INFECTION AND IMMUNITY, July 2007, p. 3571–3580
Copyright © 2007, American Society for Microbiology. All Rights Reserved.
Vol. 75, No. 7
Evaluation of the Role of LcrV–Toll-Like Receptor 2-Mediated
Immunomodulation in the Virulence of Yersinia pestis?
Kimberly Pouliot,1Ning Pan,1Shixia Wang,2Shan Lu,2Egil Lien,2and Jon D. Goguen1*
Department of Molecular Genetics and Microbiology1and Division of Infectious Diseases, Department of Medicine,2
University of Massachusetts Medical School, 55 Lake Avenue North, Worcester, Massachusetts 01655
Received 11 October 2006/Returned for modification 26 November 2006/Accepted 30 March 2007
Pathogenic members of the Yersinia genus require the translocator protein LcrV for proper function of the type
III secretion apparatus, which is crucial for virulence. LcrV has also been reported to play an independent
immunosuppressive role via the induction of interleukin-10 (IL-10) through stimulation of Toll-like receptor 2
(TLR2). To investigate the LcrV-TLR2 interaction in vitro, His-tagged recombinant LcrV (rLcrV) from Yersinia
pestis was cloned and expressed in Escherichia coli and purified through Ni-nitrilotriacetic acid column chroma-
tography. High concentrations (5 ?g/ml) of rLcrV stimulated TLR2 in vitro. Fractionation of rLcrV preparations
via gel filtration revealed that only a minor component consisting of high-molecular-weight multimers or aggregates
has TLR2 stimulating activity. Dimer and tetramer forms of rLcrV, which constitute the bulk of the material, do not
have this activity. To investigate the potential role of LcrV/TLR2 in plague pathogenesis, we infected wild-type and
TLR2?/?mice with virulent Y. pestis. No discernible difference between the two mouse strains in severity of disease
or kinetics of survival after subcutaneous challenge was observed. IL-6, tumor necrosis factor, and IL-10 levels from
spleen homogenates; bacterial load; and the extent of inflammation observed in organs from mice infected intra-
venously were also indistinguishable in both mouse strains. Taken together, our data indicate that the most
abundant molecular species of Y. pestis LcrV do not efficiently activate TLR2-signaling and that TLR2-mediated
immunomodulation is unlikely to play a significant role in plague.
Three members of the Yersinia genus are pathogenic for
humans. Yersinia pseudotuberculosis and Yersinia enterocolitica
cause self-limiting mesenteric lymphadenitis or ileitis. In con-
trast, Yersinia pestis infection results in the highly invasive and
often fatal systemic infection known as plague. All three spe-
cies elaborate a type III contact-dependent secretion system
(TTSS) that is essential for virulence and is encoded on closely
related plasmids. This apparatus allows delivery of effector
molecules directly into host cell cytosol. These intracellular
effectors, termed Yops, inhibit phagocytosis, induce apoptosis,
and inhibit cytokine induction (4, 29). Three translocator pro-
teins, YopB, YopD, and LcrV, are required for efficient intra-
cellular delivery of the Yops (4). LcrV (V antigen) is a multi-
functional protein essential for virulence. It comprises the tip
of the injection needle of the TTSS (6, 11, 17) and along with
LcrG has a regulatory role in Yop secretion (14). In addition,
LcrV has been shown to be one of only two proteins to serve
as highly effective vaccine antigens against Yersinia pestis (9,
An immunomodulatory role for LcrV has also been pro-
posed (3). In vivo studies have shown that a recombinant Y.
pseudotuberculosis LcrV-protein A fusion, produced in Esche-
richia coli, suppressed induction of tumor necrosis factor
(TNF) and gamma interferon induced by injection of lipopoly-
saccharide (LPS) into Swiss Webster mice. It also enhanced
the severity of disease and the bacterial burden following in-
fection of treated mice with attenuated Y. pestis, Salmonella
enterica serovar Typhimurium, and Listeria monocytogenes
(18). Further studies utilizing recombinant Y. enterocolitica
LcrV (rLcrV), also produced in E. coli, showed suppression of
zymosan-induced TNF production in C57BL/6 macrophages
(24). It has been proposed that rLcrV interacts with both
Toll-like receptor 2 (TLR2) and CD14 to induce interleu-
kin-10 (IL-10) in transfected HEK293 cells (25). Candidate
residues responsible for this interaction with TLR2 were iden-
tified in the N-terminal region of Y. enterocolitica LcrV based
on the activity of cognate synthetic peptides (23, 25). Lower
activity was detected in similar peptides based on Y. pestis and
Y. pseudotuberculosis LcrV sequences (23). More recently,
Overheim et al. (22) showed that His-tagged rLcrV derived
from Y. pestis and purified from E. coli induced IL-10 from
murine macrophages and human monocytes. It also sup-
pressed induction of TNF from murine macrophages. That
group also showed that deletion of either of two domains
within LcrV eliminated stimulation of IL-10 secretion but that
deletion of only one of these, near the C terminus of the
protein, eliminated both induction of IL-10 secretion and sup-
pression of TNF. Neither of these domains corresponds with,
or overlaps the sequence of, the active peptide identified by
Sing et al. in the LcrV of Y. enterocolitica (23, 25). Overheim et
al. (22) also presented evidence that a fragment of LcrV lack-
ing the C terminus, which includes the domain responsible for
suppression of TNF, was an effective vaccine against Y. pestis at
lower doses than unmodified rLcrV.
TLRs participate in many aspects of the host defense against
infections (2). Stimulation of TLR2 results in the induction of
proinflammatory cytokines such as TNF and IL-6. Like TLR4,
the receptor for LPS, TLR2 is also responsible for the release
of anti-inflammatory cytokines such as IL-10 and IL-4, al-
* Corresponding author. Mailing address: Department of Molecular
Genetics and Microbiology, University of Massachusetts Medical
School, 55 Lake Avenue North, Worcester, MA 01655. Phone: (508)
856-2490. Fax: (508) 856-3355. E-mail: email@example.com.
