The wily ways of a parasite: induction of actin assembly by Listeria

R E V I E W S
29 Buss,J.E. and Sefton,B.M. (1985)J. Virol. 53, 7-12
30 Hancock,J.F. et al. (1989) Cell 57, 1167-1177
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8655-8659
The wily ways of a parasite:
induction of actin assembly
by Listeria
Lewis G. Tilney and Mary S. Tilney
o us it is fascinating to
The intracellular pathogen Lister& has a
and phospholipase C (Ref. 2).
examine the diversity of
spectacular mode of transport within and These enzymes break down
biological organisms on
between host cells. BY inducing host cell
the phagosomal membrane so
the planet and how they funcactin to assemble from its surface, the
that the bacterium can enter
tion. Many of these organisms
bacterium forms a tail composed of many the macrophage cytoplasm,
are pathogenic and these, alshort, crossbridged actin filaments. With
where it grows and divides
though often insidious in their
this tail Listeria is propelled across the
using host cell nutrients. At
behavior towards their hosts,
cytoplasm like a comet streaking across
the same time it assembles
have evolved a variety of
from its surface numerous
the sky. Here we discuss the antics of
'special mechanisms' to enter,
Listeria and some of the bacterial genes
short actin filaments, which
instrumental in maintaining it in the host. form a polar tail up to
feed, replicate and spread that
are entertaining to any biol5 lam long (Fig. 1). Some 2.5 h
L.G. Tilney and M.S. Tilney are in the Dept of
ogist. Listeria is no exception
after infection, Listeria begin
Biology, Leidy Laboratory, University of
and to try to unravel how it
to migrate around the cytoPennsylvania, Philadelphia, PA 19104-6018, USA.
operates is a fantastic trip on
plasm of the cell at speeds
its own.
proportional to the length of
Listeria is a common pathogen throughout the
their actin tails3: up to 1 lim s-1. They always migrate
world, and one that is thought to be carried by many head first, like a comet streaking across the sky.
of us without any obvious effects; but when Listeria
When the Listeria make contact with the plasma
reaches certain levels in the body it can be, and often membrane of the macrophage a protruberance is
is, fatal. Particularly susceptible are pregnant mothers, generated (Fig. 1), which can be up to 40 ~tm long 4.
their foetuses, and immunocompromised individuals.
The membrane is applied tightly around the Listeria
Apart from its importance as a pathogen, Listeria is and its tail, like a finger in a rubber glove. What hapattractive to investigators because it provides a pens next is remarkable: when this long pseudopod
relatively simple system to probe cytoskeletal makes contact with a neighboring macrophage, the
function in actin-based motility. The movement of second macrophage phagocytoses the pseudopod of
the first. Thus within the second macrophage is a
Listeria is, for example, considerably less complex
than the movement of an entire cell such as a phagosome containing the plasma membrane that
neutrophil or tissue culture cell, or even a growth
covered the pseudopod of the first macrophage, withcone, as no membranes are involved. It is, in essence, in which is the Listeria and its tail (Fig. 1). The
a particle that is moving around the cytoplasm.
doubly encapsulated Listeria escapes into the cytoShortly after Listeria makes contact with a plasm by dissolving both membranes, again mediated
macrophage it becomes phagocytosed (Fig. 1). Once
by phospholipases and hemolysin, and the cycle is
inside the phagosome, Listeria secretes hemolysin I repeated.
T
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processing of a bacterial protein delivered into the
cytoplasm.
Fig. 1. The antics of Listeria in macrophages. Modified from Ref. 5.
Once Listeria has entered the cytoplasm of a
macrophage, it can spread from cell to cell without
ever again leaving the cytoplasm and entering an
extracellular compartment. Thus this insidious beast
cleverly avoids any detection by circulating antibodies. Although the reason why circulating antibodies
have no effect was not appreciated until recentlys, the
fact that circulating antibodies are ineffective in listeriosis has been known for many years6. Accordingly,
this pathogen has been studied extensively as an example of a disease combated by cell-mediated mechanisms such as CD8 ÷lymphocytes or cytotoxic T cells
(CTLs). Furthermore, since Listeria spreads from cell
to cell via phagosomes, cytokines such as7 interferon
can slow its spread, presumably by increasing superoxide production in the phagosomes 7. Since cellmediated immunity requires presentation of the major
histocompatibility complex type I (MHC class I) with
a fragment of the bacterial antigen by infected cells
to CD8 ÷ cells, many studies have used Listeria to
investigate CTL killing of infected cells8'9.
