
introduction of a positive charge aa in the HA fusion peptide. We
engineered combinations of these mutations into recombinant 50-92
viruses by reverse genetics. Recombinant viruses generated were tested
for their pathogenicity. Intriguingly, despite the presence of a MBCS, the
virus was only highly pathogenic when it carried G348R in the HA gene.
Preliminary results suggest this mutation altered the pH of fusion of the
HA protein. We hypothesise that 348R destabilises the HA protein to
enable fusion and increase infectivity. Indeed, other research has shown
that HA mutations altering the pH of fusion affect pathogenesis of avian
influenza viruses (Reed et al., 2010).
MA14/17
Vaccinia virus protein 169 modulates virus virulence and
immunogenicity
Pavla Strnadova1,2, Hongwei Ren1,2, Robert Valetine2,
Michela Mazzon1,2, Geoffrey L Smith1,2
1
Department of Pathology, University of Cambridge, Cambridge, UK,
2
Department of Virology, Faculty of Medicine, Imperial College London,
London, UK
Vaccinia virus (VACV) is a well characterised member of the poxvirus
family, the vaccine used against smallpox, and an expression vector
with utility as a vaccine against other diseases. VACV replicates in
the cytoplasm and encodes around 200 proteins. These include
many immunomodulators that suppress the host immune response
to infection. The study of these proteins can provide insights into
the immune response and development of vaccines with increased
immunogenicity.
VACV strain WR protein 169 was uncharacterised hitherto. Here we
show that 169 is a small, cytoplasmic protein that is expressed by 4 h
post infection. A VACV strain lacking gene 169 (vΔ169) resulted in
unaltered virus replication and spread in tissue culture, but increased
virulence in both intranasal and intradermal murine models compared to
controls. However, mice immunised with vΔ169 were better protected
against challenge with VACV. Future work will determine the function
of the169 protein and the mechanism responsible for the phenotype
observed in vivo.
MA14/18
Hepatitis C virus compartmentalisation in the liver
Ditte L Hedegaard1, Gary Reynolds2, Ian A Rowe1,2,
Stefan Hübscher2, David Adams2, David Mutimer2, Peter Balfe1,
Jane A McKeating1
1
HCV Research Group, The University of Birmingham, Birmingham, UK,
2
Centre for Liver Research, NIHR Biomedical Research Unit, The University of
Birmingham, Birmingham, UK
The process which hepatitis C virus (HCV) maintains chronic infection
in the liver is unknown. A ‘demic’ model of HCV evolution, where
quasispecies diversity arises as the result of independent evolution in
small isolated populations, is supported by recent observations that
HCV transmits via cell-to-cell route to form discrete foci in the liver
i.e. becomes compartmentalised. To investigate this we sampled the
eight segments of the liver from HCV-infected subjects undergoing liver
transplant for HCV RNA burden and sequence diversity in comparison
to plasma sampled at the same time. Comparable HCV RNA levels
were observed across all 8 samples from a single liver. However
between patients we observed up to 100 fold differences in hepatic viral
burden. Furthermore, the relative amounts of HCV RNA detected in
the liver and plasma varied significantly between subjects. Comparison of
the E1E2 region from different liver segments showed minimal genetic
heterogeneity compared to that seen in the plasma, leading us to
conclude that liver derived sequences underestimate HCV heterogeneity
in end-stage liver disease. Studying the quasispecies structure in
infected patients will have a major impact on our understanding of the
mechanism(s) by which HCV escapes from host immune responses and
anti-viral therapies.
MA14/19
Isolation and characterisation of coronavirus in Irish cattle
Lynda Gunn, Helen O’Shea
Cork Institute of Technology, Cork, Ireland
Bovine Coronavirus (BCoV) has been implicated in disease in cattle,
causing either respiratory or enteric disease, in calves it is associated with
chronic, often bloody diarrhoea while in adult bovines, it causes winter
dynasty. BCoV is a common pathogen in Cuba, Brazil, Italy, Quebec,
Korea and China. In Ireland, BCoV represents the third most commonly
diagnosed agent of calf morbidity and death, but is only identified
in less than 10% of cases. Previous studies on BCoV in Ireland have
mainly focused on immunological reactions, with little data regarding
molecular epidemiology available, In this study, 11 calf BCoV isolates
were characterised using molecular methods, with emphasis on the spike
protein. The aim of the study was to identify variations in key genes
such as the nucleocapsid, spike and hemagglutinin esterase genes when
compared to global isolates. Resultant sequence data was examined
using phylogenetic analysis and sequence alignment profiles to observe
any unique features. Phylogenetic analysis of nucleocapsid, spike and
hemagglutinin esterase genes revealed that the Irish isolates formed
distinct clades, while sequence analysis of translated proteins found
unique amino acid changes in nucleocapisid and Spike (hypervariable
region) and hemagglutinin esterase proteins. These residue changes have
not been previously described.
MA14/20
A wild-type mumps virus (MuV) rescue system illuminates potential
role of cell–cell spread in infection and pathogenesis
Connor G G Bamford1, Michael R Wilson1, Martin Ludlow1,
Linda J Rennick1, Ken Lemon1, Bertus K Rima1, W Paul Duprex2,1
1
Queen’s University Belfast, Northern Ireland, UK, 2Boston University, Boston,
Massachusetts, USA
MuV is a respiratory and neurotropic virus that causes mumps. The
mechanisms of MuV cell entry, cell-to-cell spread, pathogenesis and
attenuation are poorly understood. A wild-type MuV strain (G09) was
isolated from a buccal sample obtained during a recent mumps outbreak.
Consensus genomic sequences were determined and a plasmid,
pMuVG09, containing the complete genome was generated based on
the sequence from unpassaged material. This plasmid was modified
by inserting an additional transcription unit encoding enhanced green
fluorescent protein (EGFP) after the second gene, pMuVG09EGFP(3).
Helper plasmids expressing the nucleocapsid, phospho-and large
proteins were used to recover two recombinant (r) viruses, rMuVG09
and rMuVG09EGFP(3). These were plaque picked, grown on Vero cells
and molecular characterised. Their growth kinetics were comparable
to MuVG09. Fusogenicity is a hallmark of infection and MuVG09, rMuVG09
and rMuVG09EGFP(3) produced syncytia in a range of cell lines. Transient
expression of the hemagglutinin-neuraminidase (HN) and fusion (F)
94
Poster abstracts
glycoproteins demonstrated that the HN glycoprotein is the major
determinant. Lectin blocking and neuraminidase treatment identified
which sialic acids govern virus-to-cell and cell-to-cell. A non-fusogenic
variant of rMuVG09EGFP(3) was isolated and sequenced and a single
amino acid change in F was shown to separate virus-to-cell from cell-tocell fusion.
MA14/21
Characterisation of an ultra pathogenic strain of Schmallenberg virus
MAriana Varela1, Marco Caporale2, Ilaria Piras3, Anna Janowicz1,
Eva McGregor1, Esther Schnettler1, Claudio Murgia1, Gerald Barry1,
Melanie McFarlane1, Andrew Shaw1, Martin Beer4, Mandy Glass1,
Vanessa Herder5, Kerstin Hahn5, Wolfgang Baumgärtner5, Alain Kohl1,
Massimo Palmarini1
1
University of Glasgow, Glasgow, Scotland, UK, 2Istituto G. Caporale, Teramo,
Italy, 3Dipartimento di Medicina Veterinaria, Sassari, Sardinia, Italy, 4Institute
of Diagnostic Virology, Friedrich-Loeffler-Institut, Greifswald-Insel Riems,
Germany, 5Department of Pathology and Center of Systems Neuroscience,
University of Veterinary Medicine, Hannover, Germany
Schmallenberg virus (SBV) is a novel Orthobunyavirus of ruminants
that emerged in Europe in late 2011. SBV infection of adult animals
is characterised by mild clinical signs including diarrhea, reduced milk
production and pyrexia. However, infection of susceptible pregnant
animals is associated with musculoskeletal and central nervous system
malformations in stillborn or newborn lambs and calves.
Here we used a forward genetics approach to identify SBV determinants
of virulence. To this end SBV was serially passaged in tissue culture
(referred to as SBVp32) and its pathogenicity assessed by intracranial
infection of newborn NHI-Swiss mice. SBVp32 resulted more pathogenic
than wild type SBV (wtSBV), which was associated with a faster spread
of the virus in the brain of infected mice and the accumulation of a
variety of mutations in all three viral genome segments. Moreover,
SBVp32 was in vivo characterised in adult IFN receptor knock out mice
where, although no lethal, it was found to induce more pronounced
clinical signs than wtSBV. Importantly, the organ distribution of SBV could
be characterised in this mouse model of infection.
The development of an ultra pathogenic strain of SBV could prove useful
as a challenge model for vaccine trials in ruminants.
MA14/22
Identifying determinants of HIV-1 cell-to-cell spread at virological
synapses
Shimona L Starling, Elisabetta Groppelli, Clare Jolly
Cell-cell spread of HIV-1 between CD4+ T-cells confers many
advantages including more rapid infection kinetics, evasion of neutralising
antibodies and cellular restriction factors, and may pose a barrier
to eradicating HIV-1 from the host. HIV-1 cell-cell spread occurs at
intercellular contacts called Virological Synapses (VS) at which HIV-1
preferentially assembles and buds. We have previously shown that the
microtubule organising centre (MTOC) and associated organelles are
polarised within the HIV-1 infected cell at the VS; however the causes
and consequences of this are unknown. To address this we have used
immunofluorescence microscopy coupled with mutant cell lines and
viruses and show that recruitment of the MTOC to the VS is triggered
upon engagement of integrins on the HIV-1 infected cell. Specifically our
data implicate LFA-1 as an important component of this process. We
have also observed that HIV-1 infected cells are more prone to polarise
than uninfected cells in response to integrin engagement, indicating a
viral protein maybe responsible. We further show the VS is a site of
enriched phosphotyrosine staining, suggestive of activation of synaptic
signalling. We hypothesise that polarisation at the VS is triggered by cellcell contact and that HIV-1 infection may regulate this to promote viral
spread.
MA14/23
Emergence of HPAI from LPAI using the H7N7 HPAI Oxfordshire,
UK 2008 outbreak as a model
Amanda Hanna1,2, Wendy A Howard1, Alejandro Núñez1,
Wendy S Barclay2, Jill Banks1
1
Animal Health & Veterinary Laboratories Agency (AHVLA), Weybridge, UK,
2
Imperial College, London, UK
In June 2008 an outbreak of highly pathogenic avian influenza (HPAI)
H7N7 was confirmed in hens in Oxfordshire, UK. In addition to highly
pathogenic (HP) genotype diagnosis, sequencing also revealed a rare
‘intermediate’ cleavage site (CS) which confers a low pathotype in the
context of wild type isolates; however it has an extra basic amino acid in
the CS than is typical for low pathogenicity avian influenza (LPAI) viruses.
We hypothesised that a virus containing this ‘intermediate’ CS was the
outbreak progenitor virus.
We also hypothesised that although a multi-basic CS sequence is the
main determinant of high pathogenicity, mutations outside of the CS and
in other genes will also contribute to the HP phenotype and evolution
to high pathogenicity. We used the outbreak as a model to identify
genetic markers responsible for the HP phenotype in the H7 backbone
using reverse genetics and embryonated egg infections; we generated
two isogenic H7N7 viruses, one based on a wild type HP virus from
the outbreak and another with the ‘intermediate’ cleavage site inserted.
Preliminary data indicates that the CS is the main contributor to the
HP phenotype but mutations in the rest of the HP genome may also
contribute.
MA14/24
Effect of African swine fever virus and two of its proteins on surface
levels of MHC class I expression
Derah Saward Arav1,2, Dave Chapman1, Mike Skinner2,
Linda Dixon1
1
Pirbright Institute, Pirbright, UK, 2Imperial College, London, UK
African swine fever virus (ASFV) is a large double-stranded DNA virus
which causes fatal haemorrhagic fever in domestic swine. Previous
investigations have suggested that ASFV downregulates the host MHC
class I antigen presentation pathway, since surface levels of MHC class I
molecules decrease in ASFV-infected cell populations compared to levels
in uninfected cell populations.
In the current study, we have used flow cytometry to show that surface
MHC class I expression varies with the presence or absence of two
ASFV proteins, EP153R (which has previously been implicated in the
modulation of MHC class I expression) and CD2v. On infection of PBM
cells with avirulent ASFV isolate NHP68, which lacks functional genes
for both EP153R and CD2v due to frameshift mutations, surface MHC
class I expression increased significantly in relation to mock infected
cells (p<0.05). However, infection with recombinants of this isolate with
either restored CD2v, or restored CD2v and EP153R genes, resulted in
surface MHC class I levels unchanged from the mock. The data suggest
that these virus proteins are involved in preventing up-regulation of
surface MHC class I expression following virus infection.
Work is in progress to investigate the mechanism by which these virus
proteins exert this effect.
MA14/25
Mechanism of respiratory virus regulation of ‘cough’ receptors on
bronchial epithelial and neuronal cells
Shadia Omar, Locan McGarvey, S Louise Cosby
Queen’s University Belfast, Belfast, UK
Viruses induce cough and wheeze in asthma and other respiratory
conditions by unknown mechanisms. However, central to the process
is sensitisation of the cough reflex. Likely receptors implicated in
sensation and found on airway epithelium and nerves are: transient
receptor potential vanilloid 1 (TRPV1) and acid sensing ion channel
95
Poster abstracts
receptor (ASIC). We infected the human bronchial epithelial cell
line BEAS-2B with both measles virus (MV) and respiratory syncytial
virus (RSV) and neuroblastoma SHSY5Y cells (model for pulmonary
nerves) with MV. We found that both TRPV1 and ASIC3 mRNA and
protein levels were up-regulated at specific time points after infection
or treatment with UV inactivated virus. We examined whether direct
interaction of virions with these receptors occurred using anti-receptor
antibodies. The results indicated that virus induced soluble factors rather
than direct virus contact are involved in regulating TRPV1 and ASIC3
following virus infection. We examined correlation between selected
inflammatory mediators and TRPV1 and ASIC3 modulation in BEAS-2B
cells infected with RSV or MV by ELISA. IL8 was induced in both MV
and RSV infection in BEAS-2B cells and also IL6 by MV. We are currently
examining the effect of these cytokines on TRPV1 and ASIC as potential
receptor therapeutic targets.
MA14/26
Increase of ‘cough receptor’ expression on human sensory nerve and
human primary bronchial epithelial cells following respiratory virus
infection: studies of asthmatic and non-asthmatic subjects
Haniah Abdullah, Jeremy Parker, Michael Shields, Liam Heaney,
Lorcan McGarvey, Sara Louise Cosby
Queen’s University Belfast, Belfast, UK
Airway nerves control crucial reflexes such as cough and
bronchoconstriction. However, in asthma and other respiratory
conditions these reflexes become hyperactive causing cough and
wheeze which are exacerbated during viral infections. The novel
transient receptor potential (TRP) channel proteins, which are found
on the surface of many cell types including airway sensory nerves and
epithelial cells represent potential candidate receptors. Rhinovirus (RV)
known to cause common cold symptoms, is frequently detected in
patients with chronic asthma. Bronchial epithelial cells (PBEC), isolated
from the lung of healthy donors and asthmatic patients as well as cells
modeling airway nerves were infected with RV and the levels of TRP
proteins and mRNA measured. We found that levels of 3-Ankyrin like
protein with transmembrane like domain 1 (TRPA1) protein (one of the
TRP channels) and mRNA increase in both cell types after RV infection
or with soluble factors released from virus infected cells. Furthermore,
PBEC from some asthmatic patients had a higher response than those
from non-asthmatic subjects. These results indicate potential therapeutic
targets for asthma and other respiratory conditions. In this respect we
are currently examining the effect of TRP channel inhibitors during virus
infection.
MA14/27
Host and virus factors influencing the clinical outcome of bluetongue
virus infection
M Caporale1,2, L Di Gialleonardo2, A Janowicz1, M Golder1,
M Palmarini1
1
MRC-University of Glasgow Centre for Virus Research, Glasgow, UK, 2IZS
G. Caporale, Teramo, Italy
The clinical outcome of BTV infection is variable and is attributed to
several factors related to the virus, the host and the environment.
However, it is difficult to compare data in the literature as different
investigators have used various BTV strains in variable experimental
settings. In this study, we assessed the clinical outcome of BTV infection
in different hosts and using various BTV serotypes/strains in the same
experimental conditions. Firstly, we assessed host susceptibility to the
same strain of BTV in different species (sheep and goats) and in different
sheep breeds. In addition, we compared virulence of the original BTV-8
strain isolated in the Netherlands (BTV-8NET; known to be highly virulent)
with an Italian isolate (BTV-8IT), as the latter was not associated with
major clinical manifestation of disease. We have also tested BTV-2, in
order to compare differences in virulence displayed by distinct BTV
serotypes, and cell culture adapted BTV strains. In all infected animals,
we assessed clinical signs including fever, viremia and levels of neutralising
antibodies. We obtained data showing that clinical severity induced by
BTV infection is associated primarily to the virus and host species, while
differences between breeds are less evident.
MA14/28
Evolution, surveillance and pathogenesis of European swine H3N2
S M Brookes, B Nash, A Núñez, I H Brown
Animal Health and Veterinary Laboratories Agency, New Haw, Surrey, UK
Influenza viruses of the H3 subtype are found in both pigs and humans,
albeit evolutionarily removed across the species. In Europe the H3N2
viruses are of conventional human/avian origin and the UK has been
swH3N2 free since 1997. The ferret is accepted as a good animal model
for human disease. Pigs have some utility in this area and represent a
natural reservoir.
Pigs and ferrets were infected intranasally with 5-6 logs10 of swH3N2
(A/swine/Italy/55295/2011) virus and monitored for pyrexia, virus
shedding and respiratory pathology. There were substantial differences
in clinical presentation, lung pathology and virus shedding between
infected pigs (dpi9, 14.6 REU) and ferrets (23.3 REU). The latter had
reduced replication in the lung and mild clinical disease. Pig infection was
asymptomatic, viral shedding was moderate and lung involvement was
considerable. Transmission dynamics will also be presented.
The differences in pathogenesis of H3N2 influenza A virus in pigs and
ferrets was substantial. Both represent useful models of human disease,
with greater severity in ferrets than the mild disease of pigs, the reasons
for this continues to be a key area of investigation and is not restricted to
virus receptor distribution.
ESNIP3:FP7#259949 http://www.esnip3.com/
Antigone:FP7#278976 http://www.antigonefp7.eu/ant/
Virology workshop:
Gene expression and replication
MA15
MA15/01
Phosphorylation of Kaposi’s sarcoma-associated herpesvirus ORF57
protein controls its interactions with components of the hTREX
complex
Brian R Jackson, Belinda Baquero, Marko Noerenberg,
Adrian Whitehouse
KSHV ORF57 is a multifunctional protein involved in multiple steps of
viral mRNA biogenesis. We have previously shown that ORF57 interacts
directly with two essential cellular export factors, Aly and UIF, and via
these two factors recruits the entire hTREX complex to form an export
competent viral ribonucleoprotein particle.
Here we present new data showing that the protein-protein interactions
essential for ORF57-hTREX binding are controlled by phosphorylation
of three specific serine residues within the putative Aly binding domain
of the ORF57 protein. These phosphorylated residues were identified
by mass spectrometry and subsequently mutated to ablate the
phosphorylation status. Removal of these phosphorylation sites does not
affect ORF57 cellular localisation. In contrast, ORF57 no longer binds to
the cellular export factors upon removal of these sites and subsequently
is unable to efficiently export viral mRNAs. These data suggest that the
phosphorylation status of ORF57 is an essential regulatory requirement
for its function in viral mRNA processing.
MA15/02
Identification of differences in polymerase basic (PB1 and PB2)
proteins of avian influenza A viruses grown in chicken and duck cells
Firas Al-Mubarak
The University of nottingham, Nottingham, UK
96
Poster abstracts
MA15/05
Autophagy is not required for infectious bronchitis virus replication
Helena J Maier1, Eleanor Cottam1, Phoebe Stevenson-Leggett1,
Jessica A Wilkinson1, Christopher J Harte1, Tom Wileman1, Paul Britton1
1
The Pirbright Institute, Compton Laboratory, Compton, Newbury, Berkshire,
RG20 7NN, UK, 2University of East Anglia, 2Biomedical Research Centre,
Norwich Medical School, Faculty of Medicine and Health, Norwich, NR4 7TJ,
UK
Avian gammacoronavirus Infectious Bronchitis Virus (IBV) is an important
pathogen of poultry causing large economic losses to the global poultry
industry. During infection, coronaviruses induce cellular membrane
rearrangements to provide a platform for assembly of viral replication
complexes. Membrane rearrangements observed include double
membrane vesicles, which resemble cellular autophagosomes generated
during autophagy. Additionally, coronaviruses induce autophagy marker
protein LC3 to be become punctate during infection, indicative of
autophagy induction. Previous work has shown that IBV induces
autophagy in mammalian cells. Using new tools for studying autophagy in
avian cells, we investigated the role of autophagy during IBV infection of
avian cells. Although we observed that IBV induced autophagic signalling
in mammalian Vero cells, no induction was seen in primary chick kidney
cells or the avian DF1 cell line. Furthermore, pre-treatment of cells to
induce or inhibit of autophagy did not affect IBV replication, suggesting
that classical autophagy may not be important for virus replication.
However, expression of IBV replicase non-structural protein 6 alone
did induce autophagic signalling in avian cells, as seen in mammalian
cells. This may suggest that IBV can inhibit or control autophagy in avian
cells, although IBV did not inhibit autophagy induced by starvation or
rapamycin treatment.
Influenza A viruses are belonging to the family Orthomyxoviridae.
The virus consists of eight RNA segments: PB1, PB2, PA, HA, NP,
NA, M, and NS. Following infection of primary cells grown with avian
influenza virus, duck cells undergo rapid cell death, while in chicken cells,
death occur less rapidly. Rapid death in duck cells is associated with
reduced production of infectious virions in comparison with the longer
surviving infected chicken cells. We hypothesise that the reduction in
infectious virus titre from duck cells may be due to changes in the viral
genome. Here we investigated the molecular differences between
viruses produced in chicken and duck cells. All eight viral segments
were amplified and sequenced. Six of viral genes (PA, HA, NA, M, NS,
and NP) showed identical sequences between viruses produced from
chicken and duck cells, but some differences were found in PB1, and
PB2 sequences in virus derived from duck cells. Changes in polymerase
gene sequences, during virus replication in duck cells might disrupt the
functional polymerase protein and reduce viral RNA cap-snatching,
endonuclease or polymerase function and consequently the infectivity of
the viruses. Such viral variation may also play a role in virus evolution and
spread.