?Published ahead of print on 16 April 2007.
though these are usually detected at a later time than proin-
flammatory mediators induced simultaneously via the same
pathway (1, 21). Curiously, TLR2 has been associated with
immunosuppression in microorganisms in addition to Y. en-
terocolitica (7, 20), suggesting that exploitation of this pathway
as a means to evade innate immunity may be a common strat-
egy among pathogens. However, no clear mechanistic basis for
a predominantly immunosuppressive effect of TLR2 stimula-
tion has been established.
Here we report that Yersinia pestis-derived rLcrV, purified
from E. coli through Ni-nitrilotriacetic acid chromatography,
activates TLR2, as has been reported for Yersinia enterocolitica
LcrV. However, further purification through gel filtration in-
dicates that only a very high-molecular-weight multimer or
aggregate, which comprises a small proportion of total rLcrV,
has stimulatory activity. Stimulation with Yersinia pestis LcrV-
derived peptides corresponding to stimulatory peptides de-
rived from Yersinia enterocolitica LcrV failed to activate TLR2.
In infection experiments, TLR2 deficiency in mice had no
significant influence on the course of disease, levels of IL-10, or
degree of inflammation in infected tissue. Taken together,
these results strongly suggest that TLR2-mediated induction of
IL-10 does not contribute significantly to the virulence of Yer-
MATERIALS AND METHODS
Bacterial strains, plasmids, cell lines, and reagents. Virulent Y. pestis strain
KIM1001 (27), biotype mediavalis, was grown and quantified on solid medium
(TB) containing 10 g Bacto-tryptose, 5 g NaCl, 3 g beef extract (paste form; Difco
catalog no. 212610), and 15 g agar per liter and supplemented with 2.5 mM
CaCl2. Although this composition is identical to that given for precompounded
tryptose blood agar base, we have on occasion encountered significant problems
with plating efficiency on the latter preparation and therefore chose to work from
the individual components. Unless otherwise indicated, plates were incubated at
25°C for 48 h.
prLcrV was described by Overheim et al. (22) and was the kind gift of Olaf
Schneewind. Construction of pLcrVYpis described below.
HEK293 cells stably expressing human TLR2-yellow fluorescent protein
(TLR2-YFP) or empty vector pcDNA3 were as described previously (13).
HEK293-TLR2-YFP/human CD14 cells were generated by stably expressing
human CD14 in the vector pCEP4, selecting in hygromycin, and fluorescence-
activated cell sorting. 293 cells were maintained in Dulbecco modified Eagle
medium supplemented with 10% fetal calf serum and 10 ?g/ml ciprofloxacin
(Biowhittaker), with addition of G418 (0.5 mg/ml) (for 293-pCDNA3 and 293-
TLR2-YFP) or G418 plus hygromycin (400 U/ml) (for 293-TLR2-YFP/CD14).
Cells were seeded at 3 ? 104per well in 96-well tissue culture plates (Costar) and
stimulated for 16 to 18 h before harvest of the supernatant for cytokine analysis.
LPS, from E. coli strain 0111:B4, was purchased from Sigma and was subjected
to two rounds of phenol reextraction to remove contaminating TLR2-stimulating
lipoproteins (10). The synthetic triacylated lipohexapeptide Pam 3-CysSerLys4
(P3C) and macrophage-activating lipopeptide 2 (MALP2) were purchased from
EMC Microcollections (Tubingen, Germany). Synthetic LcrV peptides (purified
by high-pressure liquid chromatography to ?98%) were purchased from Gen-
emed Synthesis, Inc.
Recombinant plasmids expressing LcrV from Yersinia pestis. lcrV from Yersinia
pestis strain KIM (5) amplified by PCR was cloned into vector pBAD/gIIIB
(Invitrogen) at NcoI and SalI sites, resulting in pLcrVYp. This vector incorpo-
rates a signal sequence to promote export of the cloned protein to the periplasm.
The signal sequence was included both to allow extraction by osmotic shock and
to reduce inclusion body formation. Because osmotic shock proved to have little
advantage, more convenient methods of extraction were used during preparation
of recombinant protein (see below). The coding sequence of the rLcrV in this
construct is as follows (the cleaved domain of the signal sequence is italicized,
nonnative residues of mature protein are in lowercase, and native residues are in
uppercase; note the added C-terminal hexahistidyl domain used in purification):
mkkllfaiplvvpfyshstmvMIRAYEQNP. . .DTSGKvdhhhhhh.
A second rLcrV-producing plasmid. prLcrV, was the kind gift of Olaf Schnee-
wind and was described by Overheim et al. (22). This plasmid produces full-
length rLcrV with the addition of an amino-terminal decahistidyl domain.
