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Infection and Immunity, June 2000, p. 3710-3715, Vol. 68, No. 6
0019-9567/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Interaction of Shigella flexneri IpaC
with Model Membranes Correlates with Effects on Cultured
Cells
Ngoc
Tran,1
Alexa
Barnoski
Serfis,1
John C.
Osiecki,2
Wendy L.
Picking,2
Lisette
Coye,3
Rebecca
Davis,3 and
William D.
Picking2,*
Department of
Chemistry1 and Department of
Biology,3 Saint Louis University, St. Louis,
Missouri, and Department of Molecular Biosciences,
University of Kansas, Lawrence, Kansas2
Received 20 December 1999/Returned for modification 4 February
2000/Accepted 3 March 2000
 |
ABSTRACT |
Invasion of enterocytes by Shigella flexneri requires
the properly timed release of IpaB and IpaC at the host-pathogen
interface; however, only IpaC has been found to possess quantifiable
activities in vitro. We demonstrate here that when added to cultured
cells, purified IpaC elicits cytoskeletal changes similar to those that occur during Shigella invasion. This IpaC effect may
correlate with its ability to interact with model membranes at
physiological pH and to promote entry by an ipaC mutant of
S. flexneri.
| |
TEXT |
Shigella flexneri is an
important cause of dysentery with a high incidence of infant mortality
in developing nations. An early step in Shigella infection
is bacterial invasion of colonic epithelial cells (8).
Invasion is characterized by host cytoskeletal rearrangements at the
site of bacterial contact, which leads to the formation of filopodia
that coalesce and trap the pathogen within a membrane-bound vacuole
(1). The resulting phagosomal membrane is rapidly lysed following pathogen uptake (16). IpaB and IpaC have been
identified as being the effectors of Shigella invasion
(9-11) following their secretion at the host-pathogen
interface via the mix-spa secretory system (4, 12,
21). Once released within this localized area, IpaB and IpaC form
a complex (2, 14) that is reported to be responsible for
pathogen entry (10, 12, 18, 20, 21).
It was originally proposed that IpaB alone induces bacterial uptake and
promotes the subsequent escape of S. flexneri into the host
cell cytoplasm (6). This role was revised when purified IpaB
could not be shown to possess membranolytic activity in vitro (13) and when purified IpaC was found to possess both a
potential effector role in invasion (9, 19) and the ability
to cause the release of small molecules from phospholipid vesicles at
low pH (3). In the work presented here, a novel model
membrane system is used to show that purified IpaC penetrates
phospholipid membranes at neutral pH and that this activity may be
correlated with IpaC's role as an effector molecule that elicits
cytoskeletal changes in cultured cells when it is added to the
extracellular environment. The ability for IpaC to penetrate
phospholipid membranes and to promote cellular effects may provide a
significant step forward in our understanding of the mechanism by which
S. flexneri directs its own uptake by enterocytes. Moreover,
the data presented here may have a broader impact in the area of
bacterial pathogenesis since Salmonella enterica initiates
its entry into epithelial cells by an outwardly similar mechanism that
requires SipC, a putative IpaC homologue.
IpaC interacts with membranes at neutral pH.
Protein
penetration of cell membranes occurs as a part of numerous biological
processes. The incorporation of proteins into phospholipid Langmuir
monolayers (17) provides a potentially valuable model for
exploring specific protein-lipid interactions that have an important
role in the pathogenesis of S. flexneri. The Langmuir
membrane system used here is composed of a
dipalmitoyl-phosphatidylcholine (DPPC) monolayer generated on top of a
buffered aqueous subphase. This system mimics the outer faces of the
cytoplasmic membranes of most mammalian cell types, as they are
encountered by proteins secreted from S. flexneri. Although
such a monolayer is structurally different from the phospholipid
bilayer that makes up a cell's cytoplasmic membrane, it provides a
suitable model membrane for sensitively detecting specific
lipid-protein interactions such as those that occur at the surface of a cell.
When a protein is injected into an aqueous subphase and given time to
interact with an overlying lipid monolayer, changes in surface pressure
can be monitored to deduce the nature of the protein-lipid interaction.