MA15/03
The effects of KSHV on the nucleolar proteome
Christopher B Owen, Adrian Whitehouse
The nucleolus is a subnuclear structure involved in ribosome biogenesis,
cell stress response and cell cycle control. Recent studies have shown
that infection with a number of RNA and DNA viruses causes the
host cell nucleolus to undergo proteome and morphological changes.
Many members of the herpesvirus family have been shown to induce
such responses and new techniques involving nucleolar fractionation
and SILAC (Stable Isotope Labelling with Amino-acids in Cell-culture)
combined with mass spectrometry are now being used to obtain data
about the movements of different viral and cellular proteins into and
out of the nucleolus during infection. Kaposi’s Sarcoma-associated
Herpesvirus (KSHV) is a gammaherpesvirus that causes changes to the
nucleolar proteome of infected cells, however, many of these changes
are yet to be identified and their effects on viral replication are not
fully understood. Therefore, this project aims to investigate changes
within the nucleolar proteome during KSHV infection using quantitative
proteomic approaches.
MA15/04
Studies on infectious bronchitis virus accessory proteins 5a and 5b
Stuart Dent, Helena J Maier, Paul Britton
Pirbright Institute, Compton Laboratory, Compton, Berkshire, UK
Avian infectious bronchitis is a highly contagious and economically
important respiratory disease of poultry caused by the gammacoronavirus
infectious bronchitis virus (IBV). The IBV Beaudette genome encodes
common structural and replication proteins, as well as group specific
accessory proteins 3a, 3b, 5a and 5b. Recombinant viruses have been
created that lack the ability to express the accessory proteins, 5a and 5b,
in the background of Beau-R IBV, by scrambling (scr) the start codon of
each gene, demonstrating that these genes are dispensable for replication
in in vitro and ex vivo systems derived from chickens (Casais et al, 2005).
Observations noted the behaviour of the mutant viruses in Vero
cells may be different to the Beau-R parent virus. When investigated
further, the scr5b virus did not cause cytopathic effect, whereas the
scr5a and Beau-R parent virus did. Growth curves investigated virus
growth over 72hrs and found that the growth of the scr5b virus was
comparable to Beau-R, whilst the growth of the scr5a virus was impaired.
Further studies are ongoing to investigate whether there are effects
on RNA synthesis, protein expression and virus release and using coimmunopreciptation experiments of tagged 5a and 5b to look at proteinprotein interactions.
MA15/06
The KSHV lytic switch protein Rta and the cellular proteome:
potential link between viral DNA replication and the sumoylation
pathway
David J Hughes, Jennifer Wood, Stuart Macnab, Adrian Whitehouse
KSHV is the causative agent of Kaposi’s sarcoma (KS), a malignancy of
immunocompromised patients. Like all herpesviruses, KSHV latently
infects its host; however, lytic replication and the production of infectious
virions is necessary for the pathogenesis of KS. The viral RTA protein is
essential for reactivating the lytic cycle by transactivating lytic genes and
interacting with viral origins of lytic DNA replication (oriLyt). Hence,
RTA and viral DNA replication provide attractive drug targets. To
investigate the effects of RTA expression on the cellular proteome,
global quantitative proteomics was applied using a cell line with inducible
RTA expression. As expected, protein networks involved in transcription
and DNA replication were highlighted, including the entire MCM helicase
(MCM2-7), an essential component of the pre-replicative (pre-RC)
complex required for cellular DNA replication. Further analysis has
shown that RTA interacts with the MCM complex, and a promising
cancer therapeutic that inhibits Cdc7 (MCM activator) inhibited viral
genome amplification. Further bioinformatics analysis demonstrated a
striking overlap between RTA-induced proteins and recently identified
poly-SUMOylated cellular substrates, including the MCM helicase itself
and associated proteins. Current work is validating a functional link
between viral DNA replication and the SUMOylation pathway.
MA15/07
Modulation of cellular factors implicated in murine leukaemia virus
readthrough and its effect on replication
Nerea Irigoyen, Eszter Csibra, Ian Brierley
Division of Virology. Dept. of Pathology. University of Cambridge, Cambridge, UK
Ribosomal readthrough, suppression of termination at a stop codon, is
exploited in the replication cycles of gammaretroviruses and represents
a potential target for antiviral intervention. Here we investigated the
97
Poster abstracts
effect of modulating readthrough efficiency on the replication of the
retrovirus Murine leukaemia virus (MuLV) by depleting the eukaryotic
eRF1-eRF3 translation termination complex using RNA interference
technology. It has been shown that the depletion of eRF1 and eRF3
enhances readthrough at all three stop codons. Moreover the MuLV
reverse transcriptase binds to eRF1 upregulating readthrough and
creating a positive feedback loop that drives the synthesis of more
pol gene products. Readthrough efficiency was enhanced 2-fold when
it was assayed in eRF1 and eRF3 silenced cells using a dual luciferase
readthrough reporter plasmid. Virus replication in the silenced cells,
as determined by qRT-PCR, TCID50, protein expression and reverse
transcriptase activities, seems to be increased suggesting that this modest
overproduction of the Gag-Pol polyprotein of MuLV is tolerated. We are
currently interested in the interaction of the MuLV reverse transcriptase
with eRF1 and conducting infectivity assays of reverse transcriptase
mutants in eRF1 silenced cells.
MA15/08
Avian autophagy marker LC3 with a G120A mutation is not
incorporated into autophagosomes
Jessica A Wilkinson, Pheobe Stevenson-Leggett, Paul Britton,
Helena J Maier
The Pirbright Institute, Compton, Berkshire., UK
Avian infectious bronchitis virus (IBV) is a gammacoronavirus that causes
infectious bronchitis in chickens. Like all coronaviruses, IBV rearranges
cellular membranes during infection for the assembly of viral replication
complexes. One structure produced is the double membrane vesicle
(DMV) resembling the autophagosome characteristic of cellular
autophagy. Autophagy allows the breakdown of cellular components
to ensure the survival of a cell during starvation and marker protein
microtubule-associated protein 1 light chain 3 (LC3) is incorporated
into the membrane of autophagosomes and becomes punctate when
the pathway is activated. This process requires cleavage of LC3 and
subsequent lipidation at position G120. Previously, coronaviruses have
been shown to induce autophagy during infection and viral non-structural
protein 6 (nsp6) induces autophagy when expressed alone. Replication
deficient, recombinant adenoviruses expressing tagged avian LC3 were
used to investigate the role of autophagy during IBV infection in primary
chicken cells. To facilitate this work, a control recombinant adenovirus
was generated expressing EGFP-tagged avian LC3 with a G120A
substitution, preventing cleavage and lipidation. The function of EGFPLC3 G120A was confirmed by confocal microscopy and western blot. In
addition, unlike wild type EGFP-LC3, EGFP-LC3 G120A did not become
punctate when expressed with IBV nsp6.
MA15/09
Activity of influenza virus polymerase containing PB2 627E is affected
by viral RNA template length and promoter sequence but not viral
nucleoprotein
Duncan J Paterson, Ervin Fodor
Most avian influenza viruses do not replicate efficiently in human cells.
This is partly due to the low activity of the RNA polymerase of avian
influenza viruses in mammalian cells. An E→K adaptive mutation at
residue 627 of the PB2 subunit of the polymerase increases the activity
of avian-derived virus polymerases in mammalian cells. Accordingly,
viral ribonucleoprotein (RNP) reconstitution assays show that a viral
polymerase containing PB2 627E characteristic of avian influenza
viruses exhibits impaired activity in mammalian cells compared to a
viral polymerase that contains PB2 627K characteristic of mammalianadapted influenza viruses. In contrast, purified viral polymerases
containing either PB2 627E or PB2 627K show comparable levels
of activity in transcription assays that require no RNP assembly. To
reconcile these conflicting observations, we used an NP-independent cell
based transcription/replication assay to assess viral polymerase activity.
We found that PB2 627E polymerase inhibition in mammalian cells is
independent of NP expression, but is dependent on the length of the
viral RNA template. In addition, restriction of PB2 627E polymerase was
overcome by mutations to the viral RNA template promoter sequence.
Consequently, we propose that PB2 627 affects recruitment of the viral
RNA promoter by the viral polymerase in human cells.
MA15/10
Sensitivity of murine leukemia virus replication to modulation of
readthrough rate through the Gag-Pol open reading frame
Eszter Csibra, Nerea Irigoyen, Ian Brierley
Department of Pathology, University of Cambridge, Cambridge, UK
Murine leukemia gammaretrovirus (MuLV) utilises stop codon
readthrough to trigger 5% of ribosomes translating gag to continue
beyond the stop codon and to synthesise the Pol polyprotein as a
fusion with Gag. The readthrough signal comprises an RNA pseudoknot
structure located immediately downstream of the gag stop codon.
The resultant Gag:Gag-Pol ratio of 20:1 is believed to be ideal for virus
replication, but this has not been analysed in any depth. To probe the
biological importance of this ratio, readthrough efficiency was modulated,
through creation of pseudoknot variants or by treatment of infected
cells with aminoglycosides. A broad range of readthrough efficiencies
was obtained (0.1-30% as judged in in vitro and cell culture assays) and
MuLV replication assessed by Gag/Gag-Pol expression, their assembly
into virions, particle maturation and infectivity. A four-fold reduction in
readthrough led to a modest replication defect, but greater reduction
had a severe impact, with failure of particle maturation. TCID50 assays
revealed that MuLV infectivity correlates with readthrough efficiency,
suggesting that the ratio of Gag/Gag-Pol directly specifies particle
incorporation ratio, and no additional regulation is apparent. Stimulating
readthrough further than two-fold was inhibitory, though this may be
attributable to other factors such as non-synonymous codon changes.
MA15/11
RNA binding of human respiratory syncytial virus M2-1 protein
Sian Tanner1, Miles Carroll2, Julian Hiscox3, Thomas Edwards1,
John Barr1
1
University of Leeds, Leeds, UK, 2Health Protection Agency, Porton Down,
Sailsbury, UK, 3University of Liverpool, Liverpool, UK
For an indispensible component of the Human Respiratory Syncytial
Virus (RSV) polymerase complex, surprisingly little is known about
how M2-1 protein performs its role in the viral life cycle. It is a virallyencoded transcriptional antiterminator that is required for complete and
processive transcription, both inter-and intragenically. M2-1 is known
to bind RNA of as yet undetermined specificity, with both antigenomic
leader and viral mRNA sequences having been proposed. A conserved
Cys3-His1 zinc finger at the N-terminus is necessary for antitermination
function and has been recently shown to coordinate one zinc atom per
monomer, however this motif has not been attributed to the RNAbinding ability of M2-1 protein, despite Cys3-His1 zinc fingers playing this
role in other members of this domain family. Using bacterially expressed
protein and 3’-fluorescein labeled RNA in a fluorescence polarisation
assay, we demonstrate indications of length and nucleotide preference
for M2-1 RNA binding, and suggest how this may indicate a method
of action. Understanding how M2-1 binds RNA is key to explaining its
central role in the RSV life cycle.
MA15/12
Investigations into the cellular interacting partners of CCHFV N
reveals the co-immunoprecipitation of HSP70 with native N
Rebecca A Surtees1,2, Roger Hewson2, Miles Carroll2,
Cheryl Walter1, Julian A Hiscox3, John N Barr1
1
University of Leeds, Leeds, UK, 2Health Protection Agency, Porton Down,
Salisbury, UK, 3University of Liverpool, Liverpool, UK
98
Poster abstracts
of NS5A with metallothionein (MT) -a ubiquitous metal ion chelator
protein that binds gold particles creating localised, accessible clusters.
We describe the detection and preliminary imaging of these functional,
tagged complexes by whole cell cryo-EM tomography. Our second
approach involves the insertion of the bacteriophage MS2 RNA stemloop within the 3` UTR of HCV and recovery of replication competent
subgenomic replicons and virus. Using tagged copies of co-transfected
or immobilised MS2 capsid protein, we go on to describe the use of the
MS2 trap system to purify viral RNA/protein complexes.
Crimean Congo hemorrhagic fever virus (CCHFV) is the most
geographically widespread of the hemorrhagic fever viruses, and a
severe human pathogen, causing death in up to 30% of those infected.
The nucleocapsid protein (N) of CCHFV, encoded by the small (S)
genomic segment, is essential for encapsidation of the viral genome to
allow both its transcription and replication. Upon infection, N localises to
perinuclear regions in infected cells in a temporal and actin-dependent
manner and directly binds to both actin, and the anti-viral protein
Mx1. In order to identify other cellular interacting partners of N, EGFP
was fused to its N-terminus, then EGFP-N was immunoprecipitated
using a GFP-trap, and co-immunoprecipitated cellular proteins were
identified and quantified using SILAC coupled to LC-MS/MS. Two
different immunoprecipitation strategies were compared; mix after
immunoprecipitation (‘MAP’) and precipitate after mixing (‘PAM’),
then several cellular proteins with high SILAC ratios, including HSP70,
vimentin and p53, were confirmed as co-immunoprecipitating partners
of EGFP-N following repeat immunoprecipitations and western blotting.
Native, untagged N was then immunoprecipitated using serum specific
to CCHFV N, and confirms the co-immunoprecipitation of HSP70,
the cellular protein with the highest SILAC ratio in all the LC-MS/MS
analyses.
MA16Virology workshop:
Clinical virology
MA15/13
Studies on the recently discovered non-primate hepacivirus (NPHV)
Michaela J Conley1, Cheryl T Walter1, Sinead Lyons2,
Peter Simmonds2, Mark Harris1
1
University of Leeds, Leeds, UK, 2The University of Edinburgh, Edinburgh, UK
The recently discovered non-primate hepacivirus (NPHV), is the closest
relative to the important human pathogen, Hepatitis C Virus (HCV).
NPHV has been found in both horses and dogs but has not been
associated with any disease or pathology. In order to understand the
molecular biology of this virus and enable comparative studies with
HCV, we are developing a subgenomic replicon (SGR) for NPHV. We
derived a series of cDNAs from the serum of a horse that had a high
titre of NPHV (10e7 genomes/ml). From these, a cDNA spanning the
coding sequence for the NPHV non-structural proteins NS3-NS5B was
generated by PCR and substituted for the corresponding region in the
HCV SGR. Experiments to determine if the resulting chimeric construct
can replicate are ongoing.
In parallel, the predicted NPHV NS5A coding sequence has been
expressed in mammalian cell culture. The expressed NPHV NS5A
exhibited a similar punctate cytoplasmic distribution to the HCV NS5A
protein. Intriguingly, serum from the infected horse detected the NPHV
NS5A protein both in western blot and immunofluorescence, providing
a useful immunological reagent. Comparative analysis of the effects of the
NPHV and HCV NS5A proteins on cell signalling, and interactions with
host cell proteins, are underway.
MA15/14
Understanding the structure and composition of hepatitis C virus
replication complexes using both protein and RNA affinity tags
Cheryl T Walter1, Rebecca Thompson1, Cristina Risco Ortiz2,
Neil A Ranson1, Mark Harris1
1
University of Leeds, Leeds, UK, 2Centro Nacional de Biotecnologia, CSIC,
Madrid, Spain
Numerous studies have described the viral and cellular proteins
associated with crudely isolated HCV genome replication complexes
as well as binding partners for the non-structural NS5A protein (a key
component of the replicase) or 5` and 3ÙTRs. These global surveys,
although informative, often include non-specific and indirect interaction
partners leaving a gap in the knowledge on a) what binds directly to
+/-sense HCV RNA during replication and b) what constitutes the
minimal, active HCV genome replication machinery.
Here, we describe two approaches to address this issue by tagging
and isolating HCV replication complexes. The first involves the tagging
MA16/01
Influenza virus infection In Kenya
Jolynne Mokaya
University of Nairobi, Nairobi, Kenya
WHO estimates acute lower respiratory infections cause nearly 4
million deaths per year.Rates are higher in developing countries where
pneumonia is responsible for an estimated 10-25% of all deaths among
children under 5 years of age. Although influenza has been widely
studied in developed countries, little is known about its epidemiology,
seasonality and burden in developing countries. 2009 pandemic influenza
A (pH1N1) virus was responsible for at least 20,000 laboratoryconfirmed deaths globally. Respiratory viral infections are associated
with high rates of illness and make up a substantial portion of respiratory
infection. The rate of influenza-associated hospitalisation is highest among
children aged less than 5 years. The magnitude of the disease prevalence
needs to be studied with further research.
MA16/02
Hepatitis E virus and fulminant hepatitis – virus or host-specific
pathology?
Donald B Smith, Samantha Lycett, Peter Simmonds
In a minority of cases, hepatitis E virus (HEV) infection results in fulminant
hepatitis (FH). This condition is characterised by hepatic encephalopathy,
necrosis of hepatic parenchyma, coagulopathy, renal failure or coma, and
has a poor prognosis. Previous studies have suggested that specific virus
genotypes, variants or substitutions are associated with FH. However,
a survey of published information reveals that FH can arise following
infection with all four HEV genotypes that infect humans, and also
with multiple lineages within these genotypes. The possibility that FH
results from more subtle variation between HEV strains is contradicted
by the observation that not all individuals infected from a common
source go on to develop FH. However, previous statistical analyses
have demonstrated associations between particular substitutions and
infection outcomes including FH. To investigate whether these may
have been confounded by genetic relatedness of HEV variants studied,
we performed Bayesian analysis that avoids this bias and found that
the claimed associations of FH with specific virus substitutions can
be explained by epidemiological linkage. In summary, we have found
no evidence that virus genotype, variants or specific substitutions are
responsible for the development of FH and highlight a more important
contribution from differences in host susceptibility.
MA16/03
Examining the connectivity of hepatitis C virus transmission networks
between two urban centres in scotland
Anna McNaughton1, Roman Biek1,2, Sheila Cameron3,
Kate Templeton4, Carol Leitch1
1
MRC-University of Glasgow-CVR, Glasgow, UK, 2IBAHCM, University of
Glasgow, Glasgow, UK, 3West of Scotland Specialist Virology Centre, Glasgow,
UK, 4Edinburgh Specialist Virology Centre, Edinburgh, UK
99
Poster abstracts
Glasgow has significantly higher HCV seroprevalence (62%) among
its injecting drug using (IDU) population than Edinburgh does (36%),
despite geographical proximity and similar risk behaviours1. This study
investigated HCV transmission between Glasgow and Edinburgh and
quantified the linkage between these IDU communities over a decade.
As Glasgow has significantly higher HCV prevalence, we hypothesised
it was the predominant source of HCV transmission between the two
centres.
For the study, HCV genotype 1a and 3a samples were collected from
patients in Edinburgh and Glasgow. Partial NS5B sequences were
analysed using a Bayesian phylogeographic approach along with historical
sequences from the UK1 and Europe.
Analysis demonstrated that the majority of HCV currently circulating
within Edinburgh and Glasgow appeared to emerge from local
populations of virus that were present in the same city a decade ago,
suggesting that the two IDU populations are not strongly linked. Glasgow
was found not to be the dominant source of HCV in the region and
transmission into Edinburgh was only observed with genotype 3a. This
technique may have applications giving insights into patterns of drug
resistance spread and improving targeting of local intervention strategies
in Scotland.
Reference 1Cochrane A et al, J Infect Dis, 2002, 186:1212-21.
MA16/04
The study of heterosubtypic neutralising antibody responses against
HPAI influenza viruses in human subjects using a highly adaptable
multiplex neutralisation assay
Eleonora Molesti1, Francesca Ferrara1, Giulia Lapini2,
Emanuele Montomoli2, Nigel Temperton1
1
2
Department of Physiopathology, Experimental Medicine and Public Health,
University of Siena, Siena, Italy
Retroviral pseudotypes bearing influenza haemagglutinin (HA) and
neuraminidase (NA) envelope glycoproteins represent a flexible platform
for sensitive, readily standardised influenza assays for sero-surveillance
and vaccine evaluation. We describe a multiplex assay that can be
used for the study of neutralising antibodies that are directed against
both influenza H5 and H7 HA in human serum samples. This assay
permits the measurement of neutralising antibody responses against
two antigenically distinct HA envelope glycoproteins in the same human
serum sample thus increasing the amount and quality of serological data
that can be acquired from valuable sera, and potentially reducing interassay variability. A panel of serum samples collected from the Italian
population between 1992 and 2007 and previously found to be positive
for antibodies against H5N1 as determined by SRH, were evaluated in
this multiplex assay and anti-H5 and –H7 neutralising antibody responses
were studied. From the results obtained it can be concluded that this
assay is effective for the measurement of functional heterosubtypic crossreactive responses in human sera. This assay is useful as an adjunct to
the EMA approved SRH and HI assays as it measures antibodies with
different (functional) specificities contributing to comprehensive analyses
of humoral immunity to influenza viruses.
MA16/05
Comparison of automated chemiluminescence immunoassays with
capture enzyme immunoassays for the detection of measles and
mumps IgM antibodies in serum
Becky Haywood, Mauli Patel, Samantha Hurday, Ruth Copping,
Daniel P Webster, Dianne Irish, Tanzina Haque
Royal Free Hospital, London, UK
Outbreaks of measles and mumps occur regularly in the UK. Rapid
diagnosis of acute infection is important for both infection control and
epidemiological purposes. Sera previously tested for measles (n=50)
and mumps (n=40) IgM using EIA were retrospectively tested using
the DiaSorin Liaison®. Sensitivity, specificity, inter-assay variability and
intra-assay variability of the Liaison® assays were calculated. Sensitivity
and specificity of the Liaison® assay for measles IgM was 92% and 100%
respectively, with inter-assay variation of 14.1% and intra-assay variation
of 12.5%. The sensitivity and specificity of the mumps IgM Liaison®
assay was 88% and 95% respectively, with an inter-assay and intra-assay
variation of 13.9% and 5.3% respectively. Both Liaison® IgM assays gave
fewer equivocal results than EIA. Neither Liaison® IgM assay showed
any cross-reactivity with sera positive for other viral IgM, however the
measles IgM EIA cross-reacted with 1 parvovirus IgM positive sample. No
other EIA cross-reactivity was seen. The automated Liaison® assays are
more specific, cheaper and less labour-intensive compared to the manual
EIA. The Liaison® assays benefit from reduced number of equivocal
results compared to the EIA, giving definitive results for both measles
and mumps IgM. This allows clinical decisions to be made in a timely
manner.