Expression and purification of rLcrV. (i) Expression and purification from
plasmid pLcrVYp. Escherichia coli LMG194 (Invitrogen) carrying pLcrVYpwas
grown overnight at 37°C in 2? YT broth supplemented with 100 ?g/ml ampi-
cillin. Bacteria were then diluted into 4 liters of fresh medium to an optical
density at 600 nm (OD600) of 0.1 and grown at 37°C in a New Brunswick Bio Flo
2000 fermentor (with aeration at 5 liters/min, agitation at 300 rpm, and antifoam
as needed) to an OD600of 0.5. Arabinose (0.002%) was then added to induce
production of LcrV, and incubation was continued for an additional 3 h. Cells
were harvested by centrifugation at 12,000 ? g for 10 min, resuspended in 50 ml
of 50 mM sodium phosphate buffer (pH 8.0) containing 300 mM NaCl, and
sonicated on ice (Branson Sonifier 450 with 0.75-in. solid stepped horn; output
power setting, 6; run for 4 min at 50% duty cycle). Alternatively, cells were
disrupted via rapid decompression with a prechilled French pressure cell oper-
ated at 20,000 lb/in2. The extract was centrifuged at 10,000 ? g for 15 min, and
the soluble fraction was applied to a nickel nitrilotriacetic acid column (1-ml bed
volume) preequilibrated with 30 ml column buffer containing 50 mM NaH2PO4,
300 mM NaCl, and 10 mM imidazole, pH 8 (QIAGEN). The column was washed
with 15 ml wash buffer containing 50 mM NaH2PO4,300 mM NaCl, and 20 mM
imidazole, pH 8. Bound protein was eluted in buffer containing 50 mM
NaH2PO4,300 mM NaCl, and 250 mM imidazole, pH 8. Proteins were subjected
to extensive dialysis against endotoxin-free phosphate-buffered saline (PBS)
(Cambrex) and immediately frozen at ?80°C. Protein concentrations were de-
termined by absorbance at 280 nm, using an extinction coefficient of 0.515
calculated from the rLcrV sequence by the method of Gill and von Hippel (8),
and confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis
(SDS-PAGE). All buffers were prepared using endotoxin-free reagents.
Analysis of purified rLcrV treated with 5 mM dithiothreitol (DTT) by liquid
chromatography-mass spectroscopy yielded a mass consistent with removal of the
signal sequence; no material corresponding to the full-length precursor form was
(ii) Expression and purification from plasmid prLcrV. Plasmid prLcrV was
transformed into BL21(DE3), protein production was induced, and the rLcrV
was extracted and purified as described by Overheim et al. with the following
modifications: (i) rather than sonication, a French pressure cell was used to
disrupt the bacteria as described above, and (ii) Triton X-114 phase separation
and the following size exclusion chromatography on G25 Sephadex to remove
Triton were omitted. This material was analyzed identically to rLcrV from
pLcrVYpvia size exclusion chromatography on Superose 12 resin as described
Size exclusion chromatography. One-milligram samples of rLcrV in a 0.3-ml
volume of PBS were loaded on a PBS-equilibrated Superose 12 column (Amer-
sham Biosciences catalog no. 17-5173-01) and eluted with PBS at a flow rate of
0.54 ml/min. Endotoxin-free PBS (Cambrex) was used for column equilibration
and elution. Absorbance of the eluate at 280 nm was monitored. Fractions of 330
?l were collected in the wells of microtiter plates, and each was tested for the
ability to stimulate TLR2 via induction of IL-8 as described below. Following
each use, the column was washed with 1 M NaOH, followed by endotoxin-free
water and 0.1 M HCl, and stored in 20% ethanol.
Mice. Female C57BL/6 mice (6 to 8 weeks of age) were purchased from
Jackson Laboratory (Bar Harbor, ME). TLR2?/?mice were originally a gener-
ous gift of S. Akira (28) and have now been back-bred to C57BL/6 for 11
generations. All mice were bred under specific-pathogen-free conditions. All
animal studies were approved by the University of Massachusetts Medical School
Institutional Animal Care and Use Committee, and all relevant policies regard-
ing animal care, biosafety, and security were followed.
Experimental infection of mice. To prepare inocula for experimental infection,
Y. pestis KIM1001 was inoculated heavily onto TB plates from suspensions stored
at ?70°C in TB (no agar) plus 5% glycerol and incubated at 37°C for 24 h.
Bacteria were harvested from the plate with a loop and resuspended in sterile
distilled water with a vortex mixer to match a turbidity standard of an OD600of
0.3. It was important to use a low-ionic-strength medium for this initial suspen-
sion step to achieve good dispersion of the cells. This initial high-density sus-
pension was subsequently diluted as required using endotoxin-free injection-
grade PBS. For survival experiments, 10 age- and sex-matched mice per
treatment group were infected with Yersinia pestis KIM1001 by subcutaneous
(s.c.) injection of 50 ?l on the nape of the neck. For intravenous (i.v.) infection
experiments, five mice per group were injected with 500 ?l of inoculum contain-
ing the indicated doses in the tail vein. Survival was monitored every 12 h for up
to 21 days. All TLR2?/?were individually genotyped and bore uniquely num-
bered ear tags. For collection of organs, mice were sacrificed at 48 h following i.v.
infection by pentobarbital overdose followed by cervical dislocation. To deter-
3572 POULIOT ET AL.INFECT. IMMUN.
mine bacterial load, spleens were homogenized in 1 ml of PBS and titers of
bacteria in the resulting suspension were determined by serial dilution and
plating. Differences in survival were analyzed by Kaplan-Meyer survival analysis
and the log rank test.
Histopathology. Livers were fixed for in neutral buffered 4% formalin, and
sections were stained with hematoxylin-eosin.
Cytokine determination. (i) Cytokine determination from spleens. Spleens
were homogenized in 1 ml of PBS. Following removal of a sample for determi-
nation of bacterial titer, ciprofloxacin (100-?g/ml final concentration) and pro-
tease inhibitor cocktail (Roche catalog no. 11-873-580; 1? final concentration)
were added. The suspension was centrifuged at 10,000 ? g for 1 min. The
resulting supernatant was filtered to remove bacteria (0.2-?m pore, polyvinyli-
dene difluoride; Pall Acrodisc). Cytokines in the resulting supernatant were
measured using enzyme-linked immunosorbent assay (ELISA) kits from BD
Pharmingen (moIL-10) or R&D Systems (moTNFa and moIL-6) according to
the manufacturer’s directions. IL-10 levels were determined twice, each time in
triplicate from the same set of tissue samples.
(ii) Cytokine determination from tissue culture. Culture medium was removed
from the wells and diluted as required with fresh medium for use in the assays.
IL-8 was determined with a kit from R&D Systems (huIL-8). All cytokine assays
were performed in triplicate. The significance of observed differences in median
cytokine concentrations were analyzed by the nonparametric Pittman exact test.