Penetration of a protein into the phospholipid monolayer forces the
phospholipids to become more tightly packed due to the space taken up
by the protein (thereby giving rise to an increase in surface
pressure). While the phospholipid monolayer model used here does not
reproduce the entire structure of the cellular phospholipid bilayer, it
has been shown to provide an effective system for monitoring
protein-membrane interactions (17). Moreover, the
sensitivity for detecting protein penetration of membranes using
Langmuir monolayers far surpasses that seen using conventional
approaches that detect protein-membrane interactions only after they
result in the disruption of phospholipid vesicles (3).
Because IpaC-mediated effects on cultured cells have not been found to
be cytotoxic, it is important to explore this protein's interaction
with model membranes under conditions that, during invasion, may induce
only subtle changes in the structure of the cytoplasmic membrane.
For proteins that do not interact with phospholipid membranes, maximum
accumulation of the protein at the air-water interface
occurs in the
absence of phospholipids or when the phospholipids
are very loosely
packed and provide gaps at the air-water interface
for the proteins to
occupy. The ability of such a protein to accumulate
at the air-water
interface is rapidly diminished when the initial
monolayer density is
increased until, at high initial lipid densities,
the protein's access
to the air-water interface is blocked (
17).
In contrast, a
protein that possesses the ability to penetrate
a phospholipid membrane
tends to interact poorly at the air-water
interface but demonstrates an
enhanced ability to migrate to this
site as the initial phospholipid
monolayer density is increased.
This behavior is common for known
membrane-penetrating proteins
such as factor VII, a protein involved in
the blood-clotting cascade
(
17).
The
ipaC coding sequence was cloned and expressed, and IpaC
was purified as described previously (
15). The presence of a
short leader sequence containing a His
6 tag allowed
affinity purification
of IpaC in the presence of 6 M urea
(
2). When prepared with
urea, IpaC was dialyzed against 20 mM phosphate (pH 7.2)-150 mM
NaCl (phosphate-buffered saline [PBS])
containing 2 M urea. Refolding
of the protein was facilitated by rapid
dilution as described
previously (
2), and protein
concentrations were measured by
a bicinchoninic acid protein assay
(Sigma Chemical Co.). To monitor
potential interactions between
purified recombinant IpaC and a
DPPC monolayer, a NIMA model 611D
Langmuir-Blodgett trough equipped
with a model PS4 pressure sensor was
used to monitor changes in
membrane surface pressure (
17).
An aqueous subphase containing
0.1 M Tris (pH 7.4) was used, and in
some experiments, calcium
chloride was added to a concentration of 10 mM. To form phospholipid
monolayers, 100 µl of 1-mg/ml DPPC prepared
in chloroform was
dropped onto the subphase and the solvent was removed
by evaporation.
The monolayers were then compressed to predetermined
surface pressures.
Fibrinogen was used here as a negative-control
protein that does
not interact with phospholipid membranes, and
blood-clotting factor
VII was used as a positive-control protein known
to penetrate
phospholipid membranes in a calcium-dependent manner.
Fibrinogen
was prepared in 0.1 M Tris (pH 7.4), and 500 µl was
injected into
the subphase to give a final concentration of 3 µg/ml.
IpaC and
factor VII (500 µl each) were added to the subphase to give
a
final concentration of 0.02 µg/ml each. The proteins were allowed
to interact with the monolayer for 75 min, and the degree of protein
accumulation at the interfacial region was measured as a time-dependent
increase in surface
pressure.
The largest changes in surface pressure for the negative-control
protein (fibrinogen) were observed at the lowest initial
lipid
pressures (Fig.
1A), with no increase in
surface pressure
seen when the monolayer was compressed to 10 mN/m or
higher. In
contrast, IpaC showed the highest changes in surface
pressure
at the highest initial lipid pressures (Fig.
1B), with no
surface
activity seen in the absence of phospholipids. Membrane-binding
proteins typically display this type of increased surface activity
in
the presence of a compacted phospholipid monolayer (
17).
Even at initial surface pressures approaching 30 mN/m (the estimated
average density of phospholipids in the cell membrane), IpaC
efficiently
penetrated the DPPC monolayer, although at a rate that was
somewhat
lower than when the initial surface pressure was 15 mN/m (data
not shown), indicating that this activity has physiological relevance.
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FIG. 1.
