Journal of Infectious Diseases Advance Access published March 21, 2014

MAJOR ARTICLE

The Virulence Polysaccharide Vi Released

by Salmonella Typhi Targets Membrane
Prohibitin to Inhibit T-Cell Activation
Srikanth K. Santhanam, Debjani Dutta,a Farhat Parween, and Ayub Qadri
Hybridoma Laboratory, National Institute of Immunology, New Delhi, India

Keywords.

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T cells are critical to immunity against pathogenic Salmonella including Salmonella Typhi which causes systemic infection, typhoid, in humans. The strategies that this pathogen employs to keep T-cell mediated immune
responses in check during establishment of systemic infection are not completely understood. Here, we show
that the virulence polysaccharide Vi, which distinguishes S. Typhi from localized gastroenteritis-producing nontyphoidal Salmonella serovars, is a potent inhibitor of T-cell activation. Vi released by S. Typhi interacts with the
membrane prohibitin complex and inhibits IL-2 secretion from T cells stimulated through the T-cell receptor
(TCR) but does not affect PMA-activated interleukin 2 (IL-2) secretion. Treatment with Vi suppresses early
activation events including TCR down-regulation, actin polymerization, and phosphorylation of ERK. Coadministration of Vi with anti-CD3 Ab reduces secretion of IL-2 and interferon in mice. Our ndings reveal
a mechanism by which S. Typhi may target T-cell immunity during establishment of typhoid.
Salmonella Typhi; virulence polysaccharide Vi; prohibitin; T cells.

Salmonella Typhi enters the body through the oral

route, invades the intestinal epithelium, and disseminates throughout the reticuloendothelial system. It produces this systemic infection almost exclusively in
humans [1, 2]. Due to this extreme host specicity,
the pathogenesis of typhoid infection is poorly understood. Unlike S. Typhi, Salmonella Typhimurium produces a systemic infection in susceptible strains of
mice that is analogous to human typhoid. Therefore, infection of mice with S. Typhimurium has been used as a
model to understand pathogenesis of human typhoid
[3]. Even though this model does not truly represent
S. Typhi infection in humans as S. Typhimurium does
not cause typhoid in humans, it has provided important
insights into Salmonella pathogenesis in general. Studies carried out with this model have demonstrated that

Received 15 October 2013; accepted 15 January 2014.

a
Present afliation: Laboratory of Immune Cell Biology, Center for Cancer
Research, NCI, NIH, Bethesda MD 20814. debjanidutta14@gmail.com
Correspondence: Ayub Qadri, Ph.D., National Institute of Immunology, New Delhi110067, India (ayub@nii.ac.in, qadriayub@gmail.com).
The Journal of Infectious Diseases
The Author 2014. Published by Oxford University Press on behalf of the Infectious
Diseases Society of America. All rights reserved. For Permissions, please e-mail:
journals.permissions@oup.com.
DOI: 10.1093/infdis/jiu064

T cells and T-cell-derived cytokines such as interferon

(IFN-) are required for clearance of bacteria and for
long-term immunity against Salmonella [47]. Human
volunteers immunized with vaccine strains of S. Typhi
showed activation of CD4+ and CD8+ T cells, which had
protective characteristics similar to those reported for
T cells in the mouse model [8]. Collectively, these studies have suggested an important role for T cells in protective immunity against S. Typhi. How S. Typhi might
thwart T-cell immunity during establishment of
typhoid is unclear.
One of the major distinctions between S. Typhi and
S. Typhimurium is the presence of virulence polysaccharide Vi in S. Typhi [9]. This polysaccharide prevents
complement-mediated killing of S. Typhi and renders
this pathogen resistant to phagocytosis [10]. We have
previously shown that Vi can suppress inammatory responses from intestinal epithelial cells and monocytes
through its interaction with membrane prohibitin [11,
12]. Prohibitin belongs to a family of evolutionarily conserved proteins that include the ottilins, stomatin and
the stomatin-like protein, podocin, and erlins. These
proteins have been implicated in a large number of cellular functions such as protein trafcking, stabilization
of mitochondrial respiratory chain complexes, cell