NF-?B luciferase reporter assays. Cells were transfected with the NF-?B
luciferase reporter plasmid (a gift of Katherine Fitzgerald, University of Massa-
chusetts Medical School) using Genejuice (Promega) as described by the man-
ufacturer. Following stimulation and incubation as indicated, cells were lysed
with passive lysis buffer (Promega), and luciferase activity was measured by
luminometry following addition of luciferase substrate.
Preparation of Y. pestis rLcrV. The lcrV gene from Yersinia
pestis strain KIM1001 was cloned into the expression vector
pBADgIIIB (Invitrogen), resulting in pLcrVYp. (See Materials
and Methods for a detailed description of the protein pro-
duced by this construct.) rLcrV was expressed and purified by
nickel chelation chromatography as described in Material and
Methods. This method of purification yields primarily dimeric
and tetrameric rLcrV as shown by native PAGE, SDS-PAGE,
Western blotting, and gel filtration chromatography (see be-
Yersinia pestis rLcrV and stimulation of TLR2. His-tagged
rLcrV derived from Y. enterocolitica (rLcrVYe) has been re-
ported to activate TLR2 in a CD14-dependent manner (25). In
these reports, LcrV from Y. enterocolitica was cloned and ex-
pressed in E. coli with an N-terminal His6tag in the vector
pQE30 (QIAGEN), and purified by Ni2?chelation chroma-
tography. To determine if similar rLcrV preparations derived
from the Yersinia pestis lcrV gene are also able to stimulate
TLR2, we transfected HEK293 cells with TLR2 in either the
presence or absence of cotransfected CD14 and stimulated
them for 18 h with rLcrV purified as described above. As a
measure of TLR2-stimulating activity, cell supernatants were
assayed for IL-8 by capture ELISA. As shown in Fig. 1A, TLR2
is required for induction of IL-8 by rLcrV, and this induction
is enhanced by, but not dependent upon, the coexpression of
CD14. These observations are similar to the results reported by
Sing et al. (25) for rLcrVYe, except that we find enhancement
by, rather than a strict requirement for, CD14.
Because Sing et al. relied primarily on an NF-?B reporter
construct to indicate TLR2 activation in the HEK293/TLR2/
CD14 system, we also examined activation with a similar re-
porter construct. The results (Fig. 1C) were consistent with
observations made using IL-8 release.
FIG. 1. Activation of TLR2 by rLcrV. (A) HEK 293 cells stably
transfected with vector alone (pcDNA3), TLR2 alone, or both TLR2
and CD14 were stimulated with 10 ng/ml LPS, 1 ng/ml P3C, or 500
ng/ml rLcrV as indicated for 18 h. Supernatants were assayed by
capture ELISA for the presence of IL-8 as an indicator of TLR2
activation. (B) HEK293 cells stably transfected with both TLR2 and
CD14 were stimulated with 100 ng/ml LPS, 100 ng/ml P3C, 5 ?g/ml
rLcrV, or 20 ?g/ml of the indicated synthetic peptide for 18 h. Super-
natants were assayed by capture ELISA for the presence of IL-8 as an
indicator of TLR2 activation. (C) The same cells as for panel B were
transfected with an NF-?B luciferase reporter and stimulated with
P3C, MALP2, or rLcrV at the indicated concentrations for 18 h.
Relative luciferase activity is shown. Data shown are means from
triplicate assays with error bars indicating ranges and are representa-
tive of at least three experiments.
VOL. 75, 2007TLR2 AND VIRULENCE OF Y. PESTIS 3573
Yersinia pestis LcrV synthetic peptides fail to stimulate
TLR2. Peptides identical to specific domains of Y. enterocolitica
LcrV and capable of stimulating TLR2 have been previously
described (23, 25). These are derived from the N-terminal
globular portion preceding alpha helix 1 in the LcrV structure
(6). A peptide comprising residues 31 to 49, designated V7,
was most active. We tested the ability of the synthetic cognate
peptide based on the Y. pestis sequence, which differs at single
residue (K433N) from the homologous region in Y. enteroco-
litica, to activate TLR2. This peptide has identical residues at
all of the positions established by Sing et al. (23) to be required
for TLR2 activation. As shown in Fig. 1B, this peptide is
unable to stimulate HEK293 cells stably transfected with both
TLR2 and CD14. Sing et al. (23) observed only weak activity
with a longer synthetic peptide comprising residues 31 to 66 of
Y. pestis LcrV (23). A peptide comprising residues 27 through
43, corresponding to the inactive peptide designated V5 by
Sing et al. (25), was also found to be inactive.
Multimers of rLcrV in purified preparations. It has been
previously observed that in solution, rLcrV is not present in
significant amounts in monomer form but instead exists pri-
marily as a mixture of dimers and tetramers (29). Dimers can
form through disulfide bonds between the single cysteine res-
idue (C273) in each rLcrV molecule (6). To determine the
form(s) present in our rLcrV preparation, we analyzed samples
by electrophoresis, gel filtration, and immunoblotting. As
shown in Fig. 2, the major band of rLcrV seen by PAGE under
native conditions migrates at a rate expected of rLcrV tet-
ramer, and the preparation also contains higher-molecular-
weight forms. The addition of a reducing agent results in mi-
gration consistent with a dimer. Under combined reducing and
denaturing conditions, migration is consistent with a monomer.