MA16/06
Development of a recombinant nucleoprotein ELISA for detection of
antibodies to Rift Valley fever virus in wildlife
Emily Goldstein1, Elizabeth McMonagle1, Sarah Cleaveland2,
Margaret Hosie1, Brian Willett1
1
Centre for Virus Research, Glasgow, UK, 2Boyd Orr Centre for Population and
Ecosystem Health, Glasgow, UK
Rift Valley fever virus (RVFV) is an arthropod-borne Phlebovirus, which
causes severe disease in ruminants and when transmitted to humans
can cause a fatal haemorrhagic fever. Outbreaks occur following periods
of exceptionally high rainfall, but little RVFV is observed in domestic
livestock during inter-epidemic periods. Low level transmission of RVFV
may take place between outbreaks, but as serosurveillance is limited
there is currently little data available from inter-epidemic periods. High
virus neutralising antibody (VNAb) titres have been observed in many
wildlife species, suggesting wildlife may be important reservoir hosts.
The virus neutralisation test is the gold standard assay for detecting
VNAb to RVFV. As this test uses live virus and as RVFV is classified as
a category 3 pathogen, the test must be performed under high-level
containment facilities. We have developed an indirect ELISA using the
recombinant nucleoprotein of RVFV which can be used to detect RVFV
antibodies in diverse species. Using this assay we have measured RVFV
antibodies in lions from Tanzania and have tested for antibody crossreactivity to related Phleboviruses. The ELISA provides a diagnostic assay
enabling rapid serosurveys to be performed on a wide range of species,
which will help predict major wildlife reservoirs of RVFV.
MA16/07
Mapping the molecular epidemiology of hepatitis C virus (HCV)
infection among HIV-positive patients in sub-Saharan Africa
Simon King1, Kwabena Adjei-Asante1, Lambert Appiah2,
Dorcas Adinku2, David Chadwick3, Geraldine M Foster1, Stephen Sarfo2,
Sanjay Bhagani4, Richard O Phillips2, Anna Maria Geretti1
1
Institute of Infection & Global Health, University of Liverpool, Liverpool, UK,
2
Kwame Nkrumah University Of Science and Technology and Komfo Anokye
Teaching Hospital, Kumasi, Ghana, 3South Tees Hospitals NHS Foundation
Trust, Middlesborough, UK, 4Royal Free Hospital, London, UK
Background Recent studies have reported alarmingly high (>5%) HCV
antibody seropositivity among HIV-positive adults in sub-Saharan Africa
(SSA). The reliability of such EIA-based estimates remains controversial.
To gain definitive prevalence data, we screened HIV-positive adults from
Kumasi (Ghana) for the presence of HCV RNA using both plasma and
dried plasma spots (DPS).
Methods Following validation of the HCV RNA screening protocol,
pooled plasma and DPS were tested by real-time PCR, followed by
testing of individual samples of positive pools. Sanger sequencing of a
~400bp region of NS5B was used to genotype HCV RNA positive
samples. Hepatitis B surface antigen (HBsAg) status was determined by
Determine and Murex EIA.
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Poster abstracts
Results The optimised HCV RNA screening protocol for pooled plasma
and DPS showed a lower limit of detection of 110 IU/ml and 1000 IU/
ml, respectively. Overall, 8/1249 (0.64%) samples tested HCV RNA
positive, including 2/203 (0.99%) samples from HBsAg-positive patients
and 6/1046 (0.57%) samples from HBsAg-negative patients. HCV
genotypes comprised 1 and 2.
Conclusions HCV RNA positivity was 0.64% among HIV-positive adults
in Kumasi, Ghana. Published findings on HCV antibody screening appear
to have overestimated the burden of HCV infection in the HIV-positive
population of Ghana and possibly other SSA regions.
MA16/08
The incidence of medically attended respiratory virus infections in
north-west England, 2007–2012
Edward A GOKA1, Pamela J Vallely1, Kenneth J Mutton,2,
Paul E. Klapper,2
1
University of Manchester, Institute of Inflamation and Repair, Faculty of
Medical and Human Sciences, Manchester, UK, 2Department of Clinical
Virology, Central Manchester University Hospitals -NHS Foundation Trust,
Manchester, UK
Introduction: The incidence of respiratory viruses varies considerably
yet estimation of incidence is important to determine effectiveness of
health interventions, costs of disease management, and early indication
of epidemics.
Methodology: The expected number of cases was used to calculate
incidence using the age specific mid-year population figures for North
West England as a denominator in a Poisson model.
Results: Repiratory viruses peaked in summer of 2009, ILI 321 per
100,000 and 2010/2011 winter 56 per 100,000. People ≥85 years and
children ≤5 years had higher incidence than 24-39 year olds, influenza
A(H1N1)pdm09 358 per 100,000 vs. 22.92 per 100,000 among 25-39
year olds (IRR: 15.62, 95% CI: 10.24 -23.83, p = <0.0001). RSV (39.03
and 64.64 per 100,000 among the ≥85 and ≤5 years old vs. 1.40 among
≤5 year olds, IRR: 27.88, 95% CI: 5.17 -150.47, p = <0.0001 and IRR:
46.34, 95% CI: 8.68 -247.19, p = <0.0001). Similar results were observed
for seasonal influenza A viruses, rhinoviruses, parainfluenza viruses 1-3,
metapneumovirus and adenoviruses but not for influenza B viruses.
Conclusion: Laboratory surveillance of respiratory viruses is a useful tool
for prediction of incidence of respiratory virus infections and assessment
of effectiveness of public health intervention.
MA16/09
Implementation of a Pneumocystis jirovecii PCR and comparison with
indirect immunofluorescence
Emilie Sanchez, David Muir
Imperial College Healthcare NHS Trust, London, UK
Pneumocystis jirovecii (PCP) pneumonia has a high mortality rate
among HIV and non-HIV immunosupressed patients. Diagnosis can
be challenging since clinical features and imaging may not be typical.
Since early treatment is important for the patient’s outcome, sensitive
laboratory methods for PCP detection are warranted. However patients
may be colonised with PCP in the absence of disease making the choice
of an optimal diagnostic laboratory assay a matter of debate.
We will describe the introduction of a real-time PCR assay targeting
a fragment of the mitochondrial large subunit rRNA gene in our trust
as described by Totet et al.1. We will also present the clinical utility
of the assay in comparison to an indirect immunofluoresence test
(IF) from Axis-Shield Diagnostics Ltd. 67 respiratory samples from a
variety of children and adult patients from our centre were tested. We
will describe the presentation and the outcome of the patients with
a positive PCP PCR test and/or a positive PCP IF test. We will also
describe the introduction of this PCR into the routine service at another
centre and their experience.
Reference 1Totet et al. (J Eukaryot Microbiol. 2003;50 Suppl:668-9)
MA16/10
Parvovirus B19-induced haemophagocytic syndrome with HSV and
EBV reactivation
William L Irving1, Fouzia Jabeen2
1
Birmingham Heatlands Hospital, Birmingham, UK, 2Notingham University
Hospitals, Nottingham, UK
Haemophagocytic syndrome secondary to viral infection has
been described, predominantly associated with EBV infection. It is
characterised by excessive infiltration of the bone marrow and other
organ systems with activated antigen presenting cells and CD8 +T
cells resulting in the destruction of blood cells and their precursors
often with a fatal outcome. We describe a case of primary Parvovirus
B19 infection in an otherwise healthy young lady with simultaneous
HSV and EBV reactivation. The primary infection was teased out by
serological, molecular and histological testing. Supportive management
and treatment of the HSV with acyclovir led to full recovery, sparing the
need for immunotherapy or chemotherapy.
MA16/11
Evaluation of the Abbott real-time HCV PCR assay for the detection
of hepatitis C RNA in dried blood spots
Anupama Mutagi, Husam Osman, Erasmus Smit, Sowsan Atabani
Health Protection Agency,Public Health Laboratory,Birmingham, Birmingham,
UK
The use of dried blood spots (DBS) for the detection of hepatitis C virus
(HCV) RNA was evaluated using a quantitative real-time PCR assay
(Abbott Diagnostics, Weisbaden, Germany). The limit of detection,
sensitivity and specificity of HCV RNA detection was compared in DBS
and plasma. In addition, the precision, reproducibility and effect of storage
conditions were evaluated. A total of 126 DBS were prepared from 76
patient samples (62 known HCV RNA positive and 14 known HCV
RNA negative). Out of the 104 samples tested neat there was 1 falsenegative result and 2 false-positive results and 1 inhibitory result .The
sensitivity, specificity, positive predictive value and negative predictive
value for HCV RNA detection in DBS were 98.7%, 91.3%, 97.5% and
95.4% respectively. A significant correlation was observed between HCV
RNA concentration in DBS and plasma (R2 = 0.9; P<0.001). However,
the quantitative values obtained on the DBS were significantly lower
than in plasma. We established that in samples were HCV RNA is >
250 IU/ml, DBS may be a useful alternative for screening purposes in
circumstances where a large volume of blood is unobtainable.
MA16/12
The assessment of the impact of molecular diagnosis on the
management of patients with enteroviral meningitis
W M D G B Wijayaratne, P Wilkinson, L M Hesketh,
J S Cheesbrough
Lancashire Teaching Hospitals NHS Foundation Trust, UK
Introduction Enterovirus reverse transcriptase polymerase chain
reaction (EV RT-PCR) should facilitate the rapid discharge of patients
with suspected viral meningitis providing results are available
promptly.
Objective To assess the impact of on-site same day EV RT-PCR testing
of CSF on duration of hospital stay among patients with EV meningitis.
Methods and results Until June 2012 EV RT-PCR was only available
from the regional laboratory. Thereafter the test was available on-site on
a daily basis, but only undertaken on CSF from Medical Assessment Unit
patients, aged 16-50 years, with a leucocyte count of ≥20/µl without
CSF features of bacterial infection.
There were 13 EV positive patients in the pre on-site test period fulfilling
the same selection criteria (group A) and 7 in the post on-site test
period (group B). The mean duration of hospital stay was: A 78 hours, B
50 hours. The mean test turn-around-time was A 265 hours, B 17 hours.
Of 11 CSF samples tested on-site 7 were positive.
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Poster abstracts
Conclusion On-site EV RT-PCR is more effective in saving bed
days when compared with off-site testing. This saves unnecessary
antimicrobials and investigations. The higher cost of on-site testing is
contained by targeting tests that fulfil three simple criteria.
CMM
Clinical and medical microbiology
CMM/01
The construction of chimeric rabies virus glycoproteins rescues
Arctic-like rabies pseudovirus production
Ruqiyo A Ali1, Edward Wright1,3, Daniel Horton2, Ashley C Banyard2
1
School of Life Sciences, University of Westminster, London, UK, 2Wildlife
Zoonoses and Vector Borne Diseases, Animal Health and Veterinary
Laboratories Agency, Surrey, UK, 3Viral Pseudotype Unit (Fitzrovia), University
of Westminster, London, UK
Arctic-like rabies viruses (AL RABV) are a lineage of rabies viruses
circulating widely in the Middle East and Asia, with distinct antigenic and
genetic characteristics. As with other members of the RABV species they
can cause a zoonotic disease that ultimately leads to death of infected
patients. RABV glycoprotein (G) lentiviral pseudotypes (PV) have been
shown to be highly sensitive and specific when used as surrogates for
live virus in neutralisation assays. However, using wildtype AL RABV
glycoprotein failed to generate any infectious PV. Therefore, we sought
to determine if it was possible to increase the biological titre of AL RABV
PV through the construction of chimeric RABV and vesicular stomatitis
virus (VSV) G. Initial studies were undertaken to generate a chimeric G
by splicing the ecto-and transmembrane domains of an Indian AL RABV
G strain with the cytoplasmic domain of VSV G. PV were produced
using wildtype and chimeric G, revealing a 125 fold increase in titre
for the chimeric G PV. Similar mutations were undertaken with other
AL RABV allowing the efficacy of current animal vaccines to be tested
against this subset of RABV.
CMM/02
Characterisation of the fungal microbiota (mycobiome) in healthy and
dandruff-afflicted human scalps
Hee Kuk Park, Wonyong Kim
Department of Microbiology, Chung-Ang University College of Medicine,
Seoul, Republic of Korea
The human scalp harbors a vast community of microbial mutualists,
the composition of which is difficult to elucidate as many of the
microorganisms are not culturable using current culture techniques.
Dandruff, a common scalp disorder, is known as a causative factor of
a mild seborrheic dermatitis as well as pityriasis versicolor, seborrheic
dermatitis, and atopic dermatitis. Lipophilic yeast Malassezia is widely
accepted to play a role in dandruff, but relatively few comprehensive
studies have been reported. In order to investigate fungal biota and
genetic resources of dandruff, we amplified the 26S rRNA gene from
samples of healthy scalps and dandruff-afflicted scalps. The sequences
were analysed by a high throughput method using a GS-FLX 454
pyrosequencer. Of the 74,811 total sequence reads, Basidiomycota
(Filobasidium spp.) was the most common phylum associated with
dandruff. In contrast, Ascomycota (Acremonium spp.) was common
in the healthy scalps. Our results elucidate the distribution of fungal
communities associated with dandruff and provide new avenues for the
potential prevention and treatment of dandruff.
CMM/03
Full genomic analysis of a korean human group C rotavirus closely
related human group C rotaviruses from Bangladesh, India and England
Inseok Lim1, Van Thai Than2, Wonyong Kim2
1
Department of Pediatrics, Chung-Ang University College of Medicine, Seoul,
Republic of Korea, 2Department of Microbiology, Chung-Ang University
College of Medicine, Seoul, Republic of Korea
Notwithstanding increases in human group C rotavirus (GCRV) infection
worldwide, only eight GCRV strains have been characterised and
analysed by genomic sequencing. In 2011, a GCRV strain, CAU10-312,
was detected in a stool sample from a 5-year-old South Korean boy and
a complete genome sequence was obtained. The 11 gene segments of
the strain were classified as G4-P[2]-I2-R2-C2-M2-A2-N2-T2-E2-H2
genotypes. The strain CAU10-312 appears closely related to strains from
Bangladesh (DhakaC13 and BS347), India (v508), and England (Bristol),
but distinct from Chinese (Wu82 and YNR001), and Japanese (OH567
and BK0830) strains. These findings may clarify genetic relationships
among globally distributed GCRVs and suggest that two distinct strains
are persistent and widespread.
CMM/04
Culture-independent analysis of the preterm gut microbiota in twins
Christopher J Stewart1, Emma CL Marrs2, Clare Lanyon1,
John D Perry2, Nicholas D Embleton3, Stephen P Cummings1,
Janet E Berrington3
1
University of Northumbria, Newcastle upon Tyne, UK, 2Freeman Hospital,
Newcastle upon Tyne, UK, 3Royal Victoria Infirmary, Newcastle upon Tyne,
UK
Within a twin cohort, it is possible to explore how environmental
and demographic variables affect the development of the bacterial
community in the gut microbiome in a constrained genetic background.
We aimed to compare the development of the gut microbiota in
premature multiples, focusing on dysbiosis and its role in late onset
disease (NEC and sepsis).
Stool (n=173) was collected from 11 twin pairs and 1 triplets. Samples
were analysed by PCR-DGGE and 454 pyrosequencing. Bands were
excised and sequenced to obtain taxonomic classification.
Partial least squares discriminant analysis (PLS-DA) demonstrated profiles
from siblings were more similar to each other than to unrelated profiles.
Enterococcus and Actinomyces were significantly (P=0.000) more
abundant pre and post NEC, respectively. Enterococcus were found in
higher levels in stools form infants delivered by caesarean section.
Differences within the community of related twins may predispose
infants to disease. A reduced species richness and evenness was
observed pre disease diagnosis and thus dysbiosis may be causal due
to a reduction in commensal bacteria, leading to the prevalence of a
pathogen. Communities involved in NEC development may be due to
an increase of Enterococcus and Corynebacterium.
CMM/05
Molecular characterisation of the hemagglutinin gene of confirmed
cases of pandemic A H1N1(2009) in Mauritius
Mitradev Pattoo1,2, Yasmina Jaufeerally-Fakim2, Chitra Sujeewon1,
Hari Mathur1, Shaad Lallmohamood1, Sanjiv Rughooputh1,2
1
National Influenza Centre, Candos, Mauritius, 2University of Mauritius,
Reduit, Mauritius
The first case of pandemic A H1N1 (2009) was diagnosed in Mauritius
in June 2009, with approximately 150 laboratory confirmed cased.
Of these, 63 samples were selected and a temporal-spatial study was
carried out to determine whether there the virus has evolved. Molecular
analysis of the Hemagglutinin (HA) gene, crucial for the monitoring of
modification in viral genome related to pathogenesis and transmissibility
was carried out by PCR using newly designed primers to span the
whole HA gene. The amplicons were then purified and sequenced.
Sequences data obtained were analysed for homology and variations
with the reference vaccine strain A/California 07/09 using BioEdit and
MEGA 5.0 Bioinformatics software. A Phylogenetic analysis was then
carried out. The effect of positional amino acid substitutions on the mean
hydrophobicity compared to the reference sequence was investigated
using the Einsenberg mean hydrophobicity scale. Characterisation of
the HA gene revealed conserved sequences at the antigenic sites and
variations due to amino acid substitutions, potentially resulting in an
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Poster abstracts
aureus (MRSA) to β-lactam antibiotics (Stapleton et al., 2004). The
aim of this study was to develop a zebrafish embryo model for in vivo
determination of the effects of ECg on MRSA infections. An increase in
zebrafish embryo survival was found after injection of bacteria grown
in the presence of ECg. We hypothesised that ECg-grown EMRSA-16
are more susceptible to the innate immune system of the zebrafish
in comparison to untreated bacteria. Confocal microscopy revealed
modified bacteria within zebrafish granulocytes and four distinct immune
cell populations were identified using flow cytometry. An increase in
induction of the respiratory burst and a reduction in IL-1β expression
determined that bacteria pretreated with ECg are less likely to induce
an inflammatory response and are more readily eliminated by the
immune system compared to untreated MRSA cells. The data suggest
that exposure to ECg reduces the capacity of MRSA to avoid the innate
defences of the zebrafish embryo.
References Stapleton, P. D., Shah, S., Anderson, J. C., Hara, Y.,
Hamilton-Miller, J. M. T. and Taylor, P. W. 2004. International Journal of
Antimicrobial Agents, 23, 462-467.
N-linked glycosylation site. Novel mutations previously unreported
were found, some of these variations were seen to affect the mean
hydrophobicity at the concerned sites drastically. Continuous monitoring
of the evolution of Influenza is thus important and advocated.
CMM/06
The role of fructose-1,6-bisphosphate aldolase (FBA) in the
pathogenesis of Neisseria meningitidis
Fariza Shams1, Neil J Oldfield1, SiKei Lai1, Akhmed Aslam1,
Alan Berry2, Karl G Wooldridge1, David P J Turner1
1
Molecular Bacteriology and Immunology Group, Centre for Biomolecular
Sciences, University of Nottingham, Nottingham, UK, 2Astbury Centre for
Structural Molecular Biology, University of Leeds, Leeds, UK
Neisseria meningitidis is a leading cause of fatal sepsis and meningitis
worldwide. Despite the Embden-Meyerhof-Parnas (EMP) glycolytic
pathway being inactive in meningococci, three enzymes of this pathway,
namely enolase, glyceraldehyde 3-phosphate dehydrogenase (GAPDH),
and fructose 1,6 bisphosphate aldolase (FBA) have been shown to have
non-glycolytic (moonlighting) functions related to interactions with host
proteins or adhesion to host cells. This project aims to explore the
moonlighting functions of FBA in the pathogenesis of meningococcal
disease. Constructs were generated to express rFBA with mutations
in the active (cation-binding) site (D83A and H81A/H84A). Following
expression and purification, a coupled assay confirmed the aldolase
enzyme activity of wild-type rFBA. The kinetic parameters of purified
wild-type rFBA for the cleavage of FBP were calculated as Km=0.027
mM and kcat=420 min-1. In contrast, the two mutated FBA enzymes
had no detectable enzymatic activity confirming the importance of the
mutated residues in enzymatic activity. In addition, our preliminary data
also suggest that rFBA binds plasminogen and that this can be inhibited
by the lysine analogue aminocaproic acid indicating the involvement of
lysine residue(s) of rFBA in this interaction.
CMM/07
Does PCR have a role in managing Clostridium difficile infection (CDI)?
Shankar Kumar1, Timothy Planche1, Ivan Muscat2
1
St George’s Hospital Medical School, London, UK, 2Jersey General Hospital,
Channel Islands, UK
Aims To compare inflammatory markers and mortality in CD toxin
positive patients with toxin negative, PCR positive individuals.
Methods Between 2008 and 2012, 3532 stool samples were tested for
CD toxin and glutamate dehydrogenase (GDH) and 974 were tested for
CD toxin only. 131 toxin negative, GDH positive samples had PCR using
Cepheid Xpert. Patients were grouped by test result. White cell count
(WBC), C-reactive protein (CRP) and 30 day mortality was compared.
Results Patients in Group 1 (n=207) were toxin positive, Group 2
(n=92) toxin negative and PCR positive and Group 3 (n=39) were toxin
and PCR negative but GDH positive. All-cause mortality was 21.3%,
13% and 12.8% in Group 1, 2 and 3, respectively. A positive toxin test
was a significant independent predictor of death (OR 1.6, 95% 1.0-3.3,
p=0.036) but a positive PCR was not (OR 1.0, 95% CI 0.3-3.1, p=0.609).
A positive toxin test was associated with higher WBC (p=0.002) and
CRP (p=0.001). There was no statistical difference in WBC and CRP
between PCR positive and PCR negative patients.
Conclusion In toxin negative disease, a positive PCR neither predicts
disease severity nor mortality. However, it may have a role in disease
surveillance and infection control.
CMM/08
Effect of epicatechin gallate on staphylococcal virulence in a zebrafish
embryo infection model
Christina S Stevens, Robert Harvey, Peter W Taylor
UCL School of Pharmacy, London, UK
Epicatechin gallate (ECg) sensitises methicillin-resistant Staphylococcus
CMM/09
Induction of stringent response in S. aureus by mupirocin
Sari Talal Alhoufie
Slford University, Biomedical Research Center R, Manchester, UK
In most bacteria, nutritient deprivation induces the stringent response.