Vi Inhibits T-Cell Activation

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proliferation, and apoptosis and cell signaling [1315]. Prohibitin and its related members have been reported to be associated
with antigen receptors in B cells [16]. More recently, prohibitin
has been found to be important for raf-rasdependent activation of extracellular regulated kinase (ERK) in epithelial
cells [17].
In this study, we provide evidence that targeting of membrane prohibitin in T cells by Vi released from S. Typhi can
down-regulate T-cell responses. Treatment with this polysaccharide inhibited T-cell receptor (TCR)induced intracellular
signaling including activation of MAP-kinases and suppressed
cytokine secretion in vitro as well as in vivo. The expression of
Vi might therefore provide S. Typhi a strategy to interfere with
T-cell activation.

inhibition in IL-2 secretion from activated T cells was also mimicked by bacterial culture supernatant derived from Vi positive
S. Typhi but not by supernatants obtained from Vi-decient
S. Typhi or other bacterial strains (Figure 2J). The down
-regulation of IL-2 secretion required presence of acetyl groups
in the polysaccharide as this effect was not observed with deacetylated Vi (Figure 2K and 2L). It was also not seen with the capsular polysaccharide derived from Streptococcus pneumoniae
pneumococcal capsular polysaccharide (PCP) or lipopolysaccharide (LPS) isolated from Salmonella (Figure 2K and 2L), suggesting that the ability to suppress IL-2 secretion from antigen
receptor activated T cells was specic to Vi.

RESULTS

To get insights into how Vi inhibits T-cell activation, further experiments were carried out with Jurkat. Immunoprecipitation
with Vi revealed that the Vi-recognition complex in T cells
composed of prohibitin associated with its closely related homolog BAP-37, similar to what we had previously reported
with IECs and monocytes (Figure 3A) [11, 12]. Microscopic
analysis showed punctuate staining with Vi, indicating that
the membrane prohibitin complex interacting with this polysaccharide might be localized within specic membrane domains
(Figure 3B). Similar staining pattern was observed during interaction of T cells with Vi+ S. Typhi but not with Vi- S. Typhi or
S. Typhimurium (Figure 3B).
To analyze effect of Vi-prohibitin interaction on early activation events, we looked at TCR down-regulation from the
surface following activation with anti-CD3 antibody or PMA.
Vi suppressed internalization of TCR in cells activated with
anti-CD3 antibody without affecting PMA-activated TCR internalization (Figure 3C and 3D). In fact, PMA-induced TCR
internalization was increased in presence of Vi (Figure 3). Vi
also inhibited anti-CD3 Ab-induced CD69 expression and
ERK phosphorylation in Jurkat (Figure 3E and 3F ), but it did
affect PMA-activated ERK phosphorylation (Figure 3F ).
The activation of TCR-triggered proximal signaling events is
largely regulated through actin cytoskeletal rearrangements
[18]. We therefore considered the possibility that ligation of
membrane prohibitin complex with Vi might interfere with
actin polymerization. This was investigated by analyzing staining of cells with the fungal toxin Phalloidin, which binds lamentous actin. A signicant proportion of cells treated with
Vi for 1 hour at 37C showed reduced binding to Phalloidin
indicating depolymerisation of preexisting actin laments
(Figure 4). This effect was time-dependent, and recovery of
actin polymerization was observed at later time point (Figure 4).
These results suggested that ligation of membrane prohibitin
with Vi during TCR activation might interfere with intracellular
signaling through modulation of actin cytoskeletal rearrangements in Jurkat cells.