Analysis by gel filtration on a calibrated Superose 12 column
yields similar results (Fig. 3). In the absence of a reducing
agent, the elution times of the two primary peaks are consistent
with a tetramer and a dimer. In the presence of glutathione,
the dimer forms the primary peak and the tetramer peak is
dramatically reduced (Fig. 3A, inset). In some preparations, a
small peak consistent with monomer was observed under re-
ducing conditions, but in no case did we detect monomer
without addition of glutathione to the elution buffer (data not
shown). We conclude that disulfide bond formation in neces-
sary for the existence of stable tetramers but not for that of
Both native PAGE and gel filtration also revealed high-
molecular-weight species (?200 kDa). These eluted near the
void volume of the column during gel filtration. The integrated
absorbance of this early peak was reduced only slightly in the
presence of glutathione, indicating that its integrity is not de-
pendent on disulfide bonds (Fig. 3A, inset). The presence of
rLcrV in these high-molecular-weight fractions was confirmed
by immunoblotting (Fig. 3B, inset). Silver-stained SDS-poly-
acrylamide gels showed only a single band consistent with
rLcrV in these fractions (data not shown), indicating that
rLcrV is the major protein component of this material.
The high-molecular-weight species containing rLcrV is re-
sponsible for TLR2-stimulating activity. To determine which
form(s) of rLcrV contains TLR2-stimulating activity, fractions
from rLcrV preparations separated on Superose 12 were as-
sayed for TLR2-stimulating activity (Fig. 3). HEK293 cells
stably transfected with TLR2 and CD14 were treated with a
sample of each fraction, and IL-8 was measured as an indicator
of TLR2 activation. Surprisingly, neither the dimer nor tet-
ramer form was able to stimulate TLR2. Virtually all of the
TLR2-stimulating activity was associated with the higher-mo-
lecular-weight fractions, which contained less than 10% of the
total rLcrV protein. The pattern of activity among fractions
was remarkably consistent when independent preparations
were compared, even when different cell disruption techniques
(sonication versus French pressure cell) that yielded different
proportion of dimer and tetramer, but a consistent proportion
of high-molecular-weight material, were used (compare Fig.
3A and B).
Taken together, these results indicate that the bulk of the
TLR2-stimulating activity of our rLcrV resides in stable mul-
timers or aggregates substantially larger than a tetramer.
rLcrV is the major, and perhaps the only significant, protein
component of this material. However, the presence of nonpro-
tein components, or of highly active protein components in low
abundance, cannot be excluded. It is also possible that the
tetramer and lower-molecular-weight forms have some stimu-
latory activity obscured by the tailing peaks of the higher-
activity fractions, but if so, this activity must be very weak.
Analysis of an alternative rLcrV construct. Virtually all
studies of the immunosuppressive properties of LcrV are
based on recombinant constructs with somewhat different
structures. For example, the parent rLcrV fusion protein uti-
lized by Overheim et al. (22) differs from ours in two significant
ways: it has an amino-terminal decahistidyl domain as opposed
to the carboxy-terminal hexahistidyl domain in our construct,
FIG. 2. Analysis of rLcrV by PAGE. Lanes 1 and 8, molecular mass
markers formulated for use in native gels, with their sizes in kilodaltons
indicated at the left. In the absence of SDS, DTT, and thermal dena-
turation, rLcrV migrates at a rate consistent with a tetramer, with some
dimer also present (lane 2). Addition of DTT results in migration
consistent with a dimer (lane 4). The combination of DTT and dena-
turing conditions (lane 6) results in migration consistent with a mono-
mer. Lanes containing rLcrV were separated by an empty lane, as
diffusion of DTT into adjacent lanes affected migration. High-molec-
ular-mass species of greater than 200 kDa are visible under native
conditions when the gel is loaded with 5 ?g rLcrV (lane 9). The bands
are less distinct than usual for SDS-PAGE because the buffers do not
contain SDS, allowing some loss of detergent from the protein as it
3574 POULIOT ET AL.INFECT. IMMUN.
and it includes the native Y. pestis LcrV sequence with no other
additions. In contrast, our mature construct has three addi-
tional amino-terminal resides (TMV) that precede the native
LcrV domain and two additional carboxy-terminal residues
(VD) which precede the added polyhistidine sequence. To
determine if our finding that TLR2 stimulation is due to high-
molecular-weight forms was robust to such differences, we
purified and analyzed rLcrV produced by the construct used by
Overheim et al. (22). The results of these experiments are
shown in Fig. 4. As was found with our construct, all of the
TLR2-stimulating activity of this rLcrV form was associated
with fractions having much shorter elution times than those for
dimers and tetramers. Also, in agreement with results obtained
with our construct, almost all of the rLcrV was in the form of
dimers and tetramers. No monomer was present. In fact, the
preparation analyzed in Fig. 4 contained less high-molecular-
FIG. 3. Analysis of rLcrV by gel filtration. (A) rLcrV purified from sonic extracts was separated on a calibrated Superose 12 column. The
elution profile (A280) of the primary peaks indicates that rLcrV dimers and tetramers are the major species present. A high-molecular-weight peak,
eluting near the void volume of the column, is also present. The profile obtained when 1 mM glutathione is included in the column buffer is shown
in the inset. Note conversion of the tetramer peak to dimer but retention of the high-molecular-weight fraction. Samples of fractions were also
assayed for TLR2-stimulating activity (E) as for Fig. 1. Note that this activity is associated almost exclusively with material eluting prior to rLcrV
tetramer. (B) An independent preparation of rLcrV purified from extracts made via a French pressure cell was analyzed as for panel A. This
preparation contained a much higher proportion of tetramers and a similar proportion of high-molecular-weight material. Assay of fractions for
TLR2-activating activity (E) again showed the material eluting before the tetramer to be most active. The inset shows an immunoblot of selected
fractions prepared from a standard SDS-polyacrylamide gel and probed with an LcrV-specific monoclonal antibody. The selected fractions are
indicated by circled numerals. Note that the high-molecular-weight fractions contain rLcrV.
VOL. 75, 2007TLR2 AND VIRULENCE OF Y. PESTIS 3575
weight material than we had observed with our construct, and
its TLR2-stimulating activity was correspondingly lower (Com-
pare Fig. 4 and 3, noting the differences in scaling on the IL-8
concentration axis.) Thus, it appears that our basic observa-
tions are not sensitive to minor differences in rLcrV primary
structure present in the amino- and carboxy-terminal domains.