During the stringent response, bacteria start to synthesise (p)ppGpp,
an alarmone that regulates transcriptional and translational control
mechanisms which enables the cell to adapt to stress conditions. This
adaption is processed through huge alterations in gene expression which
may change the cell properties such as virulence and persistence.
Relatively little is known about the stringent response in S.aureus,
in particular the effects of long term exposure to sub-inhibitory
concentrations of mupirocin have not been studied. In this work, the
production of ppGpp, the hallmark of the stringent response, after
exposure to sublethal concentrations of mupirocin was determined by
HPLC for S.aureus strain 8325-4. Intracellular concentration of ppGpp,
reached a maximum yield of 3.5 nmole/mg cell dry weight after 1 h
decreasing to1.19 after 24 h and dropped to 0 48 h after mupirocin
treatment.
A detailed study of the stringent response and its effect on the
intracellular activity including virulence gene expression in S.aureus will
enhance our understanding. Observing (p)ppGpp production in S.aureus
might expand the knowledge of its adaption in difficult environment
which may lead to a new approach to fight the organism.
CMM/10
Genetic variation in a multi-resistance plasmid
Amira Amine1, Dan Andersson2, Linus Sandegren2
1
Alexandria University, Alexandria, Egypt, 2Uppsala University, Uppsala,
Sweden
We have studied the genetic variation in a multi-resistance plasmid
carried by a Klebsiella pneumoniae involved in an outbreak at Uppsala
University Hospital over five years. The main type of variation found are
deletions and amplifications mediated by homologous recombination,
mainly via IS26 elements. Interestingly, several plasmid isolates had
amplifications (up to 10x) of a region encoding genes for tetracycline
resistance.
To experimentally analyse the amplification dynamics in, we looked
at copy number variation of one of the clinical plasmid isolates with a
preexisting 3-fold amplification of 12.3 kbp in the resistance cassette
with tetA and tetR genes. After 500 generations, all lineages increased
the copy number of the amplification when selected on tetracycline.
Isolates showed an increasing MIC to tetracycline with the increasing
copy number of the amplification. Without tetracycline, some lineages
did amplify but most segregated back to one or two copies. Steady state
experiments show that the amplified state can stay in the population
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Poster abstracts
even without selection.The frequency of duplication/amplification of
the tet-region from a plasmid with no preexisting amplification was
approximately 10-6 .
CMM/11
Differential genetic regulation of peripheral blood monocytes in a
Schistosoma haematobium exposed population
Amir Kirolos, Norman Nausch, Laura Appleby, Francisca Mutapi
Introduction 200 million people in the developing world are chronically
infected with the water-borne parasite Schistosoma haematobium.
Infection results in monocytes producing a forceful immune response
in the urinary tract system, often causing haematuria, hydronephrosis,
bladder fibrosis and bladder cancer. Genes implicated in this response
include macrophage complement receptors, C5AR1 and C3AR1, and
Th2 associated proteins, IL-10 and LOX-15.
Methods 86 participants from a Zimbabwean village endemic with
S.haematobium had venous blood collected. CD14+ peripheral blood
monocyte cDNA, separated from blood samples, underwent real-time
PCR. Expression of the genes above were compared between each
other and with infection intensity, gender and age from census data.
Results C3AR1 was expressed significantly more with higher infection
intensities (p=0.016), particularly in those aged over 14(p=0.002). IL10 was expressed significantly more with higher infection intensities,
particularly those aged 10-14(p=0.021). In those aged 5-9, C5AR1
was expressed significantly more in those infected(p=0.048). LOX-15
expression was not related to any characteristics but correlated with
C5AR1(p<0.001) and IL-10(p=0.033), while C3AR1 and IL-10 were also
correlated(p=0.018).
Conclusions This study indentifies monocyte genes differentially
regulated based on S.haematobium infection intensity and age.
This implicates these genes as potential players in the aetiology of
complications arising from S.haematobium infection.
CMM/12
Extracellular fibrinogen binding protein Efb – a potential
antithrombotic agent?
Stefan Bagby, Giordano Pula, Bernhard Merget, Mareike Posner
University of Bath, Bath, UK
Staphylococcus aureus produces amongst others several fibrinogen
binding proteins (Fibs) which interact with platelets [1]. One of them is
the extracellular fibrinogen binding protein Efb. It has a mass of 15.8 kDa
and binding site for the Aα-chain of fibrinogen (Fg) on each terminus.
Therefore it inhibits the platelet aggregation in vitro with binding to Fg
and activated platelets [2].
We evaluated divergent platelet aggregation models in vitro using
washed platelets in buffer and plasma and compared the influence of
various concentrations of the agonists ADP, Thrombin, Thromboxin and
Collagen in different concentration. This was accomplished with dual
channel platelet aggrometer and flow cytometry. The results will be used
to evaluate further research on Efb and its antithrombotic effect.
References 1 Zhang, X., et al. (2011) Inhibiting platelets aggregation
could aggravate the acute infection caused by Staphylococcus aureus.
Platelets 22; 2 Shannon, O. and Flock, J.I. (2004) Extracellular fibrinogen
binding protein, Efb, from Staphylococcus aureus binds to platelets and
inhibits platelet aggregation. Thrombosis and Haemostasis 91
CMM/13
Potentially pathogenic bacteria in shower hose biofilms are eradicated
with a novel biocide
John Moat, Mathew Upton
The environment can be a source of pathogenic bacteria. We show here
that domestic shower hoses harbour significant numbers of potentially
pathogenic bacteria that can be eliminated with a novel biocide.
Well developed biofilms were physically removed from the internal
surface of shower hoses recovered from a hotel in NW England. Total
viable counts (TVC) were determined and bacteria identified using 16S
rDNA analysis. Metagenomic analyses of total DNA was also carried out.
Hoses were treated with an acid sequestering agent and a biocide, alone
and in combination before determination of TVC.
TVC ranged between ~1 x 105 and 1 x 106 cfu/ml. The bacteria
observed were dominated by a small number of morphotypes
identified as Comamonas testosteroni, Delftia tsuruhatensis, Acidovorax
delafieldii, Arthrobacter sanguinis and Pseudomonas fluorescence. However,
metagenomic analysis indicated dominance of alpha-proteobacteria
(55%) and members of Mycobacterium (23%). TVCs underwent a 2-3
log reduction following treatment with the sequesterant alone and were
completely removed in all cases by a combined treatment with the
sequesterant followed by the biocide.
Domestic showering may result in exposure to aerosols of bacteria that
are potentially pathogenic. It may be prudent to ensure their eradication
by the use of biocide treatments.
CMM/14
Paradoxical antibodies: IgG2 specific for the O-antigen of Pseudomonas
LPS inhibits serum killing and increases the severity of bronchiectasis
infections
Timothy J Wells1, Deborah Whitters1, Yanina R Sevastsyanovich1,
Jennifer N Heath1, John Pravin1, Douglas F Browning1,
Mathew K O’Shea2, Adam F Cunningham1, Calman A MacLennan3,
Ian R Henderson1, Robert A Stockley1
1
The University of Birmingham, Birmingham, UK, 2University of Oxford,
Oxford, UK, 3Novartis Vaccines Institute for Global Health, Sienna, Italy
Non-cystic fibrosis bronchiectasis is a pathological condition of lung
damage that often leads to recurrent and chronic colonisation by
Pseudomonas aeruginosa. These chronic infections are extremely difficult
to eradicate once established.We studied eleven patients with chronic
P. aeruginosa lung infections for the ability of their serum to kill their
infecting strains. Sera with impaired killing were analysed to determine
which factor was inhibiting serum killing. Three patients had sera that
were unable to kill their respective infecting strains. Patients whose sera
exhibited impaired-killing to their infecting P. aeruginosa had a greater
severity of disease. Each inhibitory serum failed to kill isolates from the
three patients exhibiting impaired serum killing, but could kill strains from
patients exhibiting normal serum killing. Inhibition could be removed
by extracting IgG2 specific for the lipopolysaccharide O-antigen of
the infecting P. aeruginosa. Thus, IgG2 antibodies to lipopolysaccharide
O-antigen may be associated with enhanced disease severity.
Furthermore, such antibodies may have a pathogenic role in disease
indicating that caution should be applied to developing vaccines based on
the Pseudomonas O-antigen.
CMM/15
A comparative evaluation of the diagenode multiplex PCR kit on the
BDMAX versus routine in-house assay used to diagnose Bordetella
pertussis
Juliet EM Kenicer, Alison Hardie, Naomi J Gadsby, Kate E Templeton
NHS Lothian, Edinburgh, UK
The objective of this study was to assess performance of a Diagenode
Bordetella multiplex assay on the BDMax to the well validated in house
real-time PCR method.
131 Nasopharyngeal aspirates (NPA) collected in 2012 from paediatric
patients (85% aged less than 2 years). All NPA had previously tested
with our inhouse PCR assay (easyMAG and ABI7500) were tested using
the Diagenode multiplex on the BDMax extraction and PCR platform
(Diagenode/BDMax).
38 NPAs tested positive on both the Diagenode/BDMAX and the in
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Poster abstracts
house PCR. 2 samples tested negative with Diagenode/BDMAX and
positive with the in house PCR and 91 samples tested negative with both
methods. No samples tested negative on the in house PCR and positive
with the Diagenode/BDMAX. This equates to a diagnostic sensitivity of
(lower bound 95% CI) 95% (84%) and a diagnostic specificity of (lower
bound 95% CI) 100% (95%). The Κ statistic shows excellent agreement
between the two tests (0.96). In addition, 16/38 positives (42%) were
also co-infected with a respiratory virus and 1 was co-infected with
Bordetella parapertussis.
In conclusion, the Diagenode/BDMAX performed well and the simplicity
of the assay set up would lend itself to less specialist laboratories enable
more local rapid diagnostics.
CMM/16
Persister bacteria prevent eradication of Burkholderia thailandensis by
melioidosis antibiotics
Michael E G Steele1, Claudia M Hemsley1, Helen S Atkins2,
Rick W Titball1
1
College of Life and Environmental Sciences, Biosciences; University of Exeter,
Exeter, UK, 2Defence Science and Technology Laboratory, Porton Down,
Salisbury, UK
Burkholderia pseudomallei is the causative agent of melioidosis, a disease
with diverse clinical manifestations. Salient features of the disease,
including the occurrence of chronic infection and recalcitrance to
aggressive antibiotic treatment, implicate persister bacteria in chronic
infection. Persistence is a reversible phenotype found in a subpopulation
of genetically homogenous bacteria, which confers multi-drug tolerance
(MDT) -the ability to remain viable in the presence of a variety of
antibiotics.
In this study, the antibiotic susceptibility and frequency of persister
formation of B. thailandensis, as a model for B. pseudomallei, were studied
using several antibiotics used to treat melioidosis. We hypothesised that
different persister populations would be exposed by different antibiotics,
resulting in different kill curves. In order to test this hypothesis, minimum
inhibitory concentrations (MIC) were first obtained for the melioidosis
antibiotics. Kill curves were established for each antibiotic and persister
levels were calculated after extended antibiotic treatment at 10-100x
MIC. All antibiotics are shown to generate persisters, indicating the
significance of these phenotypic variants in failure to treat melioidosis.
The difference in persister dynamics between antibiotics may reflect
different antibiotic-specific resistance mechanisms employed by
persisters.
CMM/17
Testing the specificity of Burkholderia pseudomallei toxin-antitoxin (TA)
toxins in a range of host bacteria
Harriet L Bare1, Claudia M Hemsley1, Aaron Butt1,
Timothy P Atkins1,2, Richard W Titball1
1
College of Life and Environmental Science, University of Exeter, Exeter, UK,
2
Defence Science and Technology Laboratory, Porton Down, Salisbury, UK
Toxin-antitoxin (TA) systems are found in many prokaryotes and consist
of a gene pair, where one gene encodes a toxin and the other a cognate
antitoxin. Normally, the antitoxin is able to bind the toxin to form a
stable complex but under stress conditions proteases in the cell degrade
the antitoxin, releasing the toxin from the complex. Toxins are able to
induce cell death or inhibit cell growth by targeting key cell processes,
such as DNA replication, protein synthesis and mRNA stability. These
toxins are of interest as potential antimicrobial strategies and in this study
TA toxins found in Burkholderia pseudomallei were examined.
Previous studies in our lab have shown that over-expression of some B.
pseudomallei TA toxins in Escherichia coli results in a dramatic decrease
in the number of culturable bacteria and so we hypothesised that
these toxins could be active against a broad range of bacteria. One of
these TA toxins was cloned into a broad host range vector and overexpressed in a range of different bacteria to determine if toxin activity
was host specific.
CMM/18
The Galleria mellonella larvae infection model indicates that ST69 and
ST127 strains are more pathogenic than other uropathogenic E. coli
MAjed F Alghoribi, Tarek M Gibreel, Mathew Upton
Microbiology and Virology Unit, School of Translational Medicine, University of
Manchester, Manchester, UK
Galleria mellonella (GM) larvae are an alternative in vivo model for
investigating bacterial pathogenicity. The goal of this study was to
understand the pathogenicity and the behaviour of leading uropathogenic
E. coli (UPEC) lineages ST69, ST73, ST95, ST127 and ST131 using GM
larvae.
Larvae were challenged with a range of inoculum doses of UPEC to
determine the 50% lethal dose (LD50) and survival outcome of the
infected larvae was compared using Kaplan-Meier survival analysis.
Larvae inoculated with ST127 and ST69 isolates (104 colony-forming
units) show significantly higher mortality rates than those infected with
ST131, ST73 and ST95 isolates; ST127 and ST69 isolates kill 50% of the
larvae within 24 hours. PCR based surveillance of 29 virulence factors
(VF) showed that ST127 isolates are significantly associated with a higher
VF-score than isolates of all other STs tested (P≤0.0001), including ST69
(P<0.02).
This study illustrates that GM larvae can be used to investigate the
virulence of UPEC strains. We suggest that the incidence of ST127
strains should be monitored, as these isolates have not yet been widely
reported, but they clearly have a pathogenic potential similar to that of
more widely recognised clones, like ST69.
CMM/19
Extracellular loop 4 of the meningococcal outer membrane protein
PorA affects cell cycle and migration of human epithelial cells
Matthew J Vassey, Lee M Wheldon, Akhmed Aslam, Jafar Mahdavi,
Neil J Oldfield, Dlawer Ala’Aldeen, Karl G Wooldridge
The cell surface laminin receptor (LR) receptor plays an important role
in cell adhesion to the basement membrane, signal transduction and
cell movement. We recently showed that meningococci bind LR and
identified the outer membrane porin protein, PorA, as one of two
meningococcal LR-binding ligands. Further studies utilising recombinant
PorA sub-fragments and synthetic peptides localised the LR-binding
domain to the hyper-variable, surface-exposed fourth extracellular loop
of this protein.
A linear wound healing assay was used to investigate the in vitro effect of
PorA loop 4 on human epithelial cells. Wound closure was significantly
reduced in cells treated with synthetic PorA loop 4. In contrast,
recombinant PorA or synthetic loop 1 peptide had no effect on wounds
closure.
Treatment with PorA loop 4, but not loop 1, also resulted in a significant
reduction in the number of cells that transitioned through the G1 cell
cycle checkpoint after 24 h.
PorA loop 4-mediated effects on cell migration and the cell cycle,
which are likely mediated through LR, appear to be cytostatic and not
cytotoxic, and may play an important role in the interaction between N.
meningitidis and human epithelial cells in vivo.
CMM/20
The effect of proton pump inhibitors on the survival, morphology and
motility of Campylobacter jejuni
Kareen Macleod, Amanda MacCallum, Andrew Stevenson, Mark
Roberts, Paul H Everest
The University of Glasgow, Glasgow, UK
Proton pump inhibitors (PPIs) are primarily used in the treatment of
conditions such as peptic ulcer, heartburn and gastroesophageal reflux
105
Poster abstracts
disease (GORD). PPIs actively inhibit the H+/K+ ATPase of parietal cells
and thereby irreversibly block production of hydrochloric acid in the
stomach. It is known that patients being treated with PPIs are more
susceptible to enteric infections such as Campylobacter1.
However, it has been found that at certain concentrations, PPIs are able
to affect the survival of C. jejuni in vitro. Broth microdilution was used
to determine the minimum bactericidal concentration (MBC) of the PPI
pantoprazole for three strains of C. jejuni. The median MBC for strain
81116 was found to be 0.89 mg/ml and for strains 11168 and 81-176
(pVir+) was found to be 0.94 mg/ml. Light and electron microscopy were
used to show that exposure to levels of pantoprazole above the MBC
resulted in coccal forms of C. jejuni. 0.4% sloppy agar was used to show
that the motility of C. jejuni was adversely affected in a dose dependent
manner.
Reference [1] Bavishi C, DuPont H L. Systematic review: the use of
proton pump inhibitors and increased susceptibility to enteric infection.
Alimentary Pharmacology & Therapeutics. 2011; 34 (11-12): 1269-1281.
CMM/21
bvlR (PA14_26880), a novel LysR-type transcriptional regulator with a
key role in pathogenicity and biofilm formation
Ronan McCarthy1, Marlies Mooij1, Olivier Lesouhaitier2,
Georg Neckermann3, Brian Noonan3, Fergal O’Gara1
1
BIOMERIT Research Centre, Microbiology Department, University College
Cork, Cork, Ireland, 2Laboratoire de Microbiologie du Froid–Signaux et
MicroEnvironnement (LMDF–SME EA 4312) GIP ,Centre Sécurité Sanitaire
et Environnementale de Normandie, Université de Rouen IUT d’Evreux, 55,
Evreux, France, 3AstraZeneca, 35 Gatehouse Dr, Waltham, MA 02451–
1215, Boston, USA
Infections with Pseudomonas aeruginosa are the leading cause of
mortality among cystic fibrosis patients. In order to establish these
chronic lung infections, Pseudomonas utilises a wide range of virulence
mechanisms, which are often regulated by LysR-type transcriptional
regulators (LTTRs), the most common family of transcriptional regulators
found in P. aeruginosa. In this study, we characterise a LTTR of previously
unknown function; bvlR at both a molecular and phenotypic level.
We demonstrate the role of bvlR in the microbe-host interaction,
showing that bvlR modulates a wide range of processes associated with
both acute and chronic infection such as biofilm formation and type
three secretion. bvlR expression also leads to a significant reduction in
virulence in the Caenorhabditis elegans model of infection. This might
be explained by a reduction of exotoxin production, as bvlR negatively
regulates toxA and exotoxin A is a key virulence factor in C.elegans
mortality .To characterise the underpinning mechanisms, promoter
analyses was performed. Bioinformatic analysis identified a putative bvlR
binding motif, direct binding of bvlR protein to this novel motif upstream
of target genes was confirmed by electro mobility shift assays. Altogether
this indicates that this previously uncharacterised LTTR is a global
virulence regulator in P. aeruginosa.
CMM/22
Genetic variation in phage yype (PT) 8 and PT14 strains of
Escherichia coli O157 point to underlying reasons behind strain-specific
susceptibility to the typing bacteriophages
Lauren Cowley1, Phillip Ashton1, John Wain1,2, Claire Jenkins1,
Tim Dallman1, David Gally3
1
Health Protection Agency, London, UK, 2University of East Anglia, Norwich,
UK, 3The University of Edinburgh, Edinburgh, UK
Phage-typing is a widely used method for population analysis of E. coli
O157 outbreaks but little is known about the mechanisms of interaction
between the bacteria and the typing phages or the reasons why certain
strains of E. coli O157 are susceptible to particular bacteriophages. The
only difference in the phage typing profile between PT8 and PT14 is the
resistance to typing phages 9 and 10 in PT8; both are T7 phages and the
only Podoviridae in the typing scheme. Genetic comparisons, using whole
genome sequencing and genome alignment tools such as Mauve, has
allowed for recognition of specific regions of genetic variation that correlate
with phage type and may influence susceptibility to the typing phages.
Regions of phage type correlating variance containing outer-membrane
proteins point to a protective lipopolysaccharide (LPS) that may provide
immunity against the binding of specific phages. Similarly, regions of phage
remnant genes may provide immunity against new infection by phages.
Further proof is provided for this theory in the sequencing of multiple
unrelated strains from a large pool of E. coli O157strains.
CMM/23
Assembly of recombinant empty capsids of enterovirus 71
Abigail D Cone, Geraldine Mulley, Rebecca M Willmot, Ian M Jones
University of Reading, Reading, UK
Enterovirus 71 (EV71) is a member of the Enterovirus genus of the
Picornaviridae family, one of the etiological agents of hand, foot and
mouth disease (HFMD). Several epidemic outbreaks of HFMD caused
by EV71 have been associated with severe neurological complications
and high mortality in the Asia-Pacific region. EV71 is now considered an
important neurotropic virus in Asia and there are currently no effective
anti-virals or vaccines available. We have evaluated the production of
virus-like particles or ‘empty capsids’ of EV71 to provide a potential
vaccine. We show that recombinant baculoviruses express high levels of
the structural precursor protein P1 but that only low levels of cleaved
proteins are observable unless the activity of the 3C proteinase is
attenuated. When 3C levels are reduced by lower expression or when
enzyme activity is reduced by mutation, the correct cleavage of P1 into
the structural proteins and assembly into empty capsids is observed.
Recombinant EV71 antigen was found in both cell and supernatent
fractions suggesting some particle release from insect cells without cell
lysis. These results suggest that EV71 empty capsids could represent a
potential vaccine against EV71.
CMM/24
Staphylococci in veterinary patients in Ireland – a comparison of
species identification methods
Lisa Geraghty, Andrew Fogarty, Neil Rowan, Mary Booth
Athlone Institute of Technology, Athlone, Co. Westmeath, Ireland
There is merit in definitively identifying staphylococcal veterinary
pathogens to species level in order to study evolving trends in resistance
patterns and to inform appropriate therapeutic management. This study
aims to compare three commercial phenotypic methods, API Staph
32 (Biomerieux), RapID (remel) and Staph-Zym (Rosco Diagnostica),
with a genotyping method (sequencing of a 412bp fragment of the
staphylococcal tuf gene). Genotyping of the rpoB gene, as well as PCRFRLP of the pta gene, were performed to definitively identify members
of the Staphylococcus intermedius group (SIG). Fifty two veterinary
staphylococcal clinical isolates from companion animals were tested. API
Staph 32 correctly identified all (11/11) S. aureus isolates, 83% (10/12) of
the SIG species, and 66% (19/29) of the coagulase negative species. RapID
and Staph-Zym correctly identified 61% (14/23) and 0% respectively of
the coagulase positives, and 10% (3/29) and 3% (1/29) respectively of
the coagulase negative species. Tuf gene sequencing was superior for
identification of staphylococcal species of animal origin. Further genotyping
using rpoB may be necessary to differentiate SIG species. In conclusion,
amplification and sequencing of tuf is recommended for definitive
identification of coagulase negative Staphylococci, with PCR-RFLP or rpoB
sequencing suggested as a superior identification method for SIG.