To study possible effects of Vi on T-cell activation, we rst analyzed binding of Vi to T cells. Vi showed a dose-dependent
binding to the model human T-cell line Jurkat (Figure 1A).
This binding was also observed with bacterial culture supernatant obtained from Vi positive but not Vi negative S. Typhi indicating release of this polysaccharide during growth of bacteria
in vitro (Figure 1B). Signicantly, the release of Vi and its interaction with T cells was also seen during infection of splenocytes
with Vi positive S. Typhi ex vivo (Figure 1C). Vi readily bound
to peritoneal T cells in vivo (Figure 1D).
To investigate if binding of Vi to T cells could modulate
T-cell response, Jurkat cells were stimulated through the T-cell
receptor in the presence or absence of this polysaccharide,
and interleukin 2 (IL-2) was analyzed in cell supernatants. Because our previous study had shown that the anti-inammatory
effect of Vi on human monocytes could be modulated by serum
and serum-derived hemoglobin (Hb) [12], we carried out stimulations under serum-free and serum-supplemented conditions
to see if that phenomenon existed in T cells as well. Vi inhibited
IL-2 secretion from Jurkat in a dose-dependent manner in the
absence of serum (Figure 2A). This inhibition was not due to
blockade of binding of anti-TCR antibody to cells or induction
of cell death (Figure 2B and 2C). Vi did not inhibit PMA/ionophore - activated IL-2 secretion from Jurkat (Figure 2D). The
lack of suppression with Vi in presence of serum or serumderived hemoglobin reafrmed the role of circulating Hb in
neutralizing immune-inhibitory capability of Vi (Figure 2E
and 2F ). The inhibition brought about by Vi was not restricted
to Jurkat as the polysaccharide also inhibited cytokine secretion
from peripheral blood-derived human lymphocytes activated ex
vivo with anti-CD3 antibody plus anti-CD28 antibody, as well
as from mouse T cells activated with anti-TCR antibody or specic peptide-MHC complexes (Figure 2GI). Vi-mediated
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Vi Suppresses Antigen ReceptorInduced Cytokine Secretion

From T cells

Vi Engages Membrane Prohibitin Complex and Inhibits Early

Activation Events

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Figure 1. Vi binds T cells. Jurkat cells were incubated with different concentrations of Vi (A) or with Salmonella Typhiderived bacterial culture supernatants (B) for 1 hour at 4C followed by anti-Vi MoAb and FITC-labeled anti-mouse Ig Ab. Cells were analyzed by ow cytometry. Control cells (shaded
histogram) were incubated only with anti-Vi MoAb and FITC-anti-mouse Ig Ab. Data were plotted using WinMDI software. C.S., culture supernatant.
C, Splenocytes isolated from C57Bl/6 mice were infected with S. Typhi at different MOI. One hour later, T cells were stained with PE-labeled anti-TCR
antibody and Vi followed by anti-Vi MoAb and FITC-labeled anti-mouse Ig Ab. Cells were analyzed by ow cytometry. The results are representative of 23
independent experiments. D, Mice were injected with 100 g Vi or equivalent volume of PBS intraperitoneally. One hour later, cells isolated from the
peritoneum were incubated with anti-Vi MoAb and FITC-labeled anti-mouse IgG followed by PE-labeled anti-TCR antibody for 1 hour. Cells were run in BD
Accuri ow cytometer, and data were analyzed using Flow Jo. The results are representative of 2 independent experiments of 2 mice each. Abbreviations:
Ab, antibody; IgG, immunoglobulin G; MOI, multiplicity of infection; PBS, phosphate-buffered saline; TCR, T-cell receptor; Vi, virulence polysaccharide.

Vi Suppresses T-Cell Activation in vivo

The effect of Vi on T-cell activation in vivo was examined by

analyzing secretion of cytokines upon administration of antiCD3 antibody. Our previous study had shown that depending

on how Vi was presented to mononuclear phagocytes, Vi could

either be anti-inammatory or generate inammatory responses
[12]. Therefore, to circumvent any interference from such inammatory responses on the ability of Vi to modulate T-cell

Vi Inhibits T-Cell Activation

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responses in vivo, we employed MyD88 decient mice, which

do not produce cytokine responses with Vi (authors unpublished data). Mice were injected with anti-CD3 antibody in
the absence or presence of Vi, and IL-2 and IFN- levels were
determined in sera. Mice injected with anti-CD3 antibody readily produced IL-2 and IFN- (Figure 5A and 5B). However, this
response was signicantly reduced in mice, which received Vi
along with anti-CD3 antibody (Figure 5A and 5B).