An interesting offshoot of the role that Listeria is
playing in revealing for us aspects of cell-mediated
immunity is its potential as a delivery system for
foreign proteins or peptides into the cytoplasm of
macrophages; portions of the foreign proteins could
then be processed and carried to the surface in the
jaws of MHC class I. For example, when a plasmid
containing the gene for 13galactosidase attached to a
listerial promoter, which induces hemolysin and
other molecules (see below), was introduced into
Listeria and the Listeria allowed to infect a mouse,
a CD8 ÷ response was elicited against a portion of
13 galactosidase 1°. Of course Listeria is a potentially
dangerous pathogen, but mutants are now available
that grow and divide normally in cells but are four to
five logs less virulent than wild-type organisms H.
Thus mutant Listeria with the appropriate plasmid
may provide a nontoxic delivery system for vaccinating organisms to respond to future challenge by
bacterial parasites that hide in the cytoplasm of cells.
Listeria, then, is a spectacular model to study the
TRENDS
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26
Cell biological processes
Viruses and/or viral proteins, such as Semliki forest
and vestibular stomatis virus, have provided us with
tools to dissect membrane-associated events such as
endocytosis, exocytosis, secretory pathways and lysosomal activities. Likewise, bacterial pathogens and
mutants of Listeria may be exceedingly useful in the
future in helping us uncover aspects of how the cytoplasm functions. Particularly relevant to this review is
that Listeria is an important model for understanding
actin assembly in vivo and how the filaments thus
formed may induce particle movement in cells. In fact
it has been stated 3 that more is currently known
about the actin antics associated with Listeria than
those of pseudopods or lamellopodia of cells, such as
growth cones, Dictyostelium or neutrophils, that
move over a substrate. There are other cytoplasmic
events that can potentially be 'dissected' using Listeria, including how induction of a protruberance
occurs (see Fig. 1) or how a pseudopod of a cell is
recognized by another cell.
Successful host colonization
Listeria can grow and multiply extracellularly, for
example in milk products and silage, as well as being
an intracellular invader. Thus one might expect that
to change environments and, more particularly, to
become an intracellular pathogen, Listeria would
have to turn on a number of genes to 'take advantage' of the intracellular environment. In essence it
must adapt to the environment within the host by
modifying a number of key processes so that it can
grow, multiply and spread. Thus, it would have to
secrete enzymes to escape from the phagolysosome,
induce polymerization of actin from its surface, which
it presumably does by gluing to its surface host actin
filament nucleators and/or inducers of actin assembly
(see below), induce the formation of a pseudopod,
and put on the surface of the pseudopod something that is particularly tasty to another host cell.
Although we have no idea as to the number of genes
necessary for a Listeria to prosper as an intracellular
pathogen, and it is presumptuous to predict from the
limited knowledge we have, it is theoretically possible
to carry out all of these modifications with less than a
dozen genes. Everything else would be supplied by the
host cell and by the processes going on in the host cell
cytoplasm.
There is a region of the bacterial genome containing eight open reading frames, all of which seem to be
regulated by a product (so far unidentified) of the
so-called prfA gene 12'13.Using deletion mutants, information is available as to the identity and at least partial function of five of these eight genes. These include
many of the 'essential modifying' components just
mentioned. Thus, to escape from the phagosome
Listeria secretes hemolysin1 and phosphatidylinositol
specific phospholipase C (Ref. 2); to induce actin
assembly Listeria uses the actA gene product TM, and
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to escape from the phagosome of the second macrophage a metaloprotease and phospholipase C are
used is. Since four of these five proteins are enzymes,
their characteristics - substrate specificity, optimal
conditions for activity, cofactors and so on - are being
explored. Furthermore, how these five proteins are
regulated is partially understood 16.