CMM/25
The development of methods designed to quantify plaque biofilm
metabolic processes using a re-adapted modified Robbins device
David Greenwood1, Howard Foster1, David Bradshaw2,
Richard Lynch2, Cynthia Pine1
1
University of Salford, Salford, UK, 2GlaxoSmithKline, Weybridge, UK
106
Poster abstracts
Introduction Caries (tooth decay) is one of the most prevalent diseases
of both adults and children and is characterised by the demineralisation
of tooth enamel by acidogenic biofilms. Current understanding of oral
disease is often impeded by the inability to reproduce conditions at the
interface between the oral biofilm and the tooth.
Aims To develop reproducible biofilms for physiological studies of ex
vivo oral biofilms.
Methods The Modified Robbins Device was redesigned to include a
colonisation stud which permits basal pH measurements. This device will
combine the production of multiple biofilms, pH profiling (The Plaque
Glycolysis Regrowth Method) and the analysis of biofilm homeostatic
processes within a single device.
Results Initial testing produced inconsistent biofilm sizes and this was
attributed to the tendency for cells to accumulate in greater numbers
at the device inlet port. A modification using reversed-flow peristaltic
inoculation produced biofilms with greater reproducibility.
Conclusions This new method has successfully grown reproducible
biofilms which will be deployed to study physiology of key acid (e.g.
enolase) and alkali (e.g. urease and arginine deiminase) generating
enzymes and their subsequent inhibition by fluoride.
comma-shaped rods, and even spirochetes. Yeast like Candida and
Saccharomyces cells and other types of fungi were not seen. An average
of 10 species were cultured and identified per stratum per cutting board.
Bacteria more often found in the superficial strata were Staphylococcus,
Enterococcus, Corynebacterium, Bacillus, Pseudomonas, Enterobacteriaceae
and Nocardia. The intermediate strata most commonly observed bacteria
were Enterobacteriaceae, Bacteroides, Campylobacter, Listeria, Lactobacillus,
and Acinetobacter. The deep strata commonly enclosed Porphyromonas,
Bacteroides, Enterobacteriaceae, Actinomyces, Clostridium, Propionibacterium,
Eubacterium, Veillonella, Prevotella, Fusobacterium and Treponema. Other
pathogens isolated include Yesinia enterocolitica. This is the first stratified
study of the microbial community of meat cutting boards.
CMM/26
Cross-sectional study in the effect of smoking on the periimplant
microbial ecology
MAneerat Kuptanon, Pravej Serichetapongse, Anjalee Vacharaksa
Chulalongkorn University, Bangkok, Thailand
Objective To use 16S rRNA-based PCR assay for detection of putative
pathogens, including Porphyromonas gingivalis (Pg), Prevotella intermedia
(Pi), Treponema denticola (Td), and Tannerella forsythus (Tf), in smokers or
nonsmokers.
Methods Patients with dental implants were randomly selected. Sterile
paper points were used for subgingival samples collection and DNA
was extracted using PowerBiofilmTM DNA Isolation Kit (MO BIO
Laboratories, USA). Endpoint and quantitative PCR were performed to
identify Pg, Pi, Td, Tf, and total bacteria.
Results The mean ages of smokers (n = 6) and nonsmokers (n =
7) were 49.83±8.21 and 55.71±11.61 years. The average years of
implant in function in smokers and nonsmokers were 7.18±3.68 and
5.17±3.69. Pg, Pi, Td, and Tf in samples were shown by endpoint PCR.
Some smokers, 50%, 66.67%, 83.33%, or 83.33%, and non-smokers,
42.86%, 14.29%, 14.29%, or 85.71%,were detected with Pg, Pi, Td, or Tf,
respectively. The prevalence of Td in smokers was significantly higher
than that was in non-smokers by Fisher’s exact test analysis (p<0.05).
Consistently, quantitative PCR showed the ratio of Td to total bacteria
were higher in smokers.
Conclusion The prevalence of periodontal pathogenic bacteria was
higher in smokers than non-smokers with statistical significantly difference
in Td.
ENV
Environment
ENV/01
The microflora of the butcheries’ cutting boards
M A Alqumber
Albaha, Saudi Arabia, Albaha, Saudi Arabia, Saudi Arabia
Polyethylene cutting boards used in Saudi Arabian butcheries host a
microbiological niche in their fissures that has been characterised only to
a very limited extent. Yet a good understanding of the residing microflora
can be essential to control food-borne diseases and food spoilages. The
primary objective of this study was to obtain a stratified map of the
microflora in 40 cutting boards. Sections obtained from cutting boards
were analysed by culture-based and molecular techniques. Microscopic
examination revealed a mixed microflora containing various amounts
of Gram-negative and Gram-positive cocci, rods, some fusiforms and
ENV/02
Symbiotic Nitraria retusa–rhizosphere interactions
Zainab Haji Baroon, Awatif Mohammed Yateem,
Tahani Ibraheem Al-Surrayai
Kuwait Institute for Scientific Research, Kuwait, Kuwait
Rhizosphere-revegetation interactions are excellent tools in the
enhancement of desert biodiversity. Native desert vegetations in Kuwait
have been severely depleted due to both natural and human factors,
thus facing the danger of extinction. Symbiotic rhizospheric microbial
communities can play a significant role in growth and abundance of
native plant communities. This study aimed to emphasised the enhancing
effect of microbial symbiosis on the growth of Nitraria retusa, a native
desert plant that was selected on the basis of its high potential for the
revegetation in desert ecosystems in Kuwait. A shed house study was
carried out where N. retusa seedlings were grown in a completely
designed randomised block. Seedlings were propagated in three soil
treatments; (i) soil with added mixed rhizospheric inoculum (SI), (ii)
soil with amendment (SP), and (iii) soil with added both rhizospheric
inoculum and amendment (SIP). Growth performance was monitored
in terms of shoot height and number of leaves, on monthly basis for
120 days. Results clearly showed the enhancing effect of the microbial
inoculum when combined with amendment, on the plant growth.
Apparently, N. retusa maintained a high survival rate in SI treatment.
The trends of this study outlined the future research for integrated
management and sustainability of desert ecology, from microbial and
eco-physiological issues to the biotechnological developments.
ENV/03
Use of multiple displacement amplification based approaches for
detection and analysis of environmentally significant and contaminating
bacteria in diesel-contaminated ground water samples
Omolola A. Akinbami, Leonid A Kulakov, Michael J Larkin
Queen’s University of Belfast, Belfast, UK
A great challenge of microbial detection in natural environment is
that more than 99% of microorganisms found in environments are
not culturable when standard conditions are used. To overcome this,
molecular approaches are widely used nowadays to successfully study
environmental microorganisms. Multiple displacement Amplification
(MDA) is especially useful as it could be applied to identify functional
genes associated with dominant bacterial species or even for singlecell genomic projects. The aim of this project was to study ground
water sample from diesel-contaminated site in Northern Ireland in
order to detect and identify dominant bacterial species and key genes
associated with them that likely to be involved in biodegradation of the
contaminating compounds. From this environment, we identified (by
MDA assisted PCR) dominant bacterial strains such as Pseudomonas
fluorescence, Polaromonas naphthalenivorans and Pseudomonas
putida. Functional genes likely to be involved in key processes (e.g.
biodegradation) that encoded by these species were identified. We
showed that narG gene (nitrate reductase gene) was associated with
P. fluorescens and P. naphthalenivorans; naphthalene dioxygenase gene
(nahAc) was associated with P. fluorescence and P. putida detected from
107
Poster abstracts
ground water samples. All dominant bacterial species identified by MDAassisted PCR were present in the 16S rRNA gene clone libraries.
ENV/04
Biofilm formation of bacteria isolated from fruits and vegetables and
their control using some medicinal plant extracts
Ome K Achi
Michael Okpara University of Agriculture, Umudike, Umuahia, Nigeria
Bacterial biofilms are increasingly implicated in the pathogenesis of
disease outbreaks in food processing environment. Adherent bacterial
cells resist displacement strategies including physical and chemical
procedures due in part to encasement within a polymeric matrix as
well as an altered phenotype. In this study, three organisms (Escherichia
coli, Pseudomonas aeruginosa and Staphylococcus aureus) isolated
from the surfaces of fresh produce, were investigated for their ability
to form biofilms in vitro using a microplate assay with crystal violet
staining. Biofilm formation by the isolates was observed over 48 hours
in tryptic soy broth with varying concentrations of glucose, sodium
chloride, incubation time and at different temperatures. Aqueous and
ethanolic leaf extracts of Ocimum gratissimum, Piper guiniensis and
Gongronema latifolium were also investigated to determine their
antibiofilm activities. All bacterial isolates were found to possess biofilm
forming capabilities which is dependent on the surface on which the
biofilm is forming. Biofilm formation was equally efficient under static
and agitated conditions. The strains readily attached to glass and plastic
surfaces. Biofilm production showed moderate values at pH 7 but low
at acidic and alkaline levels. There were significant differences between
investigated extracts in antibiofilm activities.
ENV/05
Rapid identification of Zygosaccharomyces yeasts with genus-specific
primers
Michelle Hulin, Alan Wheals
University of Bath, Bath, UK
The genus Zygosaccharomyces comprises 10 species: Z. bailii, Z.
bisporus, Z. gambellarensis, Z. kombuchaensis, Z. lentus, Z. machadoi, Z.
mellis, Z. rouxii, Z. sapae and Z. siamensis. Z. sapae contains the species
with the temporary name Z. pseudorouxii. The Zygosaccharomyces
clade contains some species that are important as food and beverage
spoilage organisms and thus the ability to identify them rapidly is of
significant importance. Species-specific primers have already been
developed for most species enabling PCR identification directly from
colonies in 3 hours. It would be advantageous if a single reaction could
detect any member of the clade and then species-specific primers could
be used only on those isolates that were positive. We report here the
development of the first yeast genus-specific primers designed on the
ITS1/5.8S/ITS2 rRNA region that can detect all Zygosaccharomyces
species (except Z. machadoi) with no false positives. ITS region DNA
sequences are available for virtually all yeast type species and thus,
prior to having complete genome sequences (currently only available
for all species of the genus Saccharomyces), rRNA regions may
provide opportunities for the design of PCR primers for genus-specific
identification.
ENV/06
An Investigation into the contribution of the photoreceptor Lmo0799
to the growth of Listeria monocytogenes when exposed to light
Beth O’Donoghue, Jessica Aherne-Kristensen, Conor P O’Byrne
NUI Galway, Galway, Ireland
Listeria monocytogenes is a Gram-positive food-borne pathogen, found
ubiquitously in the environment. L. monocytogenes poses a serious health
threat to immunocompromised patients, due in part to its capacity to
withstand stresses commonly used in food processing to limit bacterial
survival. The alternative sigma factor σB is responsible for regulating
the defence mechanisms of L. monocytogenes in response to physical
and energy-depletion stresses. Various stress signals are thought to be
integrated into the σB activation pathway via the stressosome, a large
molecular complex consisting of multiple copies of signalling and sensory
proteins. Research has shown that Lmo0799, one of five putative sensor
proteins found in the L. monocytogenes stressosome, is a photoreceptor
and contains a Light, Oxygen or Voltage (LOV) domain at its N-terminal
region. Experiments conducted to investigate the contribution of the
Lmo0799 protein to the growth of L. monocytogenes indicate that
compared with wild-type EGDe, the ∆lmo0799 mutant displays a
reduced growth rate in the presence of light and an osmotic stress, but
no significant difference is observed between the two when exposed
to light stress alone. This provides further evidence that there may be
a synergistic relationship between osmotic and light stress effects on L.
monocytogenes.
ENV/07
Functional potential of microbial communities of arsenic and
chromium contaminated soils
Yasir Rehman1, Fariha Rizvi1, Michael McInerney2, Shahida Hasnain1
1
Department of Microbiology & Molecular Genetics, University of the Punjab,
Lahore, Pakistan, 2Department of Microbiology & Plant Biology, University of
Oklahoma, Norman, Oklahoma, USA
Heavy metals affect structure and composition of soil microbial
communities. Bacterial Communities respond to the toxic pollutants by
shifts in their phylogenetic compositions as well as number of functional
genes. In Pakistan, many industries release their untreated wastes
including chromium and arsenic in the environment. Chromium and
arsenic polluted sites in Kasur, Sialkot and Kalashakaku were selected to
observe the behaviour of functional gene pool of microbial communities
under the contaminants stress. Functional gene array was performed and
changes in functional potential of microbial communities were analysed.
A large percentage of detected genes were common in all samples,
but the numbers were different in controls and contaminated sites.
Changes were also observed in the number of genes responsible for
metal resistance, antibiotics resistance, organic compounds degradations,
carbon, nitrogen and sulfur cycles under the stress of arsenic and
chromium.
ENV/08
Detoxification of hexavalent chromate by Amphibacillus sp. KSUCr3
cells encapsulated in silica coated magnetic alginate beads
Abdelnasser SS Ibrahim1, Mohamed A El-Tayeb1,
Yahya B Elbadawi1, Ahmad El-Toni2, Ali A Al-Salamah1,
Garabed Antranikian3
1
King Saud University, Riyadh, Saudi Arabia, 2King Abdullah Institute for
Nanotechnology, Riyadh, Saudi Arabia, 3Hamburg University of Technology,
Hamburg, Germany
A new biocatalyst for Cr(VI) detoxification was developed using
combination of cell immobilisation, sol-gel chemistry and nanotechnology.
A novel Cr(VI) reducing Amphibacillus KSUCr3 cells were entrapped in
various polymers. Magnetic Fe3O4 nanoparticles were prepared using
solvothermal synthesis with particles size of 40 nm. For preparation of
magnetic beads, the cells were entrapped in the polymers gel in the
presence of the magnetic nanoparticles. Among all matrices tested,
magnetic alginate beads showed the highest immobilisation yield (81.3%).
Magnetic beads prepared using 50 µg magnetic nanoparticles per one
ml of alginate showed a very good separation of the cell-magnetic
alginate beads from the solution using a magnet. Furthermore, in order
to improve the physical properties of the magnetic bead, it bead was
successfully coated with silica layer using sol–gel chemistry. The reduction
of the toxic Cr(VI) by Amphibacillus sp. KSUCr3 cells entrapped in the
silica coated magnetic alginate beads showed better pH and thermal
stability than the free cells. Moreover, the immobilised cells could be
reused for Cr(VI) detoxification up to 10 cycles without significant
108
loss in activity. Hence, the developed cells encapsulated in silica coated
magnetic alginate beads cells have the potential to be applied for the
detoxification Cr(VI).
ENV/09
Phosphorous and sulfur mobilising activities of bacteria isolated from
Irish soils
MAiread Scanlan, Aaron Fox, Achim Schmalenberger
Application of conventional fertilisers in agriculture to improve
phosphorus (P) and sulfur (S) status has become increasingly expensive
and non-sustainable. However, most of the P and S in alternative
fertilisers is not directly plant available and need to be mobilised by
microbes first. In this study, 10 bacterial S and P mobilising isolates
from Irish soils were tested on their carbon source preferences and
capabilities of utilising various P and S sources. Mobilisation of particulate
P was tested on tricalciumphosphate agar plates. Liquid minimal
media in a microtiter essay with toluenesulfonate, nitrophenolsulfate,
phosphonoacetate and phytate as sole S and P source were used for
growth analysis, individually and concurrently.
The tested isolates grew best with glycerol, glucose and mannose
as carbon sources. All isolates grew well with toluenesulfonate,
nitrobenzenesulfonate and morpholinopropanesulfonate as sole S
and phytate as sole P source. Two isolates of the Enterobacteriaceae
and the Burkholderiaceae showed strong growth within 48 hours
with toluenesulfonate and phosphonoacetate as sole S and P source,
exceeding growth rates of all previously known aromatic sulfonate
desulfurising soil bacteria. These bacteria might be suitable as soil
inoculants to enhance plant growth as S and P mobilisers in combination
with alternative fertilisers.
ENV/10
Polyphosphate stabilisation within bacteria
Alan P Barry1, Karen C McCarthy1, Mingzhi Liang1, Niall O’Leary1,
Stefanie Frank2, Martin J Warren2, Michael B Prentice1
1
University College Cork, Cork, Ireland, 2Univeristy of Kent, Canterbury, UK
Bacterial polyphosphate kinase (PPK1) reversibly catalyses the
polymerisation of the terminal phosphate of ATP into a polyphosphate
chain. Polyphosphate polymer accumulation inside bacterial cells
resulting in storage of phosphate is long recognised as volutin granules,
and underlies the ability of bacteria to remove phosphate from
wastewater in biological phosphate removal processes. Polyphosphate/
phosphate storage by bacteria is usually temporary, because the reaction
is reversible and other enzymes whose substrate is polyphosphate
are present in the bacterial cytoplasm (exophosphatase PPX,
endophosphatases PPN, GTP synthetase PPK2). This results in a
phenotype where bacteria take up phosphate in certain conditions, or at
certain times in the cell cycle, but excrete phosphate at other times. This
complicates procedures for biological phosphate removal. We describe
novel approaches to stabilise polyphosphate inside bacterial cells and
thereby enhance storage and reduce the cyclical excretion of phosphate
by polyphosphate-forming bacteria. These procedures are of potential
application to wastewater treatment and other situations where
phosphate removal is desired.
ENV/11
Characterisation of the SPI-1 and Rsp type three secretion systems in
Pseudomonas fluorescens F113
Frank Egan1, Matthieu Barret1, Jennifer Moynihan1,
Olivier Lesouhaitier2, John P Morrissey3, Fergal O’Gara1
1
BIOMERIT Research Centre, Microbiology Department, University College
Cork, Cork, Ireland, 2Laboratory of Microbiology – Signals and Microenvironment EA 4312, University of Rouen, Evreux, France, 3Microbiology
Department, University College Cork, Cork, Ireland
Poster abstracts
Pseudomonas fluorescens F113 is a plant growth-promoting
rhizobacterium (PGPR) isolated from the sugar beet rhizosphere.
The recent annotation of the F113 genome sequence has revealed
that this strain encodes a wide array of secretion systems, including
two complete type three secretion systems (T3SSs) belonging to the
Hrp1 and SPI-1 families. While Hrp1 T3SSs are frequently encoded
in other P. fluorescens strains, the presence of a SPI-1 T3SS in a plantbeneficial bacterial strain was unexpected. In this work, the genetic
organisation and expression of these two T3SS loci have been analysed
by a combination of transcriptional reporter fusions and transcriptome
analyses. Overexpression of two transcriptional activators has shown
a number of genes encoding putative T3 effectors. In addition, the
influence of these two T3SSs during the interaction of P. fluorescens F113
with some bacterial predators was also assessed. Our data revealed that
the transcriptional activator hilA is induced by amoeba and that the SPI-1
T3SS could potentially be involved in resistance to amoeboid grazing.
ENV/12
An investigation of the factors associated with the risk of meat as a
source of classical swine fever introduction into the UK
Lucie Cowan2,1, Felicity Haines2, Helen Everett2, Trevor Drew2,
Helen Crooke2
1
University of Bristol, Bristol, UK, 2Animal Health and Veterinary Laboratories
Agency, Weybridge, UK
Classical swine fever virus (CSFV) is a highly infectious disease of pigs,
which has devastating economic and social consequences. To minimise
the potential for future outbreaks, it is important to assess the risk of
CSFV introduction to the UK via porcine products.
Our aim was to determine the rate of CSFV inactivation in pork
products commonly imported to the UK. Previous risk assessments have
used historic data which are variable and lack quantitative values. We
have determined the rate of inactivation of CSFV in unprocessed tissues
at key temperatures. Virus survival was much lower in lymph node and
fat samples than in serum or culture media, highlighting the importance
of the sample matrix when assessing virus survival and the risk a product
may pose for virus introduction.
We also investigated CSFV survival under the temperature and humidity
parameters used in dry-cured ham and bacon production.
Pigs experimentally infected with CSF were euthanised, carcasses hung,
chilled and muscle tissues used to produce ham and bacon and virus
survival assessed.
Levels of CSFV in muscle tissues prior to curing were at the limit of
detection for the virus detection assays. However, qRT-PCR showed
that CSFV viral RNA is stable for at least three-months.
ENV/13
Isolation of Legionella spp. from UK composts
Sandra Currie, Tara K Beattie, Charles Knapp
Legionella spp are widespread environmental bacteria, of which several
species are known human pathogens. Over the past five years, a
number of cases of Legionellosis in Scotland have been associated
with compost use. Legionellosis symptoms range from a self-limiting
flu-like illness to life-threatening pneumonia, known as Legionnaires’
Disease. Water systems contaminated with Legionella spp, commonly
Legionella pneumophila, are a widely known source of Legionellosis
infection; however studies investigating alternative sources of infection
are limited. Twenty-four compost samples were tested for Legionella
spp through culture on BCYE-α and confirmation using PCR of the
mip gene. Twenty-two of the samples were re-tested after eight
weeks in suspension with sterile distilled water at 30°C. In total, 15
out of 24 samples tested were positive for Legionella spp. 10 out of
24 tested positive during initial tests, 13 out of 22 tested positive after
the eight week period. The occurrence of Legionella spp in over half of
the samples does not appear to correlate with the limited number of
109
Poster abstracts
compost-linked Legionellosis cases seen in the UK. In order to assess the
potential risk to human health posed by the presence of Legionella spp in
compost samples, further studies need to be done.
ENV/14
Use of gaseous ozone to prevent microbial post-harvest spoilage of
fresh leafy produce
Shreya B Wani, Jeremy Barnes, Ian Singleton
Newcastle University, Newcastle upon Tyne, UK
Field crops suffer from post-harvest microbial contamination and decay.