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DISCUSSION
S. Typhi causes systemic infection typhoid in humans, whereas
nontyphoidal Salmonella serovars such as S. Typhimurium produce only self-limiting gastroenteritis that is associated with a potent inammatory response in the gut [19]. To date, very little is
known about the identity of specic host-pathogen interactions
that might be responsible for different clinical manifestations

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Figure 2. Vi inhibits IL-2 secretion from activated T cells. A, 105 Jurkat cells were stimulated with plate-coated anti-CD3 antibody (10 g/mL) and soluble
anti-CD28 antibody (5 g/mL) in the absence or presence of Vi. Cell stimulations were carried out in serum-free RPMI-1640. 18 hours later, IL-2 was
determined in the cell supernatants by ELISA. B, Cells were incubated with Vi (5 g/mL) at 4C followed by anti-CD3 antibody and FITC-labeled goat
anti-mouse IgG. Cells were analyzed by ow cytometry. Shaded histogram shows staining only with FITC anti-mouse IgG. C, Cell death was analyzed
by propidium iodide uptake in Jurkat activated in serum free medium in the presence or absence of Vi (5 g/mL). Shaded histogram represents staining
with untreated cells. D, IL-2 secretion was determined in Jurkat stimulated for 20 hours with PMA and ionophore in the absence or presence of Vi. E and F,
105 cells were stimulated with plate coated anti-CD3 antibody and soluble anti-CD28 antibody in RPMI-1640 supplemented with 10% FCS, or serum-free
RPMI-1640 supplemented with hemoglobin (Hb)/BSA in the absence or presence of Vi. IL-2 was determined by ELISA.

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Figure 2 continued. G, Nonadherent cells (0.3 105 cells/well) derived from human peripheral blood were stimulated with plate-coated anti-CD3 antibody and
soluble anti-CD28 antibody in serum-free RPMI-1640 in the absence or presence of Vi (5 g/mL). 48 hours later, IL-2 was determined in the cell supernatants by
ELISA. H, Murine T-cell hybridoma was stimulated with plate-coated anti-TCR antibody in the presence or absence of Vi or LPS, or (I) with MBP - derived peptide
Ac1-11(4Y), along with I-Au - expressing B cell lymphoma line, PL-8, in the presence or absence of Vi. After 18 hours at 37C, IL-2 secretion was analyzed by ELISA.
J, T cells (mouse T-cell hybridoma) were stimulated with plate-coated anti-TCR antibody in the presence or absence of bacterial culture supernatants derived from
Vi+ Salmonella Typhi, Vi- Salmonella Typhi, Salmonella Typhimurium or Escherichia coli. IL-2 was determined by ELISA. K, Jurkat T cells or (L) mouse splenocytes
were stimulated with anti-CD3 antibody in the presence of Vi, de-acetylated Vi (DeVi), PCP or LPS and IL-2 was analyzed in the supernatants. Data are representative of 23 independent experiments. *P value < .05; hIL-2 is human IL-2, and mIL-2 is mouse IL-2. Abbreviations: ELISA, enzyme-linked immunosorbent assay; IL2, interleukin 2; IgG, immunoglobulin G; LPS, lipopolysaccharide; PCP, pneumococcal capsular polysaccharide; TCR, T-cell receptor; Vi, virulence polysaccharide.

produced by these closely related Salmonella serovars in humans.

Clearly, S. Typhi must be modulating immune activities in a
manner that allows it to establish systemic infection. How these

modulations are brought about is not completely understood. A

major difference between S. Typhi and S. Typhimurium is the
presence of Vi capsular polysaccharide in the former. Vi protects
Vi Inhibits T-Cell Activation

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Figure 3. Vi interacts with membrane-associated prohibitin family of molecules and suppresses anti-CD3 antibody-induced but not PMA-induced early
activation events. A, Jurkat cells (3 107 cells) were incubated with Vi (1 g/106 cells) in RPMI-1640 for 1 hour at 4C. Cells were washed, lysed with TKM
buffer (Tris. HCl, 50 mM, pH7.4, KCl 25 mM, MgCl2 5 mM, EDTA 1 mM, NaN3 0.02%, Triton X-100 1%, supplemented with a cocktail of protease inhibitors),
and lysates were immunoprecipitated with anti-Vi Ab loaded on Protein G-Sepharose beads. The beads were washed, boiled with Laemmli sample buffer,
and electrophoresed in a 12% SDS-PAG. Proteins were transferred to a nitrocellulose sheet and probed with prohibitin and BAP-37specic rabbit Ab. The
blot was developed using ECL. Lane 1: cell lysate, lane 2: immunoprecipitation with cell lysate from untreated cells, lane 3: immunoprecipitation with lysate
from Vi-treated cells. B, Cells incubated with Vi (1 g/mL) at 4C or infected with Vi+ S. Typhi, Vi- S. Typhi or S. Typhimurium, followed by incubation with
anti-Vi antibody and Alexauor-594-labeled antimouse Ig were analyzed by confocal microscopy. C and D, Jurkat cells were activated with plate-coated
anti-CD3 antibody or PMA in the absence or presence of Vi (5 g/mL) for 1 hour at 37C. Cells were washed and incubated with anti-CD3 antibody followed
by FITC-labeled anti-mouse Ig antibody. Cells were analyzed in a ow cytometer. Shaded histogram represents staining with FITC-labeled anti-mouse IgG
alone. E, Jurkat cells were activated with plate-coated anti-CD3 Ab for 6 hours and incubated with APC-labeled anti-CD69 antibody for 1 hour at 4C. Cells
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S. Typhi from complement-mediated killing and phagocytosis [9,