Formation of the actln tail
Within 15 min after the Listeria have escaped the
phagosome and entered the cytoplasm, they begin to
be surrounded by a thin cloud of actin filaments. One
hour later many have short tails, by 2 h many more
have short tails and some have longer tails, and by 3 h
after the initial infection some have very long actin
tails and are moving around in the cytoplasm as a prelude to entering a pseudopod and spreading s. From
these static observations it might be predicted that the
length of the tail is proportional to the time the Listeria has spent in the cytoplasm 17. Because filaments
do not assemble on newly formed surfaces such as
those resulting from septation (Fig. 2) and since septa
form near the center of the elongated bacterium, each
ensuing division amplifies the polar distribution of
actin filaments so that after four divisions some Listeria have a tail up to 5 gm long (Fig. 1). Thus we began
to suspect that the older the surface of the Listeria,
the longer its attached tail will be. This prediction
could be tested by examining the length of the tails of
all the Listeria that were present in the cytoplasm of a
macrophage after four divisions. If, indeed, the age of
the bacterial surface is related to tail length, then one
would predict (see Fig. 3) that after four divisions
there would be one with a long tail, one with an intermediate length tail, two with short tails, and four
with no tails, only clouds. This has been experimentally verified 17. These observations, made on fixed
cells in which we examined the net growth of the tail
of Listeria as it resides in the cytoplasm, are not inconsistent with video observations made by ourselves
and others TM. These show that, upon occasion, individual tails can rapidly break down and then re-form,
and that there is a continual flux of actin subunits
into and out of the tail (see below).
The next question we should address is how the
actin filaments rearrange to form a smooth contoured
tail extending from only one end of the Listeria. The
key, of course, comes from observations on the division of Listeria into two daughters, in which each
daughter cell has a newly formed end, bare of actin
filaments (Fig. 2). To form a tail, all that is necessary
is for the existing actin filaments to become interconnected and as progressively more filaments are
formed, a tail will naturally develop simply by the
mechanics of growth. Two lines of evidence indicate
that the actin filaments in the tail are crosslinked. The
first is the observation that Listeria with attached
tails (up to 5 I.tm in length) can be isolated following
detergent extraction19; the component filaments do
not drift away, nor do they become rearranged even if
the tails are left for more than 1 h at room temperature (Fig. 4). This is particularly significant as the
TRENDS
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Flg. 2. Thin sections through Listeria in the cytoplasm. (a) A Listeria with
a short tail. Actin filaments extend from the lateral and basal surfaces of
the bacterium but are conspicuously absent from the anterior pole (arrow).
(b) A Listeria in the final process of division. Whereas actin filaments
extend from all surfaces of this dividing cell, they are sparse in the area
where septation occurs. Notice that the right half of the Listeria has a
thicker actin cloud and a more extreme tail than the left half. (¢) A newly
divided Listed& Where division has just occurred the surfaces of the
Listeria are free of actin filaments. The Listeria on the right has a
much longer tail than the one on the left. Reproduced from Ref. 17, with
permission.
maximum length of the individual filaments making
up the tail does not exceed 0.3 lam. Second, using
immunofluorescent techniques, the tails are seen to
contain the crosslinking protein ot actinin 4. Further
details on the shape and consistency of the tail are
presented by Tilney et al.19, including the fact that the
tail appears 'hollow' in section with a higher density
of filaments at the periphery and few in the center
(this again just reflects the mechanics of growth).
Polymerization of actin from the bacterial surface
Before describing details of the polymerization of
actin induced by Listeria, we should mention three
facts about the assembly of actin in vitro. First, actin
filament assembly occurs in two stages: in stage I it is
thought that three monomers come together 2° to
form a trimer, in a process called nucleation; in stage
2, monomers add to the ends of the trimer or nucleus
and the filament elongates. Nucleation in vitro is a
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newly assembled filaments were unidirectionally polarized with their
barbed ends associated with the surface
of the bacterium (Fig. 5). Furthermore,
since the critical concentrations for the
0:50
polymerization of actin are different for
the two ends, we used actin concen.0
trations below the critical level for the
J:40
pointed end to show that the filament
elongation must be occurring by
( ~ ~ ~ :
( .... ~ ,---monomers adding to the barbed ends,
which are the ends associated with the
z:30
surface of Listeria. Putting all this
t o g e t h e r w e 23 concluded that Listeria
induces the assembly of actin from its
3:z0
surface by secreting a nucleator.