Infection of TLR2?/?mice. The interaction between Yer-
sinia enterocolitica rLcrV and TLR2 is reported to result in an
anti-inflammatory effect, including suppression of TNF and
induction of IL-10 (23–25), leading to decreased macrophage
activation. During Y. enterocolitica infection, this mechanism is
proposed to contribute to evasion of innate immunity. To ad-
dress the potential relevance of this mechanism to plague, we
compared the diseases produced in wild-type and TLR2?/?
mice following s.c. infection with virulent Y. pestis. A dose of
1,000 CFU of Y. pestis strain KIM1001 was uniformly lethal for
both C57BL/6 mice and C57BL/6 TLR2?/?mice, and no sig-
nificant difference in mean time of survival was observed (Fig.
5A). Subtle differences in resistance are more readily observed
at doses that do not cause complete mortality. Accordingly, we
conducted an additional comparison using a dose of 100 CFU,
again delivered s.c. (Fig. 5B). This dose yielded 80% mortality
in both wild-type and TLR2?/?animals. Again, no significant
difference in mean time of survival between mouse genotypes
Bacterial load and cytokine levels in mice infected with Y.
pestis. To determine if the bacterial load and/or the induction
of selected cytokines during infection was influenced by the
TLR2 status of the mice, we measured the numbers of bacteria
and the levels of IL-10, TNF, and IL-6 in the spleens of five
mice of each genotype at 2 days following i.v. infection with
1,000 CFU of KIM1001. The i.v. route was used in this exper-
iment because it results in essentially simultaneous infection of
internal organs; with s.c. infection, the time of dissemination of
the bacteria sometimes varies among the animals. The ob-
served differences in mean bacterial load and IL-6 titers were
not statistically significant (Fig. 6C and D). IL-10 levels were
about twofold greater (1.07 versus 0.58 ng/ml) in TLR2?/?
mice (Fig. 6A), a difference that was statistically significant
(P ? 0.04) but which is inverse to the result expected if inter-
action of LcrV with TLR2 elevates IL-10 levels. Despite the
enhanced IL-10 levels in TLR2?/?mice, their mean TNF
levels were also slightly but significantly higher than those
observed in the wild-type controls (34% [621 versus 462 pg/ml];
P ? 0.02) (Fig. 6B).
Inflammation at foci of infection. If TLR2-dependent stim-
ulation of IL-10 production makes an important contribution
to inhibition of local inflammation, we would expect enhanced
inflammation at foci of infection in TLR2?/?mice. This should
be readily observable in the livers of infected mice, because
there is little inflammatory response to wild-type Y. pestis in
this tissue (18, 27) and any enhancement of the inflammation
can be readily detected. Accordingly, we harvested livers from
the mice at 2 days after infection and examined liver sections
to determine the state of inflammation at foci of infection. As
FIG. 4. Analysis of an alternative rLcrV construct by gel filtration.
rLcrV produced by the prLcrV construct used by Overheim et al. (22)
purified as described in Materials and Methods was analyzed in exper-
iments parallel to those of Fig. 3. Note that the results are very similar,
although the content of high-molecular-weight material is somewhat
lower, as are the levels of TLR2-stimulating activity of the correspond-
ing fractions. Analysis via immunoblotting as in Fig. 3B confirmed the
presence of rLcrV in the active fractions (data not shown).
FIG. 5. TLR2 deficiency and survival. Wild-type (WT) and
TLR2?/?mice (10 animals per group) were infected s.c. with
KIM1001. Survival was monitored for 14 days, and no deaths were
observed beyond day 9. (A) Dose of 1,000 CFU. Note that survival
times are very similar (means for WT and TLR2?/?, 4.25 and 5.25
days, respectively; P ? 0.85). The results shown are representative of
three experiments. (B) Dose of 100 CFU. At this lower dose, survival
times are also very similar (means for WT and TLR2?/?, 7.25 and 6.5
days, respectively; P ? 0.75). The experiment was performed once at
this dose. (The TLR?/?group in this experiment contained 11 mice.)
3576 POULIOT ET AL.INFECT. IMMUN.
shown in Fig. 6E and F, sections from both mouse genotypes
show focal bacterial masses largely devoid of inflammatory
cells. No evidence of enhanced inflammation in TLR2-defi-
cient animals was observed.
The major goal of this work was to examine the hypothesis
that the interaction of LcrV and TLR2 contributes significantly
to Y. pestis virulence, whether via the induction of IL-10 or by
any other means. In the most direct interpretation, this model
predicts that TLR2?/?mice will show (i) enhanced resistance
to Y. pestis, (ii) enhanced inflammation at foci of infection,
and (iii) decreased IL-10 and enhanced proinflammatory cy-
tokine production during Y. pestis infection. The results of our
experiments with TLR2?/?mice did not confirm these predic-
tions. We observed no significant differences in survival, mean
time to death, or bacterial load; no evidence of enhanced
inflammation at foci of infection; and an increase, rather than
a reduction, in IL-10 titers. We did observe a slight increase in
TNF titer, but this occurred in the presence of enhanced rather
than reduced IL-10 levels. The other proinflammatory cytokine
measured, IL-6, showed no change. In comparison with sur-
vival experiments, small numbers of animals were utilized for
bacterial titer and cytokine measurements, and we may have
failed to observe modest differences. For example, for bacterial
titers and IL-6 titers, the two measurements for which no
significant differences were observed, power analysis using a
sample size of five and the measured variances of our obser-
vations yields an 80% probability of detecting differences of
about twofold relative to the mean values, with a correspond-
ingly lower probability of detecting smaller differences. An-
other limitation of these experiments is that measurements
were made at a single time point, 2 days postinfection, and we
cannot be certain that our results reflect conditions pertaining
at earlier stages of infection. However, both absolute survival
and survival kinetics were remarkably consistent at both doses
tested, indicating that any influence of TLR2 on the course of
infection must be minor. It should be noted that the virulent Y.
pestis-mouse infection model is a very sensitive one, in that
specific genetic modifications of bacteria or mice often have
large effects on virulence. For example, otherwise virulent mu-
tants lacking the Pla protease (27, 31) and mutants with defects
in iron acquisition (31) show an increase in 50% lethal dose of
several orders of magnitude, as do strains modified to produce
highly stimulatory LPS (15). TLR4-deficient mice are highly
susceptible to a strain producing stimulatory LPS, while wild-
type mice are highly resistant (15). In both of these instances,
reduced virulence (or enhanced resistance) was also associated
with marked enhancement of inflammation at foci of infection.