10]. We have previously reported that this polysaccharide can interact with membrane associated prohibitin family of molecules
in IECs and monocytes and bring down inammatory responses
[11, 12]. The results presented here show that S. Typhi releases
considerable amounts of Vi during in vitro infection, which
can readily bind T cells through prohibitin and specically inhibit
antigen receptor driven T-cell activation. These results suggest
that Vi can impair T-cell mediated immune responses during typhoid even in the absence of a direct contact between the pathogen and T cells. This impairment would be very effective at
anatomical sites which express low levels of circulating hemoglobin as the latter prevents interaction of Vi with membrane prohibitin and abolishes its inhibitory capacity [[12]; present study].
Therefore, gut, which unlike spleen does not harbor large

numbers of erythrocytes, could be a prime site where Vi would

be able to exert its effect maximally. Vi-mediated inhibition in
the gut may also be increased due to higher levels of prohibitin
at this site (authors unpublished data). Considering that gut is
the site where S. Typhi infection is initiated, the released polysaccharide would empower S. Typhi to prevent T-cell activation
right at the beginning of infection. Vi has been previously reported in circulation in typhoid patients [20, 21]. Kullas et al [22] have
recently reported that S. Typhimurium produces asparaginase,
which can also down-regulate T-cell responses. However, the expression of this molecule and its possible role during S. Typhi infection has not been tested.
The exact mechanism by which interaction of Vi with membrane prohibitin inhibits T-cell responses is not clear at the moment. Similar to IECs and monocytes, the Vi-recognition

were washed and Figure 3 continued. analyzed in a BD Accuri Flow Cytometer. Shaded histogram represents unstained cells. PE-labeled isotype matched
control antibody did not show any staining with Jurkat. F, Jurkat cells were stimulated with plate coated anti-CD3 antibody or PMA for different time points.
Cell lysates were run in a 10% SDS-PAG, transferred to a nitrocellulose membrane and probed with anti-pERK antibody followed after stripping with antiERK antibody. Blots were developed using ECL reagent. Data shown in Figure 3 are representative of 23 independent experiments. Abbreviations: Ab,
antibody; Ig, immunoglobulin; Vi, virulence polysaccharide.

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Figure 4. Vi brings about actin depolymerisation in Jurkat. Cells were incubated with PBS or Vi (5 g/mL) in serum-free RPMI-1640 for 1 hour or 5 hours
at 37C. After washing, cells were x-permeabilised and stained with Alexauor 488-labeled phalloidin. Cells were analyzed by ow cytometry. The numbers in the top half represent percentage of phalloidinhi cells. Data are representative of 2 experiments. Abbreviations: PBS, phosphate-buffered saline;
RPMI, Roswell Park Memorial Institute medium.

cytoskeletal rearrangements by Vi, might explain why Vi inhibits anti-TCR Abmediated TCR internalization without affecting PMA-activated TCR internalization as the former is
mediated primarily through tyrosine phosphorylation dependent events, whereas the latter is produced through serine phosphorylation [25]. Similar dichotomy has been previously
reported during inhibition of human T-cell activation with
cholera toxin [26]. The reasons for differential modulation of
tyrosine vs serine phosphorylation by the membrane prohibitin
complex are currently under investigation in our laboratory.
In conclusion, our ndings have revealed a very efcient
mechanism by which Salmonella Typhi could target the T-cell
arm of the immune system at hemoglobin-low locations, notably the gut that is the primary site of Salmonella infection, and
thus promote establishment of its systemic infection.
MATERIALS AND METHODS