This conclusion is probably incorrect
(
~,
4
;
for two reasons. First, mutants of LisCQ (z3
teria that lack the actA gene are unable
to assemble actin filaments on their surFig. 3. The lineage of a Listeria that had entered the cytoplasm of a macrophage. Since the
Listeria divides every 50 min, by 4 h it should have divided three times (which includes 45
faces in vivo TM, but, curiously, mutants
min for the Listeria to escape from the phagosome and enter the cytoplasm). After four
that express increased amounts of actA
divisions there should be 16 Listeria, two with a long tail, two with a shorter tail, four with
on their surfaces do not nucleate
a tiny tail, and eight with no tail. Reproduced from Ref. 17, with permission.
increased numbers of actin filaments
from their surfaces in vitro. Second, and
chancy event and thus rate limiting; in contrast, once more telling, are the observations of Heuser and
nucleated, elongation occurs rapidly. Thus, in cells Morisaki 24. They showed that by treating macrothere are likely to be proteins that nucleate actin as- phages with a combination of lanthanum and zinc,
endosomes can be induced to move around the cell
sembly. Second, actin filaments
are polar, which can be easily with an actin tail similar to that attached to Listeria.
seen in electron micrographs by Thus the formation of an actin tail does not require a
examining filaments that have contribution from Listeria, but is a characteristic of
the host cell that can be induced by bizarre conditions
been decorated with subfragment
or infection by Listeria.
1 of myosin. What one sees is an
What then does actA do, and how does Listeria
'arrowhead' configuration with
induce actin filament assembly from its surface? The
'barbed' and 'pointed' ends to
easiest explanation, and one that is consistent with
the filaments. Third, the minithe remarkable behavior of endosomes, is that actA
mum concentration of monomers
(critical concentration) that will is a 'glue' that attracts to it host cell molecules that
permit elongation is different for stimulate and/or nucleate actin assembly. The surface
the two ends, being 0.1 IIM for of endosomes, when the host cell is treated with
lanthanum and zinc, would seem to attract the same
the barbed or high-affinity end,
molecules in a similar way. The nature of these host
and 0.7-0.9 }IM for the pointed
end 2°-22.
molecules is under intense scrutiny now in several
labs. One possibility is that, because of proline-rich
We have demonstrated that
something attached to the surface repeats in its primary structure TM,actA is binding a n
actin-binding protein. Similar polyproline repeats are
of Listeria induces actin filament
assembly23. In our experiment,
found in vinculin, another actin-associated protein TM.
Using this information, Theriot and Mitchison 2s sugmacrophages were infected with
gested that actA may bind profilin, and by immunoListeria and then cytochalasin
was added to inhibit actin as- fluorescence they showed that this is indeed the case.
sembly. Three hours later the From these observations they suggested that profilin
Fig. 4. Thin section through
a macrophage that has been
may facilitate actin assembly at the surface of Listeria
macrophages were extracted with
extracted with the detergent
by increasing nucleotide exchange and thus the local
detergent and exogenous G actin
Triton X-IO0. The filaments
concentration of polymerizable actin or actin bound
was added to the preparation
making up the tail of the
to ATP. Unfortunately this hypothesis cannot acunder polymerizing conditions.
Listeria are stable for many
What happened was that each c o u n t for the observations mentioned earlier which
hours after detergent extraction and they remain
demonstrate that actin assembly in vitro takes p l a c e
Listeria resembled a tiny star
connected (crossbridged) towith actin filaments extending
on the surface of Listeria, because the actin used in
gether. Bar represents 1 ~tm.
from
its
surface
23.
Subsequent
those
experiments in vitro was ATP actin 23.
Reproduced from Ref. 17,
When we first examined the actin filaments in the
analysis using subfragment 1 of
with permission.
• 19
tail of Listeria, we were struck by the fact that the iliamyosin
demonstrated that the
©
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,.Z'.-'L
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ments that make up the tail are all short, seldom exceeding 0.2 ~tm. This observation was unexpected as
one would imagine that the tail would be made up of
a parallel bundle of actin filaments like those in the
acrosomal process of Thyone sperm 26 or those in a
microvilhs 27'2s. By decorating the filaments in the tail
with subfragment 1 of myosin we 19showed that, even
though short, the pointed ends of the filaments invariably are oriented towards the tip of the tail (Fig.