Thus, it is clear that experimental manipulations in this system
do indeed have very large effects when they are related to
functional differences in the interaction between the bacteria
and host defenses. Our failure to observe any indication of
enhanced resistance or improved inflammatory response in
TLR2 deficient mice must therefore be regarded as strong
evidence that this receptor does not play a significant role in
interactions contributing to virulence of Y. pestis during infec-
tion. Because we did not carefully examine the progress of
infection at multiple time points, it remains possible that there
are subtle differences in the development of infection between
the genotypes that do not affect survival or time to death.
The literature regarding the induction of IL-10 by the LcrV
of the yersiniae presents a somewhat confusing picture. Sing et
al. reported that specific residues are required for induction of
IL-10 by LcrV of Yersinia enterocolitica and that peptides con-
taining these residues are effective inducers (23, 25). However,
they also found that a peptide from the cognate region of Y.
pestis and Y. pseudotuberculosis LcrV has little activity (23). On
the other hand, Overheim et al. reported that regions of LcrV
entirely distinct from that defined by Sing et al. are required
FIG. 6. TLR2 deficiency has little effect during infection with Yer-
sinia pestis. Wild-type (WT) and TLR2?/?mice were infected i.v. with
1,000 CFU KIM1001. (A to C) Spleens and livers were harvested at 2
days postinfection. Spleens were homogenized, and levels of IL-10
(A) TNF (B), and IL-6 (C) were quantified by capture ELISA. No
significant differences were detected for any cytokine between geno-
types, although the higher TNF levels in TLR2?/?mice were sugges-
tive (P ? 0.056). (D) Bacterial loads in the spleens of the two geno-
types were also similar (P ? 0.2). (E and F) Liver sections from both
genotypes stained with hematoxylin and eosin showed a pattern typical
of Y. pestis infection in WT mice. Masses of bacteria occupy liver
sinusoids, with little sign of local inflammation (white arrows). At least
20 foci in the livers of each of five mice of each genotype were exam-
ined by an investigator blind to the sample source. (G) For reference,
a liver section from an uninfected WT control is shown. Comparable
sections from a Y. pestis derivative that does induce strong local in-
flammation are presented in reference 15.
VOL. 75, 2007TLR2 AND VIRULENCE OF Y. PESTIS 3577
for IL-10 induction by Y. pestis LcrV (22). While Sing et al.
provided evidence from a well-established in vivo model sup-
porting a role for the TLR2-LcrV interaction during Y. entero-
colitica infection (23, 25), these results are unfortunately de-
pendent on the mouse strain employed (26). No similar
evidence has been published previously regarding Y. pestis. For
this species, currently available in vivo data are indirect in that
all the relevant experiments involve injection of mice with
various forms of rLcrV, followed by measurement of cytokine
levels and/or challenge with LPS, attenuated Y. pestis, or other
unrelated pathogens (18, 19). While the differences in IL-10
levels that we observe are not consistent with the LcrV-TLR2
model (levels were not reduced in TLR2-deficient animals),
IL-10 is clearly elevated in mice with well-developed Y. pestis
infection. Thus, we cannot rule out the possibility that immu-
nosuppression due to elevated IL-10 levels induced by a TLR2-
independent mechanism plays an important role in plague.
Indeed, it is possible that the unusual combination of proin-
flammatory stimuli presented by Y. pestis, which as we have
shown previously activates TLR4 very poorly (15), results in an
aberrant cytokine profile that may compromise innate de-
A variety of fusion proteins have been used to demonstrate
the immunosuppressive properties of LcrV. For example,
Overheim et al. utilized an N-terminal decahistidyl tag (22).
Motin et al. fused a 34-kDa fragment of protein A to the N
terminus of a truncated LcrV lacking the first 67 residues (16).
The three-dimensional structure of LcrV was determined from
a fusion protein containing five residues fused to an LcrV N
terminus beginning at residue 28 and a C-terminal addition of
four residues plus a hexahistidyl tag (6). This structure shows
that both the N and C termini are very flexible and are located
near each other, external to one of the LcrV globular domains.
The flexibility and location of these termini are consistent with
tolerance for additions and deletions. Thus, it is unlikely that
the three-residue N-terminal and the eight residue C-terminal
additions (the latter including a six-residue His tag) present in
our LcrV construct are less reflective of the properties of
native LcrV than those employed by others. Moreover, we
have shown directly that the rLcrV protein of Overheim et al.
(22) behaves similarly to our own. It should be noted that the
cytokine-inducing properties of native LcrV purified from Y.
pestis or any other Yersinia species have not been studied.