MyD88 knockout mice on C57BL/6 background were obtained

from Jackson Laboratories, United States, and maintained in the
small animal facility of the National Institute of Immunology.
Mice were maintained in germ-free conditions. Animal experiments were carried out according to the guidelines provided by
the Institutional Animal Ethics Committee.
Cell Lines
Figure 5. Vi inhibits T-cell responses in vivo. MyD88 decient mice
were injected with Vi (100 g per mouse; 4 mice) or PBS (equivalent volume; 3 mice) followed half an hour later by anti-CD3 antibody (50 g per
mouse) intraperitoneally. 3 hours later, mice were bled and IL-2 and IFN-
levels were determined in sera by ELISA. Data are representative of 2
experiments. *P value < .05. Abbreviations: ELISA, enzyme-linked immunosorbent assay; IL-2, interleukin 2; IFN-, interferon ; PBS, phosphatebuffered saline; Vi, virulence polysaccharide.

complex in T cells was found to comprise of membrane prohibitin and BAP-37 [11, 12]. Prohibitin has been previously shown
to regulate ras-rafdependent activation of ERK in epithelial
cells [17]; therefore, it is possible that engagement of membrane
prohibitin complex with Vi modulates ERK phosphorylation in
T cells through a similar mechanism. Signicantly, engagement
of membrane prohibitin with Vi promoted actin depolymerisation,
which could abrogate activation of a large repertoire of signaling
intermediates including MAP-kinases during TCR signaling as
actin polymerization is vital to generation of signaling clusters
during T-cell activation [18, 23]. We have data to suggest that
membrane prohibitin complex might regulate activation of
the src kinase Lck (authors unpublished data); the latter has
a central role in TCR signaling [24]. The effect on Lck activation, which could well be through modulation of actin
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The human leukemic T-cell line Jurkat was obtained from

ATCC, Manassas, VA. Myelin basic protein (MBP)-specic murine T-cell hybridoma 1934.4 was provided by Prof Sally Ward,
UT, Southwestern Medical Center, Dallas, TX. Mononuclear
cells were isolated from human peripheral blood mononuclear
cells (PBMC) by Ficoll-Hypaque density centrifugation; nonadherent PBMC were used for activation purposes. Blood was collected according to the guidelines outlined by the Institutional
Human Ethics Committee.
Antibodies and Other Reagents

Polyclonal rabbit antibodies were generated against prohibitin

and BAP-37 as described previously by Coates et al, 1997
[27]. All other antibodies used in this study were from Millipore
or Cell Signaling, United States. Vi was obtained from Aventis
Pasteur Connaught, India, or Bharat Biotech Limited, India,
and dialyzed against phosphate-buffered saline (PBS) before
using in cellular studies. The de-acetylated derivative of Vi
was prepared as described by Szewczyl and Taylor, 1980 [28].
S. Typhosa LPS was from Sigma Chemical Co, United States.
Capsular polysaccharide isolated from Streptococcus pneumoniae (PCP) was kindly provided by Dr Devinder Sehgal, NII.
Alexauor 488-labeled Phalloidin was from Molecular Probes
and FITC-labeled anti-mouse Ig antibody was from Jackson
Laboratories, United States.

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Mice

Activation of T cells

Binding of Vi to Cells and Identication of Vi-recognition

Complex

The binding of Vi to cells and identication of Vi-binding molecules was carried out as described elsewhere [11].
Confocal Microscopy

Cells were incubated with Vi (1 g/mL) for 1 hour at 4C followed by anti-Vi MoAb and Alexauor-594 - conjugated antimouse Ig Ab. Alternatively, cells were infected with Vi+
S. Typhi, Vi- S. Typhi, or S. Typhimurium at a MOI 50, washed
and incubated with anti-Vi MoAb, followed by Alexauor-594
labeled anti-mouse Ig antibody. Images were acquired in a confocal microscope (Olympus FluoView FV1000) and transferred
to Adobe Photoshop for printing. Control cells were incubated
only with anti-Vi MoAb and Alexauor-594-conjugated antimouse Ig Ab.
Analysis of Actin Polymerization