Due to increasing pesticide resistance and consumer pressures over
indiscriminate use of a range of biocides, residue free alternatives, such as
ozone, are being actively explored and encouraged to curb spoilage of
crops in storage/transit to market. Previous work has demonstrated that
long-term exposure to low atmospheric concentrations of ozone can be
effective in some crops (e.g. berries and citrus) in significantly reducing
mould proliferation. This project focuses on the use of gaseous ozone
treatment to reduce post-harvest contamination and spoilage of targeted
products (leafy salads and root vegetables). Initial results have shown
several types of leafy produce have significant microbial phyllosphere
communities and that ozone gas can significantly reduce bacterial and
fungal loads (as determined by viable counts and confocal microscopy).
Importantly, ozone exposure levels must be carefully controlled to avoid
damage to produce. Further work will focus on scaling up the technology
to apply in a commercial environment.
Fermentation and bioprocessing
(industry)
FB
FB/01
Salt reduction in bread – sourdough as a promising solution
Markus C E Belz1,2, Emanuele Zannini1,2, Deborah M Waters1,
Michael Czerny3, Elke K Arendt1
1
1School of Food and Nutritional Sciences, University College Cork, Cork,
Ireland, 2Bio Transfer Unit, University College Cork, Cork, Ireland, 3IVV
Fraunhofer Institute, Freising, Ireland
Dietary intake of sodium chloride (salt) has increased considerably and is
intimately linked with elevated blood pressure and other cardiovascular
illness markers. Global food authorities recommend a maximum daily
intake of 5-6 g salt, approximately half the average current consumption
level. Baked goods are a major source of salt in the human diet and
therefore, a salt reduction in these staple foods would have major
global health impact. However, salt plays critical roles in cereal foods by
influencing bread properties including shelf-life, volume, and flavour, as
well as dough handling. Thus, a reduction of the levels of salt used must
be accompanied by functional replacer technologies, such as specifically
chosen sourdough. In this study, rheological and aroma profiling analyses
were used to assess the quality of reduced salt (0-1.2% NaCl) sourdough
breads. Decreasing levels of salt were associated with reduced dough
strength, extensibility, viscoelastic behaviour, and gas holding ability.
However, the incorporation of Weisella cibaria MG1 and Lactobacillus
reuterii FF2 sourdough, at levels of 10-20%, compensated the changes
in bread volume and structure. Additionally, the 20% sourdough
addition prolonged the bread shelf-life to 14 days, independent of salt
concentration, whilst concurrently improving texture and flavour.
FB/02
An investigation into the effect of malt type on the subsequent
growth, protein expression and volatile metabolite formation in
brewing yeast
Rachael Dack, St John Usher, Georgios Koutsidis, Gary Black
Northumbria University, Newcastle upon Tyne, UK
High levels of dark speciality malt in fermentations can lead to reduced
attenuation and alter levels of certain flavour active metabolites. Axenic
culture work, 2D gel-electrophoresis and GC-MS are being used to
investigate the effect different proportions/types of malt (of the same
nutrient content) have on yeast growth, gene expression and metabolite
formation during fermentation. Thus far results have indicated that the
use of dark malts, subject to greater heating and kilning during the malting
process, inhibit the growth of different strains of Saccharomyces cerevisiae.
This suggests the presence of inhibitory compounds in dark malts. The
inhibitory effect of these compounds may also be strain specific. Future
investigation to obtain a greater understanding of the biochemical
effects different malts have on yeast metabolism during the fermentation
process will be established through proteomic and metabolomic analysis.
This will enhance knowledge pertinent to choice of malt variety and
yeast strain with regards to new product development.
FB/03
Production of medium chain length poly-3-hydroxyalkanoates
(mcl-PHAs) from unrelated carbon source by locally isolated
Pseudomonas sp.
Iftikhar Ali1,2, Nazia Jamil1, Bruce A Ramsay2, Juliana A Ramsay2
1
University of the Punjab, Quaid-i-Azam Campus, Lahore, Punjab, Pakistan,
2
Queen’s University, Kingston, ON, Canada
Poly-3-hydroxyalkanoates (PHAs) are the biodegradable biopolyesters
that resemble in their properties with petroleum based non-biodegradable
plastics. Bacteria can produce these PHAs using different carbon
sources under unfavorable conditions. Medium chain length poly-3hydroxyalkanoates (mcl-PHAs), a class of PHAs, production has been
optimised by newly isolated Pseudomonas sp. These bacteria were
grown on varying ratios of C/N under nitrogen or phosphate limitation
in shake flask. Polymer was extracted from bacteria and purified before
its characterisation by GC-FID and/or GC-MS. This work resulted the
production of mcl-PHAs from unrelated carbon source i.e., glucose, instead
of short chain length (scl) PHAs that are characteristic when used glucose
as carbon source. Gas chromatography confirmed the presence of mclPHAs i.e. 3-hydroxyoctanoate (3-HD), 3-hydroxydecanoate (3-HD) and
3-hydroxydodecanoate (3-HDD). The isolated bacteria have shown great
interest in their biopolymer production at higher levels using unrelated
carbon source, glucose. The limitation of phosphate or nitrogen to produce
unfavorable conditions has been addressed in this work as well.
GM
GM/01
Site-specific activity of htrb1 and htrb2 in Pseudomonas aeruginosa lipid
A biosynthesis
Lauren E Hittle, Daniel A Powell, Majid Tofigh, Jace W Jones,
Robert K Ernst
University of Maryland, Baltimore, Baltimore, Maryland, USA
Pseudomonas aeruginosa (PA) is a Gram-negative pathogen well known
for its ability to grow and persist in a multitude of environments.
Many factors contribute to these survival advantages, one of which
being the alteration of LPS structures in response to environmental
cues. Lipid A, the membrane anchor of LPS, can be modified by the
addition or removal of fatty acids as well as the addition or removal of
phosphates and sugars. This work focused on identification of the lipid A
modification enzyme LpxL, a lauryl transferase, in PA. Mining of the PA
genome revealed the presence of 2 such genes, PA0011 (htrb1) and PA
3242 (htrb2). Characterisation of these enzymes demonstrated them to
be site specific and non-redundant. ESI and Gas Chromatography analysis
confirmed the acyl-oxo-acyl addition of the 2 position 2-OH C12 and
the 2’ position C12 fatty acid were mediated specifically by HtrB1 and
HtrB2 respectively. Deletion of either htrB1 or htrB2 resulted in a more
permeable membrane as well as altered susceptibility to polymyxins and
antibiotics. An impaired growth phenotype indicated htrB2 as necessary
for robust growth at multiple temperatures thus indicating the enzyme as
a target for future treatment of PA infections.
110
General microbiology
Poster abstracts
GM/02
The infiltration of OXA-48 like carbapenemase producing Klebsiella
pneumoniae in Kuwait
Ali A Dashti, Leila Vali, Mehrez Jadaon, Sherief El-Shazly,
Shorooq Al-Inizi
Kuwait University, Kuwait, Kuwait
The aim of this study was to investigate the characteristics of Klebsiella
spp in Kuwait. 832 non-duplicate Enerobcteriaceae isolates were
acquired from isolates recovered from the clinical microbiology
laboratories of three major governmental hospitals from between 20102012. Antibiotic susceptibility testing was performed by automated broth
microdilution method (Vitek2) for 15 antimicrobials according to the
CLSI guidelines. Klebsiella spp resistant to ciprofloxacin and for which the
ceftaxidime or cefotaxime MICs were >8mg/L were screened for ESBLs
and plasmid mediated quinolone resistant (qnr) genes. The isolates
were screened by PCR, sequenced and analysed for the presence of
SHV, TEM, CTX-M, NDM, PER, VIM, GEM, OXA 48,gyrA, parC, KPC
and qnrA, qnrB & qnrS. Pulsed-field gel electrophoresis was used for
typing. Twenty eight (16.1%) of 174 ESBL-producers contained plasmid
mediated qnr B or S and were associated with high level antibiotic
resistance. Nine isolates harboured CTXM-2 like genes and two
intermediate carbapenem resistant isolates contained OXA-48 like gene.
In this study, we have demonstrated for the first time the infiltration of
OXA-48-like carbapenemase in Kuwait. The dissemination of qnr and
carbapenmases among ESBLs-producer K. pneumoniea isolates raises
grave concern and should be monitored diligently.
GM/03
Studying the inhibitory effect of garlic extract on some bacteria of
multi-resistance to antibiotics
Rashad R Alhindi
King Abdulaziz University, Jeddah, Saudi Arabia
Garlic samples were collected to study their effect on Pseudomonas
aeruginosa, Staphylococcus aureus, Acinetobacter baumani, Klebsiella
spp, E. coli and Candida albicans. The Staphylococcus aureus was the
first cause of antibiotic-resistant hospital acquired bacterial infection
[25.15% of isolates], while Candidia albicans was found to be the least
causative one [8.05% of isolates]. Results showed that garlic have clear
effect at higher concentrations [1000, 500 and 250ug/ml], while the
suppressive effect was less at lower concentrations and was completely
absent at concentrations of 31.25 ug/ml. The staphylococcus aureus
was the most affected bacteria by the watery extract of garlic. Effect of
different types of garlic in the dilution method on the bacteria was varied,
as well the sensitivity of the bacteria. It was clearly apparent that the
garlic concentrations and the duration of its use have a proportionately
increasing effect on the decreasing number of the bacteria in the media
and had a suppressing effect on bacteria during incubation. Effect of
garlic combinations with some antibiotics, vancomicin and imipnem, was
also studied. Results showed that the number of colonies and the mean
survival rate were too much diminished when compared with the use of
the drug or the garlic extract alone.
GM/04
Widespread occurrence of putative bacteriocin genes in phage
genomes
Byung C Cho1, Ga H Kim1, Gwang I Jang1, Dzung B Diep2
1
Seoul Natioal Univ., Seoul, Republic of Korea, 2Norwegian University of Life
Sciences, As, Norway
Widespread distribution of bacteriocin genes in bacteria has been
well recognised. However, distributions of bacteriocin genes in phage
genomes have not been investigated. Using the genome mining tool,
BAGEL2, and phage genome sequences available at NCBI database,
we found that 57 putative bacteriocin genes occurred in 675 phage
genomes examined; 50 in dsDNA, 1 in ssDNA, and 3 in ssRNA
phage genomes and 53 in bacterial and 2 in archaeal phages. Major
host bacterial groups for the phages carrying most (44) of them are
Enterobaceriales, Bacillales, Lactobacillales, and Pseudomonadales. Two
putative archaeocins found out of 36 dsDNA archaeaphage genomes
were from Acidianus and Methanothermobacter phages, suggesting wider
distribution of archaeocins in Archaea than known thus far. Occurrences
of bacteriocin genes including both putative and potential (=interesting)
candidates in phage genomes seemed to be widespread. In addition to
the structural bacteriocin genes, immunity genes were found in phage
genomes at reduced frequency. Possibly, the presence and expression of
bacteriocin gene(s) in phage genomes might increase their fitness.
GM/05
Nocardioides salbiostraticola sp. nov., a new member of the genus
Nocardioides isolated from biofilm formed in coastal seawater of the
Norwegian sea
Yirang Cho1, Gwang I Jang1, Chung Y Hwang2, Eun-Hye Kim2,
Byung C Cho1
1
Seoul National Univ., Seoul, Republic of Korea, 2Korea Polar Research
Institute, Incheon, Republic of Korea
A Gram-positive, non-motile, aerobic, non-spore-forming and short
rod-shaped bacterial strain, PAMC 26527T, was isolated from biofilm
formed in coastal seawater of the Norwegian Sea. Analysis of the 16S
rRNA gene sequence of strain PAMC 26527T revealed a clear affiliation
with the genus Nocardioides. Based on phylogenetic analysis, strain
PAMC 26527T showed the closest phylogenetic relationship with the
species Nocardioides caricicola YC6903T with 16S rRNA gene sequence
similarity of 96.3%. Strain PAMC 26527T grew in 0 to 5.0% sea salts.
The optimum temperature and pH for growth were 20 °C and pH
7.5, respectively. The major cellular fatty acids of strain PAMC 26527T
were iso-C16:0 (44.5%), C17:1ω8c (20.0%) and C18:1ω9c (10.4%) and the
major menaquinone was MK-8(H4). The cell-wall analysis showed that
strain PAMC 26527 T contained LL-diaminopimelic acid. The genomic
DNA G+C content was 69.3 mol%. The combined phenotypic,
chemotaxonomic and phylogenetic data showed that strain PAMC
26527 T could be distinguished from validly published members of the
genus Nocardioides. Thus, strain PAMC 26527T should be classified as
representing a novel species in the genus Nocardioides, for which the
name Nocardioides salbiostraticola sp. nov. is proposed. The type strain is
PAMC 26527T (=KCTC 29158T).
GM/06
Changes in mouse gastrointestinal microbial ecology by the ingestion
of kale
Yutaka Uyeno, Shigeru Katayama, Soichiro Nakamura
Shinshu University, Minamiminowa, Nagano, Japan
Kale, a cultivar of Brassica oleracea, has received much attention because
of its health-promoting effects, including anti-obesity and anti-aging.
Considering the nutritional characteristics of kale, it is supposed that
the effects are exerted through modulation of the intestinal microbiota.
The aim of this study was to investigate the effect of kale ingestion
upon the gastrointestinal (GI) microbial ecology of mice. Twenty-one
male C57BL/6J mice were divided into three groups and housed in a
specific pathogen-free facility. They were fed either a control diet or
one of two experimental diets supplemented with different kinds of
commercial kale products for 12 weeks. Contents of the cecum and the
colon sampled from the sacrificed mice were processed for microbial
composition analysis by a bacterial rRNA-based quantification method
and short-chain fatty acid analysis by HPLC. The ratio of Firmicutes to
Bacteroidetes was higher in the colon samples of a kale diet group than in
the control. Colon butyrate level was also higher with the kale-amended
diet. Overall, the ingestion of kale affects mouse GI community structure,
possibly by inducing a change in the nutritional flow through the digestive
tract, by acting as a prebiotic, or both.
111
Poster abstracts
GM/07
Phenotypic plasticity and effect of selection on protein aggregation in
E. coli
Ulfat I Baig1,2, Milind G Watve1
1
Indian Institute of Science Education and researchR Pune, Pune,Maharashtra,
India, 2Abasaheb Garware College, Pune,Maharashtra, India
Asymmetric segregation of aggregated proteins has been shown to be
associated with cellular aging in bacteria. A point of debate has been
whether asymmetric distribution of protein aggregates leading to aging
is inevitable for a growing cell or whether it is an evolved strategy of the
cells. Earlier mathematical models and empirical results have shown that
caloric restriction facilitates symmetric cell division and delay aging in
bacteria. We examine here whether the nutritional environment affects
protein aggregates in short term and on prolonged selection. Using a
fluorescent marker for protein aggregates in E coli we show that protein
aggregation was significantly lower when grown under low concentration
of substrate. Further after selection for 1800 generations in low nutrient
concentration the cells accumulated substantially lower levels of protein
aggregates even after exposure to high substrate concentration but the
segregation is still asymmetric. Protein aggregation was thus affected
by both current nutritional environment and long term selection. This
indicates that there is some inevitability in the asymmetric division of
protein aggregates. However the significant difference brought about by
selection indicates that there is also some strategic control of the cell on
the extent of protein aggregation and thereby aging.
GM/08
Characterisation of a putative RAYT family transposase from Neisseria
meningitidis
Charlie Gilbert, Mirka Woermann, Rachel Exley, Christoph Tang
Sir William Dunn School of Pathology, University of Oxford, Oxford, UK
Neisseria meningitidis type IV pili are surface polymers consisting mainly
of PilE (Pilin) protein subunits. Analysis of pilE sequences from clinical
isolates revealed that certain isolates harbour a class II pilE gene, which
is highly conserved in terms of sequence and genomic location. In
all these strains, a gene encoding a putative REP Associated tyrosine
Transposase (RAYT) is found directly downstream of pilE. In addition,
the pilE locus is abundant in repetitive extragenic palindromic (REP)
elements and their higher-order structures, BIMEs. RAYTs are relatively
novel class of transposases which have been shown to bind and cleave
BIMEs in vitro and are believed to mediate their dissemination throughout
bacterial genomes. We have successfully expressed and purified the
putative Neisseria RAYT-transposase for structural studies and in vitro
assays. Simultaneously, to assess the activity of the transposase in vivo,
we have designed a transposon activity assay in a heterologous system.
Understanding the expression and activity of this putative transposase
will provide valuable information about its potential role in the evolution
of Neisseria strains with class II pilin and, more generally, in bacterial
genome dynamics.
GM/09
LspA mutants of Clostridium difficile are involved in lipoprotein
processing
Edward C Farries1, Andrea Kovacs-Simon1, Nigel Minton2,
Rick Titball1, Stephen Michell1
1
University of Exeter, Exeter, UK, 2University of Nottingham, Nottingaham,
UK
From the genome sequence of C. difficile, it appears that this bacterium
encodes two versions of lipoprotein signal peptidase, LspA and LspA2
. LspA2 is a putative functional homolog of LspA and contains the key
set of functional amino acids found in type II signal peptidases. We
have used targeted insertional mutagenesis via the ClosTron system to
generate mutant strains in either lspA or lspA2 and demonstrated that
both strains display increased sensitivity to malachite green thus indicating
their involvement in lipoprotein processing. It is thought that these two
enzymes may act on different sets of the C. difficile lipoproteome with
LspA being the primary signal peptidase due to its greater sequence
homology to LspA’s from other bacteria. As a result, it is suggested that
LspA and LspA2 are potential drug targets, in particular for globomycin
and homologs. Work will be presented on the characteriation of the lspA
and lspA2 mutants of C. difficile.
GM/10
Characterisation of a novel membrane protein VanJ conferring
resistance to teicoplanin
Wanchen Liu, Hee-Jeon Hong
Department of Biochemistry, University of Cambridge, Cambridge, UK
Teicoplanin resistance, and the exact mode of action of this drug, shows
some important differences compared to vancomycin but these are
currently poorly understood. Streptomyces coelicolor possesses a cluster
of seven genes (vanRSJKHAX) which when expressed confer resistance
to both vancomycin and teicoplanin however expression is only induced
in response to vancomycin. In this cluster, vanJ encodes a membrane
protein with unknown function which has previously been reported not
to be essential for resistance to vancomycin. We now have preliminary
data to show that vanJ confers resistance to teicoplanin by a mechanism
which is completely different from the conventional mechanism of
glycopeptide resistance. We have also identified many homologues to
VanJ, all residing in actinomycetes. Homologues with lower sequence
identity are ubiquitous, while those with higher identity tend to occur only
in strains possessing vanRS two-component system. The aim of this study
is to characterise VanJ, identify its function and to compare this to the
functions of other vanJ homologues selected from the lower and higher
identity groups. We thus aim to define the novel mechanism of resistance
encoded by VanJ, improve our understanding of the mode of action of
teicoplanin, and characterise the functional diversity of vanJ homologues.
GM/11
Potato extract: a potential treatment for Helicobacter pylori infection
Temitope A Adeyemi, Ian S Roberts
Helicobacter pylori is a Gram-negative bacterium that is the major
cause of many upper gastrointestinal diseases such as gastritis and peptic
ulcer disease. Failure in eradication of H. pylori infection, mainly due
to antibiotic resistance, has necessitated the development of better
therapeutics, especially from natural sources. In this study, potato extract
was evaluated for antibacterial activity against antibiotic-sensitive and
clinical antibiotic-resistant H. pylori strains, as well as a range of Gramnegative bacteria. The effect of the extract on the morphology of H.
pylori was also observed by transmission electron microscopy. Result of
the antibacterial assay indicated that potato extract is bactericidal against
H. pylori at a more rapid rate than conventional antibiotics, amoxicillin
and clarithromycin, used in treatment of H. pylori infection; with MIC90
at 3.9 mg/ml. Potato extract showed minimal antibacterial activity against
other bacteria tested, with MIC90 at 250 mg/ml. Transmission electron
microscopic analysis of potato extract-treated H. pylori cells showed
disruption of the morphology of H. pylori, characterised by loss of cell
shape. The results obtained suggest the potential use of potato extracts
as a source of anti-H. pylori agents; and stimulate further studies into
determining the mechanism of action of potato extract.
GM/12
Effect of lemongrass essential oil on growth and aflatoxin production
by Aspergillus parasiticus IMI 102566 in rice
Dusanee Thanaboripat1, Nathatida Nathamploy1,
Ratthiya Laowong1, Wanvisa Numnim1, Yaowapa Suvathi2
1
King Mongkut’s Institute of Technology Ladkrabang, Bangkok, Thailand,
2
Government Pharmaceutical Organisation, Bangkok, Thailand
112
Poster abstracts
Lemongrass (Cymbopogon citratus (D.C.) Staph.) has been reported
to have an antimicrobial activity against aflatoxin producing fungi.
The objective of our study was to compare the effect of lemon grass
essential oil extracted in our laboratory (KMITL lemongrass extract)
with commercial essential oil on the growth of Aspergillus parasiticus
IMI 102566 in rice, cultured for 7 days. KMITL lemongrass extract was
prepared by hydrodistillation using Clevenger apparatus. Dried powder
of lemongrass leaves was distilled for 2 hrs and essential oil was collected
and kept at 4￮C prior to use. KMITL lemongrass extract and commercial
lemongrass essential oil at concentrations of 0.5, 1, 1.5 and 2% (v/v),
dissolved in ethanol and normal saline were used in this study. The
results show that KMITL lemongrass extracts at all concentrations had
better inhibition on fungal growth than commercial lemongrass essential
oil. KMITL lemongrass extracts were further tested for an inhibition effect
on growth and aflatoxin production by Aspergillus parasiticus IMI 102566
in rice. KMITL lemongrass extracts at concentrations of 1, 1.5 and 2%
completely inhibited fungal growth for 21 days and significantly reduced
aflatoxin production from 42 ppb to 10, 4.6 and 2.8 ppb on day 28,
respectively.