IpaC penetrates model DPPC membranes. (A) Fibrinogen
(used here as a negative-control protein that does not penetrate
phospholipid membranes) was injected into the aqueous subphase of a
Langmuir-Blodgett trough at different initial DPPC pressures ( , 3.0 dynes/cm;  , 4.0 dynes/cm;  , 5.6 dynes/cm). No interaction of
fibrinogen with the DPPC monolayer is seen once the initial lipid
pressure is 10 dynes/cm or greater (data not shown). (B) IpaC was
injected into the aqueous subphase at different initial DPPC pressures
( , 3.0 dynes/cm; , 5.0 dynes/cm; , 10.0 dynes/cm; , 15.0 dynes/cm). In both panels, single datum points are given at each time;
however, the data sets shown are representative of those generated in
at least three independent experiments that gave nearly identical
results. The magnitude of the surface pressure changes at low initial
lipid densities in panel A were greater than the surface pressure
changes caused by IpaC (B) because of the relatively small size of IpaC
and the much lower subphase concentration used for IpaC.
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The magnitude of the increased surface pressure caused by IpaC was
comparable to that seen with the same concentration of
factor VII,
which also displays an enhanced ability to penetrate
DPPC monolayers as
the initial surface pressure is increased (Table
1). These data indicate that IpaC
interactions with phospholipid
membranes at neutral pH are similar to
that of a known membrane-penetrating
protein. Unlike factor VII, whose
penetration of phospholipid
membranes is calcium dependent
(
17), IpaC interacts with the
DPPC membranes in the absence
or presence of added calcium (Table
2).
Interestingly, while the absolute value of the maximum surface
pressure
change for the phospholipid monolayers caused by IpaC
is greater in the
presence of 10 mM calcium, the ratio of IpaC
penetration at high
initial surface pressures relative to that
at low initial surface
pressures is greater in the absence of
calcium (Table
2). This
indicates that IpaC penetration of phospholipid
membranes, unlike that
of factor VII, is not calcium dependent.
Interestingly, when IpaC is freshly diluted from solutions containing
urea, the observed protein-induced surface pressure
changes are higher
(Table
2). In control experiments, urea alone
(at concentrations up to
50 mM) did not cause changes in surface
pressure when it was injected
beneath compressed phospholipid
monolayers at any initial lipid
pressure (data not shown), indicating
that urea in the IpaC samples did
not contribute directly to the
larger observed surface pressure
changes. From Table
2, it is
clear that IpaC interacts more extensively
with phospholipid membranes
when it starts out in a partially unfolded
state. This is interesting
since, like SipC from
Salmonella
(
7), IpaC appears to be secreted
via a supramolecular
"needle complex" that spans the inner and
outer membranes of the
pathogen (
5). In
Salmonella, the needle
complex
is proposed to have a relatively narrow inner diameter
(
5),
indicating that SipC is probably released in a partially
unfolded
state. It is anticipated, therefore, that IpaC and SipC
rapidly
generate their final tertiary and quaternary structures
at the
host-pathogen interface (concomitant with formation of
IpaC
[SipC]-containing protein
complexes).
De Geyter and coworkers suggest that IpaC possesses membrane-lysing
potential based on its ability to release calcein trapped
in vesicles
composed of phosphatidylserine (an acidic phospholipid
typically found
at the inner face of the cytoplasmic membrane)
and phosphatidylcholine
(a neutral phospholipid) (
3). This
lytic activity is largely
pH dependent, with little activity seen
at neutral pH and with most
efficient membrane lysis occurring
below pH 6.0 (
3). This is
distinct from the penetration of
DPPC membranes at neutral pH as
described here. Moreover, it was
shown in previous work that IpaC
enhances the invasive capacity
of
S. flexneri without lysing
host cells (
9) and IpaC does
not appear to possess a
pH-dependent hemolytic activity in vitro
(data not
shown).
Because of the sensitivity of the assay described here for monitoring
protein-membrane interactions under a variety of conditions,
it is now
possible to monitor the effect that IpaC and IpaC-containing
protein
complexes have on membranes composed of different phospholipids.
This
will provide a convenient model for deducing the events occurring
at
the host-pathogen interface immediately prior to
S. flexneri entry into host cells. It is difficult at this point to determine
whether IpaC penetration of DPPC monolayers is comparable to that
of
pore-forming toxins as previously proposed by De Geyter and
coworkers
(
3); however, it is important to note that IpaC does
not
lyse and is not cytotoxic for cultured Henle 407 cells
(
9;
data not shown). Therefore, if IpaC does form a
pore following
interaction with a host cell membrane, this pore does
not result
in the rapid death of the host cell. Alternatively, it is
possible
that IpaC penetrates phospholipid membranes at neutral pH but
forms a pore only as the pH is lowered to that found in early
endosomal
compartments. This would be consistent with the work
reported by De
Geyter et al. (
3). It should be possible to
determine
whether IpaC possesses properties consistent with pore
formation by
monitoring protein-protein interactions (see reference
2) involving membrane-imbedded
IpaC.