Cells were incubated with PBS or Vi (5 g/mL) at 37C in

serum-free RPMI-1640 for 1 hour or 5 hours at 37C. Cells
were washed, xed with 4% paraformaldehyde for 20 minutes
at room temperature, and stained with Alexauor 488-labeled
phalloidin. Cells were analyzed in a ow cytometer.
Effect of Vi in vivo

MyD88 decient C57Bl/6 mice (34 per group) were injected

intraperitoneally with Vi (100 g/mouse) or the vehicle (PBS)
followed half an hour later with anti-CD3 antibody (50 g/

mouse). Mice were bled prior to and 3 hours after injecting

the antibody and cytokine levels were determined in the sera
by ELISA. All animal experiments were carried out according
to the guidelines provided by the Institutional Animal Ethics
Committee of the National Institute of Immunology.
Statistical Analysis

Values are expressed as meanSD. Statistical analysis was performed using paired Student t-test except for the in vivo experiment in which unpaired Student t-test was employed. A P value
of < .05 was considered statistically signicant in all analyses.
Notes
Acknowledgments. We would like to thank Drs Rahul Pal and Devinder
Sehgal for critical reading of the paper and members of the Ayub laboratory
for insightful discussions.
Financial support. This work was funded through the National Institute of Immunology by the Department of Biotechnology, Government of
India.
Potential conicts of interest. All authors: No reported conicts.
All authors have submitted the ICMJE Form for Disclosure of Potential
Conicts of Interest. Conicts that the editors consider relevant to the content of the manuscript have been disclosed.

References
1. Ruby T, McLaughlin L, Gopinath S, Monack D. Salmonellas long-term
relationship with its host. FEMS Microbiol Rev 2012; 36:60015.
2. Bhan MK, Bahl R, Bhatnagar S. Typhoid and paratyphoid fever. Lancet
2005; 366:74962.
3. Jones BD, Falkow S. Salmonellosis: host immune responses and bacterial virulence determinants. Annu Rev Immunol 1996; 14:53361.
4. Hess J, Ladel C, Miko D, Kaufmann SH. Salmonella Typhimurium
aroA infection in gene-targeted immunodecient mice: Major role
for CD4+ TCR cells and IFN- in bacterial clearance independent
of intracellular location. J Immunol 1996; 156:33216.
5. Lalmanach AC, Lantier F. Host cytokine response and resistance to Salmonella infection. Microbes Infect 1999; 1:71926.
6. Pie S, Truffa-Bachi P, Pla M, Nauciel C. Th1 response in Salmonella
typhimurium-infected mice with a high or low rate of bacterial clearance. Infect Immun 1997; 65:450914.
7. Monack DM, Bouley DM, Falkow S. Salmonella typhimurium persists
within macrophages in the mesenteric lymph nodes of chronically infected Nramp1+/+ mice and can be reactivated by IFNgamma neutralization. J Exp Med 2004; 199:23141.
8. Sztein MB. Cell-mediated immunity and antibody responses elicited by
attenuated Salmonella enterica Serovar Typhi strains used as live oral
vaccines in humans. Clin Infect Dis 2007; 1:S159.
9. Felix A, Pitt RM. Virulence and immunogenic activities of B. typhosus
in relation to its antigenic constituents. J Hyg (Lond) 1935; 35:42836.
10. Wilson RP, Winter SE, Spees AM, et al. The Vi capsular polysaccharide
prevents complement receptor 3-mediated clearance of Salmonella
enterica serotype Typhi. Infect Immun 2011; 79:8307.
11. Sharma A, Qadri A. Vi polysaccharide of Salmonella typhi targets the
prohibitin family of molecules in intestinal epithelial cells and suppresses early inammatory responses. Proc Natl Acad Sci U S A 2004;
101:174927.
12. Garg R, Qadri A. Hemoglobin transforms anti-inammatory Salmonella typhi virulence polysaccharide into a TLR-2 agonist. J Immunol 2010;
184:59807.
13. Mishra S, Murphy LC, Nyomba BL, Murphy LJ. Prohibitin: a potential
target for new therapeutics. Trends Mol Med 2005; 11:1927.