5). Putting these two observations together we presume that the tail grows by the nucleation of actin
from the surface of the bacterium and the subsequent
crossbridging of the elongating filament to neighboring filaments. The elongating filament is released
from the surface of the bacterium and a new one is
nucleated and crossbridged and so forth. The question then becomes, what is controlling the short
length of the filaments and why is the tail built in this
way?
At this point all we can do is to speculate, as we
have only limited information on exactly what the
host cell cytoplasm is doing and why it is doing it.
What we think is occurring is that the host cell has a
substantial store of unpolymerized actin, which has
been estimated to be 100-200 ktm (Refs 26, 29). To
control spontaneous assembly the host must be suppressing extraneous actin filament nucleation and
growth using a variety of actin-binding proteins. We
propose that, to overcome the host controls, the bacterium secretes on its surface the protein actA which,
by binding to other host proteins, generates sites of
nucleation that initiate actin assembly. The host cell
stops the elongation of the filaments by using 'barbed
end cappers' such as cap Z (Ref. 30) or gelsolin3L At
this point the bacterium would be out of business but
it avoids this problem by releasing the short recently
assembled filaments and nucleating new ones that
are, in turn, terminated by the host. The result is a tail
of many short filaments that gradually increases in
length by the nucleation of more filaments. A similar
proposal has been recently suggested by Theriot and
Mitchison 32 for fibroblast motility.
How Listerla move
A number of investigators have become fascinated by
the movement of Listeria in the cytoplasm of eucaryotic cells 3'4,14,18'33,34 and it has been studied by a number of techniques. Given the polarity of the actin filaments in the tail and the fact that Listeria always
moves like a comet with the bacterium leading and
the tail trailing behind, conventional myosin (myosin
II) located in the cytoplasm proper could not account
for the movement because the polarity of the actin
filaments is inappropriate. It would be as if skeletal
muscle actually elongated during contraction. Having eliminated this possibility, we can think of three
possible explanations for the movement. The first requires that the assembly of actin itself (and the release
of these newly assembled filaments) provides a
propulsive force comparable to that suggested for the
elongation of the acrosomal process of Thyone
sperm 27. The second invokes a motor protein, such as
TRENDS
IN MICROBIOLOGY
29
Fig. 5. On the left is a thin section through a Listeria with a tail whose
actin filaments have been decorated with subfragment 1 of myosin. The
filaments are all short, not more than 0.3 lam long, and the lengths of the
filaments at the tip and base are comparable. On the right is a light print
on which we have indicated in ink those filaments whose polarity we can
unequivocally determine from the left-hand micrograph. Note that in all
cases the filaments have their pointed ends nearest the tip of the tail
and their barbed ends nearest the Listeria. Reproduced from Ref. 17,
with permission.
a myosin which is attached to the surface of the bacterium; the action of this motor must be coupled to
the assembly of actin. The third is the use of a cytoplasmic motor that operates in the reverse direction
to myosin. Since no such reverse motor has thus far
been described, we do not know how seriously to
take this possibility. We will therefore confine our
discussion to the first two possibilities.
The most dramatic evidence for the first possibility
comes from an experiment in which G actin covalently
coupled to caged resorufin (CR) was injected into a
Listeria-infected potoroo kidney epithelial (PtK2) cell
line3. CR actin is nonfluorescent and is incorporated
readily into actin-containing filaments. Upon illumination with ultraviolet light the CR is readily and
efficiently converted to the bright red fluorescent
parent compound, resorufin. Using this probe, short
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ment, is also conceivable although there is no evidence to support it. In this model the bacterium
would be continuously moved forward relative to its
associated short filaments. What is essential in this
model is coupling of myosin motor antics with the
assembly and disassembly of actin filaments.
Function of the actln cytoskeleton of Listerla
One might assume that the major function of the
actin tail is to move the Listeria to the cell surface as
a prelude to dispersal. This is certainly reasonable.