It has been shown that IpaB-IpaC complexes being immunoprecipitated
onto the surfaces of latex beads is sufficient for promoting
the uptake
of these beads by cultured cells, suggesting that both
IpaB and IpaC
have important roles in cellular invasion by
S. flexneri
(
10). It is therefore possible that IpaB influences
the
action of IpaC at the host-pathogen interface and that it
has a
profound effect on IpaC's ability to interact with phospholipid
membranes. It will be important to compare the interactions that
IpaC
has with phospholipid membranes before and after its recruitment
into
protein complexes containing IpaB. Because Ipa complexes
presumably
represent IpaC in its natural extracellular context,
their formation
can be expected to enhance the membrane interactions
seen here. It is
also possible that recruitment into Ipa complexes
may diminish IpaC's
interaction with phospholipid membranes while
increasing its ability to
interact with cellular integrins, which
has been described as being a
potential receptor for the Ipa complex
by Terajima et al.
(
18) and Watarai et al. (
20). In addition
to
using Langmuir films to explore the action of IpaC, it will
be
important to explore the same parameters for
Salmonella
invasion
protein C (SipC), which is a putative homologue of IpaC
(
7).
A comparison of the membrane-penetrating potentials of
these two
proteins would be enlightening since
Salmonella
invades epithelial
cells in much the same way as
S. flexneri, but without lysing
the resulting membrane-bound vacuole
(
7).
Addition of purified IpaC to Henle 407 cells promotes cytoskeletal
changes.
Because IpaC interacts with DPPC membranes and has been
shown to promote the uptake of virulence plasmid-cured S. flexneri when added at high concentrations, its effect on the
cytoskeleton of Henle 407 cells was explored. Henle 407 cells (ATCC
CCL6) were grown in Eagle's modified minimal essential medium (MEME;
Fisher Scientific) containing 10% calf serum (Gibco-BRL) and incubated in 5% CO2. When incubated in 2 µM IpaC in serum-free
medium, Henle 407 cells become rounded after 1 h, with some of the
cells detaching after even longer incubations (data not shown). At no
point during exposure to IpaC do the attached cells display
IpaC-related cytotoxicity as monitored by their ability to exclude
trypan blue or their failure to release lactate dehydrogenase into the
culture supernatant (data not shown). These morphological changes
suggest that IpaC induces cytoskeletal rearrangements in Henle 407 cells. To confirm this, Henle 407 cells were grown on coverslips and
incubated with 2 µM IpaC in serum-free MEME. Actin staining was the
carried out by simultaneously fixing, permeabilizing, and staining the
cells for 15 min in a solution containing 3.7% formaldehyde, 1%
palmitoyl-lysophosphatidylcholine, and rhodamine-phalloidin. The
stained cells were washed with PBS and overlaid with PBS containing
50% glycerol and 1 mg of n-propylgallate per ml. Stained
actin was viewed by fluorescence microscopy on an Olympus BX60
microscope equipped with a charge-coupled device camera (Optronics) and
videocapture card (Truevision).
After a 7-min incubation with IpaC, f-actin appeared to accumulate at
the cell edges with the appearance of numerous microspikes
(Fig.
2). After 30 min, the cells started to
show signs of rounding
up while appearing to have less overall f-actin
content relative
to that seen at 7 min as judged by a decrease in
overall fluorescence
intensity (Fig.
2). These time points correlate
reasonably well
with the reported times for cytoskeletal changes
observed during
S. flexneri invasion (
1). It was
recently reported that IpaC
induces actin polymerization in (i) 3T3
cells permeabilized with
saponin, (ii) 3T3 cells microinjected with
IpaC, and (iii) HeLa
cells expressing
ipaC (
19).
In this work, IpaC added to the
extracellular environment elicits a
similar effect except that,
unlike with permeabilized cells
(
19), IpaC does not cause detectable
cytotoxic effects (at
low IpaC concentrations) at times exceeding
1 h (data not shown).