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To investigate the effect of Vi on IL-2 secretion, T cells were

activated with anti-CD3 Ab (10 g/mL) plus anti-CD28 Ab
(5 g/mL) in the absence or presence of Vi, DeVi, PCP, or
LPS (Jurkat & mouse T-cell hybridoma for 1820 hours and
ex vivo human T cells for 48 hours) at 37C after which the supernatants were analyzed for IL-2 by enzyme-linked immunosorbent assay (ELISA). Murine T-cell hybridoma was also
activated with a specic peptide along with antigen presenting
cells. Splenocytes isolated from C57Bl/6 mice were activated
with anti-CD3 antibody for 48 hours. In some experiments,
Jurkat cells were activated with PMA (100 ng/mL) and ionophore (100 ng/mL), and IL-2 was determined in the supernatants after 8 hours. Cell death was examined by propidium
iodide uptake using ow cytometry.
To analyze effect of Vi on TCR down-regulation, T cells were
activated with plate coated anti-CD3 antibody or PMA in the
absence or presence of Vi for 1 hour, washed and incubated
with anti-CD3 antibody, followed by PE-labeled anti-mouse
Ig antibody. Subsequently, cells were washed and analyzed in
a ow cytometer (BD, LSR). Data were plotted using WinMDI.
The effect of Vi on TCR-induced expression of CD69 in Jurkat was also investigated by ow cytometry. Cells were analyzed in BD, Accuri, and the data were plotted using Flow Jo.

14. Morrow IC, Parton RG. Flotillins and the PHB domain protein family:
rafts, worms and anaesthetics. Trafc 2005; 6:72540.
15. Osman C, Merkwirth C, Langer T. Prohibitins and the functional
compartmentalization of mitochondrial membranes. J Cell Sci 2009; 122:
382330.
16. Terashima M, Kim KM, Adachi T, et al. The IgM antigen receptor of B
lymphocytes is associated with prohibitin and a prohibitin-related protein. EMBO J 1994; 13:378292.
17. Rajalingam K, Wunder C, Brinkmann V, et al. Prohibitin is required for
Ras-induced Raf-MEK-ERK activation and epithelial cell migration. Nat
Cell Biol 2005; 7:83743.
18. Barda-Saad M, Braiman A, Titerence R, Bunnell SC, Barr VA, Samelson
LE. Dynamic molecular interactions linking the T cell antigen receptor
to the actin cytoskeleton. Nat Immunol 2005; 6:809.
19. Young D, Hussell T, Dougan G. Chronic bacterial infections: living with
unwanted guests. Nat Immunol 2002; 3:102632.
20. Barrett TJ, Snyder JD, Blake PA, Feeley JC. Enzyme-linked immunosorbent assay for detection of Salmonella typhi Vi antigen in urine from
typhoid patients. J Clin Microbiol 1982; 15:2357.
21. Pandya M, Pillai P, Deb M. Rapid diagnosis of typhoid fever by detection of Barber protein and Vi antigen of Salmonella serotype typhi.
J Med Microbiol 1995; 43:1858.

22. Kullas AL, McClelland M, Yang HJ, et al. L-asparaginase II produced by

Salmonella typhimurium inhibits T cell responses and mediates virulence. Cell Host Microbe 2012; 12:7918.
23. Campi G, Varma R, Dustin ML. Actin and agonist MHC-peptide complex dependent T cell receptor microclusters as scaffolds for signaling.
J Exp Med 2005; 202:10316.
24. Salmond RJ, Filby A, Qureshi I, Caserta S, Zamoyska R. T-cell receptor
proximal signaling via the Src-family kinases, Lck and Fyn, inuences
T cell activation, differentiation, and tolerance. Immunol Rev 2009;
228:922.
25. Lauritsen JP, Christensen MD, Dietrich J, Kastrup J, Odum N, Geisler
C. Two distinct pathways exist for down-regulation of the TCR. J Immunol 1998; 161:2607.
26. Anderson DL, Tsoukas CD. Cholera toxin inhibits resting human T cell
activation via a cAMP-independent pathway. J Immunol 1989; 143:
364752.
27. Coates PJ, Jamieson DJ, Smart K, Prescott AR, Hall PA. The prohibitin
family of mitochondrial proteins regulate replicative lifespan. Curr Biol
1997; 7:60710.
28. Szewczyk B, Taylor A. Immunochemical properties of Vi antigen from
Salmonella typhi Ty2: presence of two antigenic determinants. Infect
Immun 1980; 29:53944.

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