However, equally important is the use of the actin tail
in pseudopod formation. This is essential for spreading because the Listeria must end up in the tip of a
pseudopod in order to be phagocytosed by a neighboring host cell (Fig. 6). Since projections of animal
cells such as microvilli, microspikes, pseudopods and
stereocilia all contain a core of actin filaments necessary for growth and maintenance of these asymmetric
processes, it seems reasonable to conclude that the
actin tail of Listeria could provide a core for the generation of a pseudopod. All that is required is for the
actin filaments at the margins of the tail to bind to the
plasma membrane and by zippering these actin filaments to the plasma membrane, a pseudopod would
be generated with the Listeria at the tip. In microvilli,
the connections that extend from the lateral surface
of the filament bundle to the membrane are myosin I
(Refs 35-37). It is undoubtedly not an accident that
the polarity of the actin filaments in all these ceil'extensions, such as microvilli, microspikes, stereocilia
and listerial tails, is identical (barbed ends located at
the tips of the process) 2s'29. The common feature is
that this polarity may regulate the attachment of~
these connections to the plasma membrane; this in
turn allows formation of a projection which, in the
case of Listeria, has the bacterium at its tip.
Rg. 6. The spread of Listeria from one macrophage to'another. Extending
from the macrophage at the base of the micrograph is a pseudopod
containing a Listeria near its tip. A second macrophage, above, is
phagocytosing the pseudopod of the first macrophage. Bar represents
I pm.
segments of the Listeria tail could be photoactivated
and, by combining the fluorescent image with that of
the moving bacterium by phase contrast video microscopy, it was shown that the photoactivated mark
on the tail remained stationary in the cytoplasm
as the bacterium moved away from the mark. It
was thus concluded that the filaments must appear
(assemble) continuously at the bacterial surface and
be released from the bacterium as it moves on, so the
rate of bacterial movement is equal to the rate of actin
filament appearance. Furthermore, the length of the
tail is linearly proportional to the rate of movement.
Putting these two observations together, it was concluded that the motility of Listeria involves the continuous polymerization and release of actin filaments
at the bacterial surface, and that the rate of filament
generation is related to the rate of movement 3. Thus
the data are consistent with actin polymerization providing the driving force for propulsion. A similar conclusion was reached by Sanger e t al. 18'34, who halted
listerial propulsion by cytochalasin D treatment,
and re-initiated it by microinjection of rhodaminelabelled monomeric actin.
The second possibility, in which myosin molecules
attached to the surface of the bacterium induce move-
TRENDS
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30
More questions
In our review we have left many unanswered questions. Some revolve around the control and function
of the eight genes turned on by the single promoter
that is induced by prfA. Others have to do with identifying the components 'donated' by the host that
allow Listeria to form actin tails, to move, and to
enter and bind to the inner surface of the membrane
of a pseudopod. And still others are concerned with
how Listeria regulates the expression of the genes
essential for surviving in the cytoplasm.
Although these are interesting problems, to us
there are two questions that appear to be the most
important in developing an understanding of the
biology of Listeria. The first is the general question of
what is the minimum number of genes that must be
expressed by Listeria in order for it to become an intracytoplasmic parasite. Are they only the eight genes
turned on by prfA, or are there others, some of which
may be regulated by other inducers? The second is,
how does a macrophage located adjacent to a Listeria-infected neighbor recognize and phagocytose a
pseudopod containing a Listeria? What makes it
tasty? Is it a product synthesized and somehow put on
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(in press)
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the cell surface by the Listeria or is it a host molecule
that the Listeria has somehow induced the host cell to
concentrate on the tip of the pseudopod?
Acknowledgement
Supported by a grant from the National Institutes of Health
HD 14474.
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in M
In file n e x t issu e o f
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journals
A selection ~ofrecently ~ t i s h e d articles of interest to T/M reack~rs
phenotl~pt¢ switching in Canaidaalbicans, by D.R. Soil, B. Morrow and T. Srikantha -
•
Trends/n Gene~Jcs9 (2), 6 1 - 6 5
=The role of host
Trends in
Ohospt'mrylation In~bacteri~ O ~ ~ i s ,
9 (3), 8 5 - 8 9
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by J.B. ~ s k a ~
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• Secretory transport in P/asmod/um, by H.G. Elmendorf .and K. Haldar - Paras
The May issue of Parasltolo~ Todaywill ~ U S ~
TRENDS
IN MICROBIOLOGY
drug r e s i ~
31
VOL.
TOa~ 9 (3), 9 8 - 1 0 2
in parasitic
1
NO.
I
APRIL
s.
1993