This observation suggests that permeabilization
introduces cellular
changes that ultimately disrupt ongoing functions
and that such
disruptions do not occur when IpaC is added to the
extracellular
milieu.
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FIG. 2.
Extracellular IpaC promotes cytoskeletal changes in
Henle 407 cells. The cells were incubated at 37°C in serum-free MEME
and immediately fixed (A) or fixed after 7 or 30 min in the serum-free
MEME (B and C, respectively). Alternatively, the cells were incubated
in serum-free MEME containing 2 µM IpaC for 7 min (D) or 30 min (E)
andstained for analysis of polymerized actin by fluorescence microscopy
(at a magnification of ×400). 1/8, 1/8-s exposure.
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The ability of IpaC to elicit changes in cultured cells suggests that
it interacts with the host cell surface to trigger a
cascade of events.
An earlier study implicated
5
1 integrins
as potential receptors for Ipa protein complexes (
20). In
previous
work from our laboratory, the need for high IpaC
concentrations
to promote overt cellular effects may indicate that
there is a
large number of possible IpaC binding sites (
9),
perhaps because
the IpaC interaction with host cells is rather general.
Such an
interaction could be explained if IpaC was able to interact
directly
with the host cell membrane in addition to, or instead of,
integrin
receptors.
Exogenously added IpaC promotes uptake of a S. flexneri
ipaC mutant.
In a recent report, it was shown that
expression of ipaC in HeLa cells leads to a fivefold
enhancement in the uptake of S. flexneri (19).
Earlier work showed that exogenously added IpaC enhances invasion over
fourfold for IpaC prepared in 20 mM phosphate (pH 7.2) with 150 mM NaCl
(PBS) (9) and eightfold for IpaC prepared in PBS containing
2 M urea (2). Moreover, high concentrations of IpaC promote
the uptake of small numbers of plasmid-cured S. flexneri
(9). To determine the potential importance of IpaC-induced cytoskeletal changes and its potential relationship with IpaC-membrane interactions, the protein was added to Henle 407 cells and the subsequent effect on the uptake of different strains of S. flexneri was monitored.
S. flexneri 2a strain 2457T was provided by A. T. Maurelli (Uniformed Services University of the Health Sciences,
Bethesda,
Md.), and
S. flexneri strain SF621 (carrying a
nonpolar null mutation
in
ipaC) (
11) was provided
by Philippe Sansonetti (Unité de
Pathogénie Microbienne
Moléculaire, Institut Pasteur, Paris,
France).
S. flexneri entry into Henle 407 cells was quantified
using a
gentamicin protection assay as described previously (
9)
except that the bacteria were centrifuged onto the surfaces of
the
Henle 407 cells to promote efficient contact between the bacteria
and
the host cells.
S. flexneri strain 2457T was used at a
multiplicity
of infection (MOI) of 1.0, while the noninvasive strains
BS103
and SF621 were used at an MOI of 10 or greater. Bacteria were
added to the monolayers in serum-free MEME and incubated for 30
min at
37°C. The monolayers were then washed with MEME containing
5%
newborn calf serum and 50 µg of gentamicin per ml, rinsed with
serum-free MEME, and overlaid with 0.5% agarose and 0.5% agar
containing 2× Luria-Bertani medium. The plates were incubated
overnight at 37°C, and the resulting colonies were
counted.
Exogenously added IpaC does not induce uptake of the plasmid-cured
S. flexneri strain BS103 at high nanomolar concentrations,
even at a high MOI, while purified IpaC does promote uptake of
strain
SF621 (data not shown). While an
ipaC null mutant of
S. flexneri is noninvasive and avirulent (
11),
exogenously added
IpaC (50 nM) restores a significant portion of its
invasive capacity
(up to 5% of wild-type activity in some
experiments). An important
observation here is that because IpaC is a
relatively efficient
extracellular effector for the uptake of SF621 but
not for BS103,
there is a factor in addition to IpaC, perhaps IpaB
and/or IpaD,
that is an important participant in the invasion
process.