Our results with His-tagged Y. pestis rLcrV are consistent
with those of others in that we do observe stimulation of TLR2
in vitro. However, we also show that the ability of this material
to stimulate TLR2 is unexpectedly complex at the biochemical
level. The major forms of rLcrV in our preparations, dimer and
tetramer, have no TLR2-stimulating activity. Such activity is
detected only in high-molecular-weight multimers or aggre-
gates. Although the data from experimental infections dis-
cussed above argue strongly against a role for this activity in Y.
pestis virulence, we consider three alternative hypotheses re-
garding TLR2 stimulation by LcrV, one of which implies phys-
First, the stimulatory activity may result from the presence
of a potent TLR2-activating contaminant (e.g., a lipoprotein,
lipopeptide, peptidoglycan, etc.) constituting a small propor-
tion of the aggregate and not from rLcrV per se. Such con-
tamination is both common in material purified from whole-
cell extracts and notoriously difficult to exclude. TLR2-
stimulating activity initially attributed to what were thought to
be highly purified materials has later been shown to result from
such contamination (for example, see references 10, 12, and
33). There is no general method to ensure freedom from such
contaminants in protein preparations. Note that vulnerability
to such contaminants is greatly increased when cells expressing
a variety of TLRs, such as mouse macrophages, are the targets
A second possibility is that the stimulatory activity is indeed
due to rLcrV in the aggregates/multimers but that this material
is nonphysiological and is an artifact of overexpression and
purification techniques. The tendency of recombinant proteins
expressed at high levels in E. coli to form high-molecular-
weight aggregates is well established. Exposure of mature
rLcrV to cell extracts, as occurs during purification, could also
be critical to formation of the stimulatory material. In Y. pestis
the level of LcrV expression is much lower, and exposure of
mature LcrV to concentrated cell extracts does not occur. In
this view, the stimulatory aggregates/multimers are purely an in
vitro artifact and have no physiologic relevance.
A third possibility is that the stimulatory fractions contain
structures that are, or resemble, physiological multimeric spe-
cies that are normally detected by TLR2 as an indicator of
pathogens with TTSS machinery. Micrographs of LcrV ar-
ranged at the tips of secretion needles (17) suggest to us an
octamer composed of four dimers. Perhaps such structures are
occasionally released from bacteria in vivo and elicit proin-
flammatory responses via TLR2. However, there is no evi-
dence to support this idea from infection experiments with
yersiniae. Also, PcrV, a related protein from Pseudomonas
aeruginosa, does not stimulate TLR2 (25).
The association of TLR2 stimulation with aggregates/mul-
timers of rLcrV suggests that individual rLcrV molecules may
interact weakly with TLR2 and that activation results from
clustering of the receptor. This would explain the failure of
low-molecular-weight forms to cause activation. Other groups
have not reported the molecular sizes of the active fractions in
their rLcrV preparations under nondenaturing conditions.
Consequently, the presence of a similar stimulatory high-mo-
lecular-weight aggregate in their experiments cannot be ex-
cluded. The specific activity of our preparations is similar to
that reported by others; if the activity that they observe is not
due to aggregates, which we find to constitute a small fraction
of total protein, then the activity of rLcrV in their preparations
on a per-molecule basis must be correspondingly low. The
clustering hypothesis suggests that rather than disrupting a
specific TLR2-LcrV interaction, mutations which give rise to
inactive rLcrV preparations may interfere with formation of
aggregates capable of activating TLR2.
Overheim et al. (22) have presented data suggesting that
LcrV deletion mutants failing to stimulate IL-10 production
are more effective immunogens, presumably due to the elimi-
nation of immunosuppressive activity. Our results show that
highly purified LcrV preparations containing only dimers
and/or tetramers lack TLR2-stimulating activity and hence
may also have improved performance in vaccine applications.
However, we have previously shown that with DNA vaccines,
production of LcrV multimers in vivo was critical to providing
an effective protective response and also biased the response
3578 POULIOT ET AL.INFECT. IMMUN.
toward TH1 compared with the response produce by con-
structs producing only rLcrV monomers (30). While we do not
know the extent of multimerization occurring in vivo, the
association of multimerization with TLR2 stimulation sug-
gests the possibility that large multimers form in vivo and
provide adjuvant activity in the context of the live vaccine
through stimulation of TLR2, rather than immunosuppres-
sion. This adjuvant effect is more consistent with current
understanding of TLR2 function than is an immunosuppres-
In summary, our investigation provides no support for the
hypothesis that activation of TLR2 by LcrV contributes to the
virulence of Y. pestis via immunomodulation. In a sensitive
infection model using virulent Y. pestis, elimination of TLR2
has no effect on the course of disease and little on cytokine
levels observed in vivo. The bulk of rLcrV protein has no
TLR2-stimulating activity in vitro, and such activity is re-
stricted to high-molecular-weight aggregates/multimers which
contain LcrV but are of undetermined composition. Given the
well-established sensitivity of the Y. pestis mouse infection
model, its lack of response to TLR2 deficiency must be re-
garded as strong evidence that TLR2-induced immunomodu-
lation does not have a significant role in plague. The early
observations suggesting a direct immunosuppressive role for
LcrV were based on direct injection of rLcrV preparations into
mice, resulting in immunosuppression and elevated levels of
IL-10 (19). A TLR2-independent mechanism of IL-10 induc-
tion would be consistent with these early observations.
Detailed infection experiments have also been conducted
with Yersinia enterocolitica and Yersinia pseudotuberculosis by
Victoria Auerbuch and Ralph Isberg (2a). They also observed
no differences in the course or pattern of disease, or in cyto-
kine levels, between TLR2-sufficient and -deficient mice. The
conflicting results of Sing et al. imply that LcrV-TLR2-medi-
ated immunosuppression may operate under certain limited
circumstances (i.e., with specific combinations of Y. enteroco-
litica strains and mouse strains), but, given the present weight
of evidence, it is unlikely to be a phenomenon of general
importance to virulence in the yersiniae.
We thank Nancy Deitemeyer and Chrono Lee for general technical
assistance and Neal Silverman and Eicke Latz for advice regarding
purification of protein with minimal contamination by TLR-activating
substances. We also thank Olaf Schneewind for use of his rLcrV
construct and Stephen Baker for help with statistical analysis.
This work was supported by Project 2 of grant AI057159 to J.D.G.
and by grant AI057588 to E.L. from the National Institutes of Health.
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