GM/13
Effect of crude extracts from the root of Stemona tuberosa Lour on
the replication of Autographa californica multiple nucleopolyhedrovirus
Ounruan Petcharawan, Dusanee Thanaboripat
Crude hexane, dichloromethane and ethanol extracts from the
root of Stemona tuberosa Lour. were tested for cytotoxicity against
Spodoptera frugiperda cell line (Sf9) using MTT assay. The cytotoxic effect,
represented as CC50 was observed after 48 and 96 h. Dichloromethane
extract was most toxic with 96-h CC50 of 1,708.98 µg/ml. Crude
dichloromethane extract (31.25 µg/ml) was tested on Autographa
californica multiple nucleopolyhedrovirus (AcMNPV) when the extract
was added after 1 h post-infection of AcMNPV at the multiplicity of
infection of 2, in Sf9 cell line cultivated in vitro. There was no significant
difference (p>0.05) between the percentage of infected cells in the
control (95.54%±3.18) and the test sample with crude dichloromethane
extract (98.51%±1.34). However, there was significant difference
between the average virus titer in the control (2.06x108±0.71 PFU/ml)
and the test samples (2.65x108±0.79 PFU/ml). The average number of
polyhedra in the control (5.11x106±0.63 OBs/ml) was not significantly
different from the test sample (4.19x106±0.31 OBs/ml). However,
there was significant difference between the percent reduction of virus
titer in the control (0%±0) and the test sample (-27.45%±4.36). Crude
dichloromethane extract could not inhibit AcMNPV infection in Sf9 cells
but it significantly increased efficacy of AcMNPV against insect cells.
GM/14
Survey of aflatoxin contamination of chilli powder in 8 canteens of
Dusanee Thanaboripat, Ounruan Petcharawan,
Sujitra Sukonthamut
Chilli powder from 8 canteens serving various faculties of King Mongkut’s
Institute of Technology Ladkrabang (KMITL) was surveyed for aflatoxin
contamination during January, May and August, 2011. Seventy-two
samples of chilli powder from 24 food stalls were collected during these
three months. These samples including 9 control samples belonging to
the same commercial brand were analysed for aflatoxin using DOAAflatoxin Test Kits. All chilli powder collected from all food stalls was
contaminated with aflatoxin. Chilli powder collected from food stalls at
5 canteens (Prathep building, Engineering Faculty, Industrial Education
Faculty, KMITL building and Agricultural Technology Faculty) contained
the highest average amount of aflatoxin (8.94±1.10, 8.43±1.32,
8.37±1.13, 8.21±0.65 and 8.12±0.96 ppb, respectively) and chilli powder
collected from all food stalls contained significantly higher amounts of
aflatoxin when compared to commercial chilli powder (5.44±0.15 ppb)
used as control (p<0.05). When the average aflatoxin content in chilli
powder collected during January, May and August, 2011 was compared,
the average amount of aflatoxin in August was highest (8.59±1.56
ppb), followed by May (7.64±0.99 ppb) and January (7.55±1.14 ppb),
respectively. However, the aflatoxin levels of all chilli powder samples did
not exceed the legal limit (20 ppb) prescribed by Thai Ministry of Public
Health.
GM/15
Investigation of the effects of light on the growth of Listeria
monocytogenes in food-related environments
Kerrie NicAogain, Beth O’Donoghue, Conor O’Byrne
Bacterial Stress Response Group, Microbiology, School of Natural Sciences,
National College of Ireland, Galway, Ireland
Listeria monocytogenes is a gram positive bacterium which has become
a major concern within the food processing industry. Its ability to
withstand harsh environments within food processing plants can lead
to the contamination of food products. L. monocytogenes can cause a
severe gastrointestinal infection called listeriosis which is a rare infection
but has a high mortality rate among immunocompromised people
and pregnant women. In preliminary studies conducted by our group,
white light was shown to have an inhibitory effect on the growth of
L. monocytogenes, but only when osmotic stress was also present.
To investigate whether light could be used to control the growth of
L. monocytogenes in foods, this study will investigate the effect of
light in the presence of different environmental factors such as pH,
salt concentration and temperature, all of which are conditions which
L. monocytogenes s may have to overcome in the food processing
environment. The role of the light sensor Lmo0799 in the presence
of light will also be investigated with other food-related environmental
factors.
GM/16
Thermoregulated expression of the region 1 promoter of the
Escherichia coli K5 capsule gene cluster: identification of multiple
transcriptional start sites
Jia Jia, Ian Roberts
Expression of Escherichia coli capsules (typified by the K5 capsuloe) is
temperature regulated, being expressed at 37°C inside the host but
repressed at 20°C. Expression is regulated at the transcriptional level
by two convergent promoters PR1 and PR3. Global regulatory proteins
H-NS and IHF have been shown to regulate transcription from PR1. In
this study, a combination of lacZ reporter gene fusions and 5’ RACE
analysis confirmed the existence of three additional transcription start
points downstream of the original mapped +1 within PR1. By analyzing
the levels of transcription from different fragments of PR1 in hns::kan
mutants we showed that H-NS is invovled in regulationg transcription at
the 5’ UTR both at 37°C and 20°C. Overall this study will be helpful to
decipher the complex regulation of capsule gene expression in E.coli.
GM/17
Increased glucosylation of the lipopolysaccharide O-antigen subunit in
Salmonella Typhi
Erica N Kintz1, Edwin Kaptein1, Mark R Davies2,
Marjan W van der Woude1
1
University of York, York, UK, 2Wellcome Trust Sanger Institute,
Cambridgeshire, UK
Glycosyltransferase (gtr) operons modify the O-antigen subunit of
Salmonella lipopolysaccharide with a glucose residue. S. Typhi O-antigen
contains a higher level of glucose modification compared to other
serovars with similar O-antigen subunits (Hellerqvist, CG, et al. 1969.
Acta Chem Scand, 23(5), 1588-1596). High levels of this glucose
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Poster abstracts
modification have been correlated to increased virulence in S. Enteritidis
(Rahman, MM, et al. 1997. J Bact, 179(7), 2126-2131). Previously, we
showed that expression of many gtr operons in Salmonella phase vary
using an epigenetic mechanism (Broadbent, SE, et al. 2010. Mol Micro,
77(2), 337-353). Here we examined the molecular mechanisms leading
to the reported high levels of glucosylation in S. Typhi. The expression
of the gtr operon (t2303-2305 in Ty2 strain) was compared to related
operons from other serovars. Instead of phase varying, this operon
exhibited constitutive expression. Visualisation of the LPS from S. Typhi
strains further confirmed that a consistent percentage of O-antigen
subunits are glucosylated. The constitutive nature of this glucose
modification compared to other Salmonella serovars suggests it is an
important component to S. Typhi’s virulence, which will be investigated
further using our S. Typhi gtr mutant strains.
GM/18
INFANTMET – infant nutrition for progamming the gut microbiota in
neonates
Cian J Hill1, Kiera Murphy2, Tony Ryan3, Catherine Stanton2,
Paul O’Toole1
1
University College Cork, Cork, Ireland, 2Teagasc, Moorepark, Cork, Ireland,
3
Cork University Maternity Hospital, Cork, Ireland
Establishment of the intestinal microbiota commences at birth. The
microbiota has a major role in protection against pathogens, maturation
of the immune system and metabolic welfare of the host. In terms of
infant health, it is important to understand how infant nutrition influences
the development of the microbiota. Breast Milk is the gold standard
feeding regime for newborn infants and represents a baseline for the
functional performance of infant formula. The objective of this study is to
define the composition and functional performance of these breast fed
infants over time, using 454 sequencing technology. It aims to assess the
microbiota of 150 (50 pre-term, 50 C-section full term, and 50 natural
delivery full term) infants at different time points. The study encompasses
both culture dependent and culture independent techniques for
investigating this developing microbial profile. This will provide infant
milk formula manufacturers with a baseline composition, with which to
compare different formulations and ingredients. The project will provide
new opportunities for optimisation of infant milk formula composition,
with appropriate new bioactive ingredients such as milk fractions,
probiotics and prebiotics to effectively influence the early infant gut
microbiota to resemble a breast fed infant.
GM/19
Antibacterial activity of some medicinal herb and spice extracts
Hoda A El Shamy1, Mona H Hashish1, Amani F Abaza1,
Neamat H Dorra2
1
High Institute of Public Health-Alexandria University, Alexandria, Egypt,
2
Faculty of Pharmacy-Alexandria University, Alexandria, Egypt
Consumers are concerned with the safety of foods containing synthetic
preservatives. Therefore, there has been increasing interest in using
natural antibacterial compounds for food preservation. This study aimed
at testing a variety of naturally occurring, medicinal and potentially foodcompatible herb and spice extracts for their antimicrobial potential
against a group of food borne bacterial pathogens. A total of 5 herbs
and spices collected from different markets in Alexandria namely: garlic,
thyme, cinnamon, marjoram and clove were tested on four types of food
borne bacterial isolates representing Gram positive and Gram negative
bacteria obtained from food samples referred to the Central Lab of
High Institute of Public Health for investigation by broth dilution method
for determination of Minimum Inhibitory Concentration, disc diffusion
method and synergy assay using checkerboard method. All the selected
aqueous plant extracts exhibited antibacterial activities against all tested
organisms with varying degrees. Garlic extract showed the maximum
activity with MIC values ranging from 18.75 to 37.5 mg/ml, also it caused
inhibition of all tested bacterial isolates using the disc diffusion method.
Staphylococcus aureus and Shigella spp. were the most susceptible to
crude aqueous extracts, while Salmonella was the most resistant. Almost
all garlic combinations showed limited synergistic capacity.
GM/20
A rapid and real time microbiological testing method: the development
and validation of five-lux-based Esherichia coli ATCC 8739
Melissa. Y Y Choong, David. C Naseby, Timothy Aldsworth
University of Hertfordshire, Hatfield, UK
A plasmid borne transcriptional fusion between five Escherichia coli
ATCC 8739 constitutive promoters and Photorhabdus luminescence
lux operon was used as a Rapid and Real Time Microbiological Testing
Method. Five lux based biosensors were validated in accordance to the
European pharmacopeia guidelines, demonstrating accuracy and precision,
linearity, equivalence, and the range of detection between bioluminescent
method and compedial plate count method. The bioluminescent
method was compared with three bacterial quantitative methodologies;
Epifluoroscence microscopy, ATP enzymatic measurements and viability
fluorescence staining. The bioluminescent method showed a minimum
limit of detection of 3.16-4.46 x 103cfu/ml with 6 orders of magnitude
detection range, supported with strong positive correlations compedial
enumeration method (R2: 0.999-1.000, P≤0.05), ATP measurements (R2:
0.996-1.000, P≤0.05), and viability fluorescence staining (R2: 0.961-0.985,
P≤0.05). Statistical F values obtained indicated there is no significance
difference (P≤0.05) exists between bioluminescence method and
the compedial plate counting method. This bioluminescent method
has significant potential and is applicable for future testing involving
of minimum inhibitory concentration (MIC), minimum bactericidal
concentration (MBC) and preservative efficacy testing (PET). The
application of the bioluminescence method could be a good alternative
for detection enumeration, instead of the laborious and time consuming
conventional method.
GM/21
Genetic adaptation of Escherichia coli to a non-host niche: an
investigation into fitness trade-offs between stress protection and
growth
Yinka Somorin, Florence Abram, Conor O’Byrne
Microbiology, National University of Ireland, Galway, Ireland
Escherichia coli is widely used as an indicator of faecal contamination in
the environment based, amongst other factors, on the assumption that it
only survives transiently outside of the host gastrointestinal tract. However,
recent studies suggest that E. coli can persist and grow in the external
environment, a conclusion that questions its continued use as indicator
organism. Soil persistent E. coli strains have shown genetic diversity and
possess unique growth and metabolic characteristics, suggesting adaptation
to conditions present in soils. This project aims to determine whether
the general stress response controlled by alternative sigma factor rpoS
(σs) remains intact and functional in soil-persistent strains or it has been
inactivated during the trade-off of survival for growth arising from adaptation
to soils. Preliminary results show presence of rpoS in 5 genetically distinct
soil persistent isolates and the rpoS loci are currently being sequenced.
GABA assay have shown that the GAD system in E. coli is under the
influence of rpoS and this assay will be applied to determine the response
of soil isolates to acid stress. Several RpoS-dependent phenotypes will
be investigated so as to better understand the role, if any, rpoS plays in
adaptation of E. coli to soil environments.
GM/22
Chemotaxis signal transduction in Campylobacter jejuni
Paul Ainsworth, Julian Ketley
University of Leicester, Leicester, UK
Motility and chemotactic movement have been shown to be important
elements in Campylobacter infection, as cells lacking either functioning
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Poster abstracts
flagella or key chemotaxis proteins fail to colonise human or chicken
hosts. C. jejuni chemotactic molecules are detected by surface receptors
(Tlps), this signal is then transduced by a two component system (CheACheY), into modulation of the flagella’s rotation, resulting in directed cell
locomotion. The chemotaxis signal transduction system is well studied
in a number of model prokaryotes, but the accessory proteins present
within the C. jejuni system are different, indicating novel features and
operation. Our work involves assaying in vitro interactions of expressed
and purified constituent proteins, in combination with previous incell studies, to build a more complete picture of C.jejuni chemotaxis.
We found the C. jejuni homologues do interact to form a chemotaxis
signal transduction system, that the predicted response regulator
domains of CheY, CheV and CheA are phosphorylated by CheA, and
preferentially in that order. CheA response regulator phosphorylates
and autodephosphorylates slowly in relation to CheY and CheV. The
phosphorylation state of CheV alters its affinity for Tlp cytoplasmic
domains, with possible implications for Tlp raft formation and control
over effective concentration of adaption proteins at those rafts.
GM/23
Insights into transport and assembly module (TAM) complex
formation and autotransporter biogenesis in proteobacteria
Inokentijs Josts1, Justyna Wojdyla2, Colin Kleanthous2,
Olwyn Byron1, Daniel Walker1
1
University of Glasgow, Glasgow, UK, 2University of Oxford, Oxford, UK
Bacterial proteins destined to function extracellularly must efficiently
cross two layers of membrane covering the cells. To do so, numerous
transporter systems are employed by specific protein types to ensure
the delivery of the proteins to their final destination in their fully
functional state. Autotransporters are outer membrane-bound proteins
that act in the extracellular milleau of the cells. Recent evidence indicates
that autotransporters utilise at least two bacterial transporter systems
to make their way to the outside of the cell. Transport and assembly
module (TAM) complex is one such transport system, involved in the
transport of the passenger domain of autotransporters across the outer
membrane. By utilising several biochemical and biophysical techniques
we investigate TAM complex formation, as well as try to delineate its
mode of substrate recognition and specificity.
GM/24
Antibiotic susceptibility profile of Staphylococcus species isolated from
a full-scale municipal wastewater treatment plant in Dubai
Munawwar A Khan, Javeria Mohsin
Department of Natural Science and Public Health, College of Sustainability
Sciences & Humanities, Zayed University, Dubai, United Arab Emirates
This study evaluated the prevalence of antibiotic-resistant Staphylococcus
species isolated from a full scale municipal wastewater treatment plant
in Dubai. Antibiotic susceptibility profile was determined using the
disc diffusion method, and polymerase chain reaction (PCR) assay was
employed for the detection of mec-A for methicillin resistance gene
& NUC gene for the confirmation of Staphylococcus aureus species.
A total of 57 Staphylococcus species were isolated from wastewater
treatment plant and tested for antibiotic sensitivity against ten antibiotics.
All 57 isolates were found to have NUC gene of size 270 bp which
confirmed Staphylococcus aureus species. Out of these 57 isolates 26
were found positive for the presence of 310 bp mecA gene. Almost half
the isolates (46%) were found resistant to oxacillin, 35% to methicillin,
and 12% to vancomycin while about 23% of the isolates were resistant
to streptomycin, gentamycin and other antibiotics. Out of the 57 isolates
analysed 36 were found resistant to three or more antibiotics, which is
63% of the total isolates. In conclusion, a high incidence of multiple drug
resistance observed in Staphylococcus aureus isolates from wastewater
environment suggesting wastewater treatment plant an important source
of multidrug resistant bacteria in the UAE.
GM/25
Evaluation of P. aeruginosa quorum sensing diversity within wound
isolates
Josie V Mckeown1, Matthew P Fletcher1, John English2,
Jonathan Nosworthy3, Paul Williams1, Miguel Camara1
1
University of Nottingham, Nottingham, UK, 2Nottingham University Hospital,
Nottingham, UK, 3Advanced Medical Solutions Ltd., Cheshire, UK
Quorum sensing (QS)-mediated gene expression plays an important role
in the outcome of wound infections caused by Pseudomonas aeruginosa.
QS controls virulence factor production via two signalling systems-Nacylhomoserine lactones (AHLs) and alkyl-quinolones (AQs). The aim
of this study was to investigate the correlation between the production
of QS signal molecules (QSMMs) in P. aeruginosa wound isolates, the
production of virulence determinants and antibiotic resistance. Fifty-six P.
aeruginosa wound isolates were characterised and whilst all were found
to attach strongly to surfaces, elastase and protease activity, pyocyanin
production and swarming ability differed. Some of the isolates studied
were unable to produce at least one of the main AHLs and others the
AQ known as the Pseudomonas quinolone signal (PQS). This is the
first time we have encountered PQS negative wound isolates from
this organism. Isolates unable to produce these QSSMs were found
to produce much lower levels of virulence determinants. Studies have
found that inactivation of the QS systems in P. aeruginosa lab strains
results in an increased susceptibility of this organism to antibiotics. We
will present the relationship between the production of QSSMs and
the susceptibility of the P. aeruginosa wound isolates from this study to
antibiotics.
GM/26
Identification of Campylobacter jejuni heparin-binding proteins
Deborah R Cantu, Dennis Linton
The bacterium Campylobacter jejuni is the one of the most important
causes of bacterial food-borne disease worldwide. Although C. jejuni
is generally found as part of the normal flora of many animals, it is
pathogenic to humans. Infection with C. jejuni results in self-limiting
diarrheal illness however, complications resulting in neurological
autoimmune disorders can occur. Despite being a highly prevalent
human pathogen many aspects regarding the pathogenic process are still
poorly understood. One aspect yet to be investigated is the ability of C.
jejuni to adhere to glycosaminoglycans (GAGs). GAGs are carbohydrates
expressed on the host cell surface that can serve as receptors for
bacterial proteins. We have identified several cell surface proteins of C.
jejuni that bind to the GAG, heparin, in vitro. For one of these proteins,
PEB3, the location of the heparin-binding region was investigated by
site-directed mutagenesis and two clusters of positively charged residues
were shown to be involved. This data suggests that GAG-protein binding
may play a role in the pathogenesis of C. jejuni.
GM/27
ScdA is responsible for the biosynthesis of cis-2-decenoic acid in
Pseudomonas aeruginosa
Ye Chen, Stephan Heeb, Paul Williams, Miguel Cámara
P. aeruginosa is a ubiquitous Gram-negative bacterium responsible for
a high frequency of nosocomial opportunist infections in humans. In P.
aeruginosa, quorum sensing signals are dominantly utilised to modulate
bacterial behaviors and interspecies communications. Recently, a novel
signal was discovered in P. aeruginosa, characterised as cis-2-decenoic
acid (CDA). CDA was reported to disperse the mature biofilm and
inhibit the biofilm formation from a range of Gram-negative and
positive bacteria. In a search for genes encoding CDA, we identified
scdA responsible for the synthesis of cis-2-decenoic acid in P. aeruginosa.
Mutation of scdA resulted in the attenuated CDA production, as well as
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Poster abstracts
the inhibited swarming and the subsequent enhanced biofilm formation
in P. aeruginosa. The contrary phenotypes could be restored by scdA
complementation. In addition, the data suggested that scdA was not
the only gene synthesising CDA, since the production of this signal
was compensated through time course in scdA mutant. However, the
identities of the other genes remained unclear yet. In conclusion, we
have discovered scdA as the biosynthetic gene of CDA in P. aeruginosa
and confirmed its function by phenotypic validation.
GM/28
High-affinity substrate binding by ScpA
MAlgorzata Tecza, Maurice R O’Connell, Jakki C Cooney,
Todd F Kagawa
ScpA, a multi-domain cell envelope protease produced by Streptococcus
pyogenes, is a virulence factor involved in the evasion of the host
immune response that contributes to the persistence of the bacteria in
the host. C5a is the only known substrate of ScpA and little is known
on how the substrate specificity is achieved. Recently proposed model
of the interactions between the ScpA and human C5a predicted large
contribution from exo-site type interactions with C5acore and salt bridges
with Arg74 (Kagawa et al., 2009). Surface plasmon resonance was used
to examine the importance of the C5acore (1-67 residues), C5atail (6874 residues), Arg74 and long-range interactions in the hC5a binding.
ScpA binds hC5a with high affinity (KD=34nM) with minor contribution
of electrostatic steering (19% of ΔGbind). The high affinity binding is
achieved mainly due to interactions with C5acore (89% of ΔGbind). The
Arg74 provides 90% of the binding free energy contributed by all 7 ‘tail’
residues. The results confirmed the computed ScpA-hC5a complex
model. Additionally, it was shown that ScpA bound mouse C5a with
reduced affinity (KD=1.62μM) and was incapable of cleaving the protein.
Reference KAGAWA, TF., O’CONNELL, MR., MOUAT, P., PAOLI, M.,
O’TOOLE, PW, COONEY, JC. 2009. J Mol Biol, 386, 754-72.
GM/29
Acinetobacter: a common guest in ICU, Alexandria, Egypt
Walaa A Hazzah1
1
High Institute of Public Health-Alexandria University, Alexandria, Egypt,
2
Beirut Arab University, Tripoli, Lebanon
The emergence of multi-drug resistant (MDR) Acinetobacter spp. among
intensive care unit (ICU) patients is increasingly recognised as a public health
threat worldwide. Moreover, prevelance rates of carbapenem resistance,
mediated by production of Metallo-Beta-Lactamases (MBL), is an arising
issue in many countries. This work aimed to study the occurrence of MDRA
among critically ill patients in a health care setting in Alexandria. During a 12
months period, 150 samples (sputum, endotracheal aspirates, blood, urine,
and pus) obtained from ICU patients were cultured on both routine and
selective Chromagar media for isolation of Acinetobacter spp and further
identification for suspected isolates was performed using biochemical tests
and API 20NE. All strains were screened for their antimicrobial susceptibility
patterns. In addition, Polymerase chain reaction (PCR) assay was performed
to detect VIM and IMP MBL genes. Acinetobacter was the most frequently
encountered pathogen in respiratory tract and urinary tract infections. Of
the 150 samples, 50 Acinetobacter strains were isolated, 90% were found
to be MDR, and 80% were ESBL. PCR assay revealed that 45% strains
possessed VIM gene and none for IMP gene. MDR Acinetobacter strains
among critically ill patients in this study was an evident problem that should
be tackled.