Taken together, the data indicate that purified IpaC penetrates model
phospholipid membranes, induces cytoskeletal changes
in cultured
epithelial cells, and promotes uptake of an
ipaC null
mutant
of
S. flexneri by cultured cells. It is not yet clear
whether
penetration of phospholipid membranes by IpaC is directly
related
to the observed changes in actin polymerization in Henle 407 cells;
however, invasion data may support this possibility. As shown
in
Table
3, the uptake of wild-type
S. flexneri is enhanced over
eightfold by exogenously added IpaC when
the IpaC protein is present
in its fully folded state prior to its
addition to the reaction
mixture of the modified invasion assay used
here. In contrast,
IpaC enhances invasion by wild-type
S. flexneri invasion by nearly
15-fold when it is freshly refolded
from a stock solution containing
2 M urea (Table
3). These data are
consistent with earlier results
(
2), and they parallel
results from experiments designed to
explore IpaC's ability to more
efficiently penetrate phospholipid
membranes when starting out in a
partially folded state (Table
2). Therefore, it appears likely that
there is a correlation
between IpaC-mediated effects on epithelial
cells and IpaC-dependent
penetration of phospholipid membranes.
The data presented here provide the first evidence that purified IpaC
elicits cytoskeletal changes in cultured cells when
it is presented as
part of the extracellular environment. The
ability for IpaC to carry
out this activity, and thus its role
in
Shigella
pathogenesis, may be related to its ability to interact
with and
possibly integrate into the cytoplasmic membranes of
host cells. This
is a significant observation that should contribute
greatly to our
understanding of the events responsible for the
early steps in
pathogenesis. Clearly the potential relationship
between IpaC-membrane
interactions and IpaC-mediated changes in
the cytoskeletons of cultured
epithelial cells warrants continued
investigation.
Interestingly, the invasive phenotype of
S. flexneri
requires the properly timed secretion of IpaB and IpaD along with IpaC.
Elimination of the gene encoding any of these proteins does not
prevent
secretion of the others (
12), but it does completely
eliminate the invasive phenotype (
11). The importance of
IpaD
has been suggested to be at the level of secretion
(
12); however,
IpaD has also been described as being part of
a complex that (i)
associates with
51
integrins (
20) and (ii) is involved in
the uptake of
noninvasive
Escherichia coli (
18). Other evidence
suggests that an IpaB-IpaC complex is the effector of
S. flexneri invasion (
10), while data from our laboratory
and that of others
indicate that IpaC alone has a central role in the
entry of
S. flexneri into cultured cells (
9,
19).
In its soluble form,
IpaC exists as part of a complex that involves
both IpaC-IpaC
and IpaC-IpaB interactions (
2). It is
therefore important to
consider the potential effect that IpaB may have
on IpaC's ability
to penetrate the host cell membrane following the
release of both
proteins at the host-pathogen interface. As mentioned
previously,
IpaB may have a profound influence on IpaC's ability to
penetrate
phospholipid membranes; however, continuing work will be
needed
to determine what form these effects will take. Determining the
regions on IpaC that are important for membrane penetration and
those
important for the protein-protein interactions involving
IpaC may
provide a great deal of insight into the effects that
IpaB may have on
the results presented here. Moreover, the prominent
hydrophobic domains
identified on IpaB may indicate that this
protein, in concert with
IpaC, has an important role in phagosomal
escape by
S. flexneri. This would be consistent with the fact
that IpaB appears
to be important for phagosomal escape (
6)
while purified
IpaB is not hemolytic (
14). Such a scenario suggests
that
the IpaB-IpaC complex retains the ability to penetrate phospholipid
membranes. Indeed, a tremendous amount of work remains; however,
the
membrane penetration assay described here and the protein-protein
interactions described in a previous report from this laboratory
(
2) should provide pertinent information on the detailed
mechanism
of
S. flexneri entry into epithelial
cells.
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ACKNOWLEDGMENTS |
We acknowledge valuable discussions with M. E. Marquart
(Louisiana State University School of Medicine, New Orleans, La.) and
technical assistance from W. E. Goldman (Washington University School of Medicine, St. Louis, Mo.).
This work was supported by PHS grant AI34428.
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FOOTNOTES |
*
Corresponding author. Mailing address: Dept. of
Molecular Biosciences, 8047 Haworth, University of Kansas, Lawrence, KS
66045. Phone: (785) 864-3299. Fax: (785) 864-5294. E-mail:
picking{at}eagle.cc.ukans.edu.
Editor:
J. T. Barbieri
| |
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Infection and Immunity, June 2000, p. 3710-3715, Vol. 68, No. 6
0019-9567/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
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