GM/30
Interaction of Escherichia coli α-macroglobulin (ECAM) with the
penicillin binding protein PbpC
Cameron D Fyfe, Richard Burchmore, Daniel Walker
Escherichia coli α-macroglobulin (ECAM) has been shown to have similar
function to eukaryotic α-macroglobulins which are able to cross-link
to and inactivate proteases. The gene encoding the penicillin binding
protein, PbpC, is often found within the same operon as the ECAM
gene and both proteins are found within the periplasm suggesting they
may form a complex. PbpC is a protein which has been suggested
to have peptidoglycan repair function. Consequently, it has been
hypothesised that ECAM and PbpC work together in defence and
repair against potentially harmful proteolytic activity. Using isothermal
titration calorimetry and size exclusion chromatography we have
shown an interaction between ECAM and PbpC providing further
evidence towards their suggested defence and repair mechanism. We
plan to further investigate their interaction using crosslinking and mass
spectrometry and define the physiological roles of ECAM and PbpC.
GM/31
Development of a robust plate assay for screening and subsequent
characterisation of two metagenome-derived metalloproteases
isolated from a milk waste treatment plant
L S Morris1, J R Marchesi1, C E Codling2, D Jones1
1
Cardiff University, Cardiff, UK, 2University College Cork, Cork, Ireland
Metagenomics is a powerful tool for unearthing novel microbial functions.
A lactose and fat free milk agar was developed to assess protease activity
in pure cultures and metagenomic libraries. The assay was used to assess
the activity of two lactic acid bacteria and two protease sequences
previously isolated from a metagenomic library. Streptococcus thermophilis,
Lactobacillus bulgaricus strains and false protease positives from a
previously constructed metagenomic library were grown on standard
skimmed milk agar and Valio™ lactose free and fat free milk powder
to screen for protease activity. This lactose free agar was a much more
robust media for protease screening and was implemented to screen a
fosmid metagenomic library. Two putative protease sequences (M1-1
and M1-2) were deduced following bioinformatic analysis of a fosmid
clone that could degrade milk agar.
Both sequences were classified as zinc-metalloproteases. M1-1 showed
homology to the aminopeptidase-like family and M1-2 shared homology
with the thermolysin-like family. Only M1-2 showed a proteolytic
phenotype. The optimum temperature and pH of M1-2 was 42°C and
8.0 respectively. Activity was inhibited by EDTA. One protease has been
successfully isolated and characterised. However, the lack of phenotype of
M1-1 indicates that current metagenomic techniques need to be improved.
GM/32
Culture-independent analysis of the colonic bacteriota during a longterm probiotic feeding study
James M Evans1, Sue Plummer2, Iveta Garaiova1,
Eshwar Mahenthiralingam1, Julian R Marchesi1
1
Cardiff University, Cardiff, UK, 2Cultech. LTD, Port Talbot, UK
Background It is becoming increasingly evident that the gut bacteriota
extend an influence on the host far beyond their niche; with the
potential to affect host health. It therefore follows that modulation of
the host bacteriota could promote an individual’s health. Modulation
of the host bacteriota in many studies has been achieved through
dietary supplementation with probiotics, but for only short periods, e.g.
weeks. Here we report the results of culture independent methods
for detecting changes in colonic bacteriota during a 6 month probiotic
feeding study.
Methods A cohort of 18 healthy males were fed a probiotic mixture (2
strains of Lactobacillus acidophilus, Bifidobacterium lactis and B. bifidum
at 109 cfu per capsule) for 6 months and stool samples taken before,
during and after feeding. DNA was extracted from all samples and
subjected to LHPCR analysis to generate individual bacteriota fingerprints
and metabonomic analysis.
Results Gross LHPCR and metabonomic profiles were not affected by
probiotic supplementation.
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Poster abstracts
Conclusions Although gross LHPCR and metabonomic profiles
were not affected by probiotic supplementation, further analysis will
need to be carried out in order to determine the effect of probiotic
supplementation on an individual, due to potential effect masking by
whole cohort analysis.
GM/33
Draft genome sequence of the causative agent of farmer’s lung disease,
Saccharopolyspora rectivirgula
Florence E Pethick1,2, Alexandra Ntemiri1, Vartul Sangal1,
Paul A Hoskisson1, Iain S Hunter1, Paul R Herrron1
1
University of Strathclyde, Glasgow, UK, 2Moredun Research Institute,
Penicuik, Midlothian, UK
Farmer’s Lung Disease (FLD) is a hypersensitivity pneumonitis caused by
antigens present in organic dust. One of the main organisms associated
with FLD, Saccharopolyspora rectivirgula, is an actinomycete of the
Pseudonocardiaceae family. As such this organism has a type IV cell
wall, produces chains of spores in both aerial and substrate mycelium
and can grow on a variety of carbon sources at an optimal temperature
of 55°C. As a first step in the identification of the FLD-causing antigen
we have sequenced the genome of this organism. Surprisingly, the
genome sequencing revealed a large difference in genome size with
previously sequenced members of this genus (Saccharopolyspora
eryhrea and Saccharopolyspora spinosa), which might be reflective of the
thermophilic habitat of S. rectivirgula.
GM/34
Genomic, metagenomic and functional characterisation of a novel
RelBE type toxin–antitoxin addiction module located within the
human gut microbiome
Benjamin McCutcheon, Wendy McFarlane, Lesley Ogilvie,
Brian Jones
University of Brighton, Brighton, East Sussex, UK
Toxin-antitoxin addiction modules (TAMs) are ubiquitous in prokaryotes
and consist of a proteinaceous toxin and its cognate antitoxin. Several
of their functions have been elucidated, such as plasmid stabilisation,
however they are also hypothesised to be involved in numerous
other functions. We recently identified a RelBE type TAM (p22-TAM)
potentially enriched in the human gut microbiome. To understand
the distribution, abundance and function of p22-TAM, we conducted
comparative metagenomic and genomic analysis using 1911 prokaryotic
chromosomes, 1191 plasmids, 710 phage, and 162 metagenomes
encompassing 7 distinct habitats including the human gut as well
as marine and terrestrial environments. This analysis revealed p22TAM homologs were enriched within the human gut, being carried
predominantly on plasmids but generally absent from phage genomes.
COG analysis of the gene neighbourhoods surrounding p22-TAMs
homologues in gut metagenomic sequences revealed a potential
enrichment of functions associated with DNA replication and repair, but
an inverse correlation with other metabolic functions common in the gut
microbiome, such as carbohydrate metabolism. Subsequent experiments
aimed at understanding the function of p22-TAMs in gut microbes
indicated a potential role in bacterial stress response, and suggested
these modules may help gut bacteria cope with environmental stresses
encountered in the human intestinal tract.
GM/35
Bacteroides phage eco-location
Lesley A Ogilvie, Lucas Bowler, Jonathan Caplin, Cinzia Dedi,
David Diston, Elizabeth Cheek, Huw Taylor, James Ebdon,
Brian V Jones
University of Brighton, Brighton, UK
High-throughput sequencing studies are revealing that bacteriophage
(phage) encode a vast and novel functional repertoire, including functions
key for ecosystem functioning. Despite these leaps in knowledge,
information regarding the ecological relevance of phage infecting
prominent gut bacterial species is still lacking. Our previous work
reported the genetic characterisation and ecological profiling of B124-14,
a phage infecting a restricted set of Bacteroides fragilis strains, showing
it and related phage occupy a distinct and as yet uncharted ecological
landscape within the human gut. Here we broaden these analyses and
further explore the ecological niche of this Bacteroides phage within
an extended set of microbial metagenomes encompassing 11 distinct
ecosystems, to provide insight into the prevalence and distribution of
B124-14-like phage and their encoded functions. These analyses revealed
that although B124-14 encodes a number of cosmopolitan genes, key
for functioning within a number of ecosystems, it carries a functional
complement signature of the human gut environment, albeit with high
inter-individual variation. Overall, these data further define the ecological
niche of B124-14 and its evolutionary trajectory, and provide functional
insight into the role of phage infecting a prominent member of the
human gut microbiome with significant medical impact.
GM/36
Ion torrent next-generation sequencing technology: the revolution of
the revolution
Peter Holden, Andy Felton, Manfred Lee
Life Technologies, EMEA, Glasgow, UK
Ion torrent is a revolutionary way to do Next Generation Sequencing,
characterised by a number of features and benefits which do not belong
to classical, light based, detection methods.
The fundamental principle of Ion torrent detection method is to use
the signal of protons generated by the reaction, differently from all
other molecular biology techniques which use photons. This was
made possible by the introduction, for the first time in history, of
semiconductors technology into molecular biology, as a substitute for
traditional optical detection systems. Among the practical benefits
associated to this innovation we can list the simplification of the whole
process, the scalability of the microchips based system, the speed of real
time detection, the great reduction of costs.
Ion Torrent technology is today still in its second year of existence
and has already proven to be effective in multiple applications, from
microbiology to transcriptomics, from targeted resequencing of gene
panels to de Novo sequencing of whole genomes. With the introduction
of 100-fold higher production level chips on the Proton system,
the number of available applications will grow to include ultra high
throughput sequencing of whole exomes, whole transcriptomes, ChipSeq and, finally, whole genomes.
GM/37
A study of the potential of diatomaceous earth to reduce
Campylobacter spp. infections in poultry
Szymon T Calus1, David K Leemans1, Adrian S Horton1,
Helen Edwards2, Michael R F Lee1, Justin A Pachebat1
1
Aberystwyth University, Aberystwyth, Ceredigion, UK, 2Natural Feeds and
Fertilisers, Aberystwyth, Ceredigion, UK
Pathogenic microorganisms such as Campylobacter spp. are one of the
most common causes of food borne infections in developed countries.
The food industry is searching for alternative methods to suppress
bacterial growth in order to improve meat quality without the use of
medicinal antibiotics. In this study we present data from trials to evaluate
the action of Diatomaceous Earth (DE), as a natural antibacterial
product, on Campylobacter. DE is finely ground diatomite rock. In vitro
studies were performed to find the most efficacious dose of DE to
inhibit Campylobacter growth. Results suggest that at low concentrations
DE has an impact inhibiting growth of Campylobacter spp. However,
high concentrations of DE seem to have a protective effect for the
bacteria. Wang et al suggest that DE has a strong capacity to absorb to
the bacterial cell surface, causing dehydration of the microorganism, but
117
Poster abstracts
at high concentrations they showed that DE provides a protective effect
by forming a microenvironment around the bacteria (Wang, 2012).
We discuss the effects of DE on the inhibition of Campylobacter and
potential effects DE on the chicken microbiome.
Reference J.Y.Wang, N. D. B., W. Verstraete (April 2012). Journal of
Industrial Microbiology Biotechnology 39(4): 567-577.
GM/38
Comparison of different sets of primers for the detection of
differentiated Escherichia coli by rep-PCR genomic fingerprinting
methods
Joao Fernandes1,2, Michael Sweet2, Ian Singleton2
1
Laboratorio de Analises of Instituto Superior Tecnico, Lisbon, Portugal,
2
School of Biology, Newcastle University, Newcastle Upon Tyne, UK
The aim of this work was to isolate and differentiate a wide variety
of wild E. coli strains from different water samples (raw and treated
wastewater and seawater from different geographical regions in Portugal)
and to evaluate the performance and specificity of existing E. coli primers
amplifying different E. coli genes available in the scientific literature by
PCR. Primer specificity was tested with bacterial species other than E.
coli. Identification of E. coli was carried out by 16S rRNA sequencing
and biochemical methods. E. coli was identified and confirmed by 16S
rRNA sequencing and biochemical methods. Two genomic fingerprinting
methods (Rep-PCR and BOX-AIR) were used for E. coli differentiation,
which revealed until 75% of genome differences from the 32 E. coli
isolates tested. All primer sets tested (targeting uidA, 16S rRNA, tuf and
gadA genes) showed good specificity for all isolates tested and did not
give PCR products with bacterial isolates other than E. coli. The two
genomic fingerprinting methods also indicated to be a good tool for
bacterial differentiation. The results indicate the potential for developing
a rapid molecular based method to detect and quantify E. coli strains
present in a wide variety of water samples.
GM/39
Microbiologically influenced corrosion of stainless steel 304 (SS304)
in the presence of Bacillus subtilis and Pseudomonas aeruginosa
Hafiz Z Wadood1, Aruliah Rajasekar2, Yen P Ting2,
Anjum N Sabri1
1
University of the Punjab, Lahore, Pakistan, 2National University of Singapore,
Singapore, Singapore
The microbial production of extracellular polymeric substances (EPS)
forms a biofilm on metal surface that may either inhibit or accelerate
corrosion of underlying metal surface. In the present study, the
biocorrosion behavior of stainless steel 304 (SS304) in the presence of
aerobic bacteria Bacillus subtilis strain S1X and Pseudomonas aeruginosa
strain ZK was investigated in minimal salt medium with 1.5% sodium
chloride as electrolyte. Biocorrosion was evaluated using Tafel polarisation,
electrochemical impedance spectroscopy (EIS), scanning electron
microscopy—energy dispersive spectrum analysis (SEM-EDAX), atomic
force microscopy (AFM) and Fourier transform infrared spectroscopy
(FTIR). The corrosion rate and corrosion current were lower in
the presence of both bacteria when compared to control medium.
Electrochemical data showed that both bacteria inhibit corrosion of
SS304 through the formation of a passive layer on metal surface, scanning
electron microscopy—energy dispersive spectrum analysis (SEM-EDAX)
also supported this observation. Atomic force microscopy (AFM) and
Fourier transform infrared spectroscopy (FTIR) showed the formation of
thick biofilm on SS304 surface. pH values of bacterial inoculated systems
decreased with increasing incubation time which showed the production
of some acidic metabolic products by bacteria.
GM/40
Identification of bacterial transporters for hydroxyalkylcysteinylglycines: a novel target for reducing axillary malodour
Daniel Bawdon1, Diana Cox1, Gordon James2, Gavin Thomas2
1
Department of Biology, University of York, Heslington, York, YO10 5DD,
UK, 2Unilever Discover Colworth, Colworth Science Park, Sharnbrook, Bedford,
MK44 1LQ, UK
The production of malodour on various sites of the human body
is caused by the microbial biotransformation of odourless natural
secretions into volatile odorous molecules. Distinctive odours emanate,
in particular, from the underarm (axilla), where a large and permanent
population of microorganisms thrives on secretions from the eccrine,
apocrine and sebaceous glands. Dipeptide-conjugated thioalcohols
form an important group of compounds secreted by the axilla with a
known role in malodour when metabolised to free thioalcohols. In this
study, we assess the molecular basis for uptake of these compounds by
the model organism Escherichia coli K-12 using a resting cell thioalcohol
detection assay. We identify that two poorly characterised secondary
peptide transporters, DtpB and DtpD, are responsible for uptake of a
model malodour precursor, S-benzyl-L-cysteinylglycine, but not more
physiologically relevant substrates, including S-[1-(2-hydroxyethyl)-1methylbutyl]-(L)-cysteinylglycine (Cys-gly-3M3SH), which passively
diffuses across the E. coli inner membrane. We also present thioalcohol
generation data from a library of axillary isolated corynebacteria and
staphylococci and report on significant variations in thioalchol production
across and within Corynebacterium species, which appears to be mediated
by an active transport process, the molecular basis of which is as yet
unidentified.
GM/41
Characterization of secreted sialate-O-acetylesterases in gut
commensal bacteria
Fatima Nadat, Gavin Thomas
University of York, Department of Biology, Heslington, York, YO10 5DD, UK
Sialic acids are widely distributed amongst mammals and are found
typically linked to the distal end of glycan chains in the gastrointestinal
tract. N-acetyl neuraminic acid or Neu5Ac, the most commonly found
sialic acid, is often found with an additional O-linked acetyl group when
located on gut epithelial cell surfaces. In E. coli the enzyme, NanS,
functions as a sialate O-acetylesterase to remove the additional acetyl
groups. Although NanS has a cytoplasmic location in E. coli, human
faecal extracts have been found to contain extracellular sialate-Oacetylesterase activity, which is thought to correlate to the presence
of bacteria from the Bacteroides genes. The molecular characterisation
of a sialate O-acetylesterase is of particular interest as changes in levels
of gut epithelial acetylated sialic acids are associated with GI cancers.
We have established the presence of potential sialate O-acetylesterase
across a range of Bacteroides species. Preliminary expression experiments
show that the proteins are highly insoluble. We are now using vectors
engineered at the university of York technology facility to create fusion
proteins which increase solubility.
Systems and cells
SC
SC/01
From conflict to mutualism in polymicrobial communities
Sylvie Estrela1,2, Christopher H Trisos3, Sam P Brown1,2
1
Institute of Evolutionary Biology, The University of Edinburgh, Edinburgh, UK,
2
Centre for Immunity, Infection and Evolution, The University of Edinburgh,
Edinburgh, UK, 3Department of Zoology, University of Oxford, Oxford, UK
118
Microbial cells excrete metabolites as a result of their metabolism
and this sets the stage for the emergence of complex interspecific
interactions observed in polymicrobial communities. Whenever one
organism uses metabolites produced by another organism as energy or
nutrient sources, this is called cross-feeding. The ecological outcomes of
cross-feeding interactions are poorly understood and potentially diverse,
spanning competition, mutualism, exploitation, or commensalism. To
address this issue, we develop a theoretical model that links microbial
metabolism to microbial ecology and explores the dynamics of a oneway interspecific cross-feeding interaction in which food can be traded
for a service (detoxification). Our results show that diverse ecological
interactions can emerge from this simple cross-feeding interaction and
can be predicted by the metabolic and environmental parameters that
govern the balance of the costs and benefits of association. In particular,
our model predicts stronger mutualism for intermediate by-product
toxicity because the resource-service exchange is constrained to the
service being neither too vital (high toxicity impairs resource provision)
nor dispensable (low toxicity reduces need for service). These results
support the idea that bridging microbial ecology and metabolism is a
critical step toward a better understanding of the factors governing the
emergence and dynamics of polymicrobial interactions.
SC/02
Gingipains, proteases from Porphyromonas gingivalis induce neutrophil
extracellular traps formation
Danuta T Florczyk1, Izabela Glowczyk1, Jan Potempa1,2,
Joanna Koziel1
1
Department of Microbiology, Faculty of Biochemistry, Biophysics and
Biotechnology, Jagiellonian University, Krakow, Poland, 2University of Louisville
Dental School, Department of Oral Health and Rehabilitation, Louisville, KY,
USA
Neutrophil extracellular traps (NETs) are neutrophil-derived DNAcomposed networks of extracellular fibers covered with antimicrobial
molecules, which are recognised as a physiological microbicidal
mechanism of innate immunity. The formation of NETs is also classified
as a model of a cell death called NETosis. Despite intensive research
on the NETs formation in response to pathogens, the role of specific
bacteria-derived virulence factors in this process, although postulated,
is still poorly understood. Recently it was shown, that a major
periodontopathogen, Porphyromonas gingivalis, is a potent inducer of
the NETs formation. The aim of our study was to determine the role of
gingipains, cysteine proteases responsible for the virulence of P. gingivalis,
on the NETosis process. We showed that NETosis triggered by P.
gingivalis is gingipain dependent since in the stark contrast to the wildtype strain (W83) the gingipain-null strain only slightly induced the NETs
formation. Furthermore, the direct effect of proteases on NETosis was
documented using purified gingipains. Notably, the induction of NETosis
was dependent on the catalytic activity of gingipains, since proteolytically
inactive forms of enzymes did not induce the NETs formation. Taken
together, our data indicate that gingipains are first well-characterised
bacterial virulence factors, which are directly engaged in NETosis.
Poster abstracts
shock response’ in which selective expression of cold shock proteins
(CSPs) aids acclimation. The major cold shock protein, CspA, and
some of its homologues function as RNA chaperones and play critical
roles during cold acclimation by destabilising aberrant RNA secondary
structures that form at reduced temperatures. However, the precise
roles and targets of this protein family remain unclear. To address this,
a genome-wide map of protein-RNA interactions was generated by
performing in vivo UV cross-linking and cDNA analysis (CRAC) using the
cold-induced protein CspA, and constitutively expressed CspE.
CRAC results reveal remarkable diversity in the RNA targets of these
CSP. CspA and CspE target mRNAs encoding proteins involved in
motility, stress, cell division and RNA turnover, as well as a number of
mRNA that are themselves cold shock-inducible. The evidence suggests
that these cold shock proteins are crucial for more processes than their
name implies.
SC/04
Construction of small nitrogen species sensing systems active in both
bacterial and mammalian cells: proof of principle
Richard Bowater, Rachel Dobson, Joy Edwards-Hicks,
Russell Gritton, Lukas Harnisch, Pascoe James Harvey, Richard Kelwick,
Rebecca Lo, Khadija Ouadi
School of Biological Sciences, University of East Anglia, Norwich Research
Park, Norwich, UK
The International Genetically Engineered Machine (iGEM) competition
challenges teams of undergraduates to produce novel systems with
real world applications. The 2012 Norwich Research Park-University of
East Anglia (NRP-UEA) team used modular systems and quantitative
logic to address problems with small nitrogen species specificity and
modularity of existing promoters and detection systems. Modularity is a
central principal of synthetic biology; hybrid promoters with analogous
activity in bacterial and mammalian chassis were created, allowing
functionality in a broad host spectrum. The PYeaR promoter was fused
to the CarG promoter, both of which transcribe in the presence of small
nitrogenous species. The promoters were fused to CFP and RFP which
demonstrated concentration dependent expression in Escherichia coli.
The hybrid promoter, when transfected into MCF7 cells, showed nitric
oxide dependent expression of CFP. Results confirmed the analogous
activity of the promoter. To enhance sensing specificity, quantitative logic
and transcriptional integration through RNA interactions were explored.
This lays the ground work for the creation of the first nitric oxide specific
sensor. Additionally there is potential for use in a broad host range. This
project was awarded a gold medal at the 2012 European championships,
held in Amsterdam.
SC/03
Characterising RNA regulators of the cold shock response
Louise McGibbon, Jai Tree, Sander Granneman, Katy Woodall,
David Gally, Garry Blakely, David Tollervey, Maurice Gallagher
RNA-dependent control of gene expression is crucial for bacterial
adaptation to environmental stresses, such as fluctuations in ambient
temperature. In the enteric pathogen Salmonella Typhimurium, a drastic
downshift in temperature immediately triggers the onset of the ‘cold
119
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Abstracts - Microbiology Society

Transcription

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