Cell Host & Microbe
The Shigella flexneri Type Three Secretion System
Effector IpgD Inhibits T Cell Migration
by Manipulating Host Phosphoinositide Metabolism
Christoph Konradt,1,2Elisabetta Frigimelica,1,2Katharina Nothelfer,1,2Andrea Puhar,1,2Wilmara Salgado-Pabon,1,2
Vincenzo di Bartolo,3,4Daniel Scott-Algara,5Cristina D. Rodrigues,6Philippe J. Sansonetti,1,2,7and Armelle Phalipon1,2,*
1Unite ´ de Pathoge ´nie Microbienne Mole ´culaire
3Unite ´ de Biologie Cellulaire des Lymphocytes
5Unite ´ des Re ´gulations des Infections Re ´trovirales
6G5 Dynamique des Interactions Ho ˆte-Pathoge `nes
Institut Pasteur, 25–28 Rue du Dr Roux, 75724 Paris Cedex 15, France
7Chaire de Microbiologie et Maladies Infectieuses, Colle `ge de France, 11 Place Marcelin Berthelot, 75005 Paris, France
Shigella, the Gram-negative enteroinvasive bacte-
rium that causes shigellosis, relies on its type III
secretion system (TTSS) and injected effectors to
modulate host cell functions. However, conse-
quences of the interaction between Shigella and
lymphocytes have not been investigated. We show
that Shigella invades activated human CD4+T
lymphocytes. Invasion requires a functional TTSS
and results in inhibition of chemokine-induced
T cell migration, an effect mediated by the TTSS
effector IpgD, a phosphoinositide 4-phosphatase.
Remarkably, IpgD injection into bystander T cells
can occur in the absence of cell invasion. Upon
IpgD-mediated hydrolysis of phosphatidylinositol
4,5-bisphosphate (PIP2), the pool of PIP2 at the
plasma membrane is reduced, leading to dephos-
phorylation of the ERM proteins and their inability
to relocalize at one T cell pole upon chemokine stim-
ulus, likely affecting the formation of the polarized
edge required for cell migration. These results reveal
T cell function.
Pathogens have evolved strategies to avoid or resist host
defense mechanisms. Although there is ample information on
the mechanisms used by pathogenic bacteria to subvert the
innate immune defense system, very little is known about how
they affect the adaptive immune response (Hornef et al., 2002).
This is also the case for Shigella, a facultative intracellular
Gram-negative enteroinvasive bacterium. Shigella is the causa-
tive agent of shigellosis or bacillary dysentery and accounts for
about one-third of the total annual deaths due to enteric infec-
tions (von Seidlein et al., 2006). Shigella’s virulence relies on
theexpression of atypeIIIsecretion system (TTSS)andthe rapid
injection of several effector proteins upon cell contact to modify
host cell functions (Parsot, 2009). As recently exemplified for
malaria (Weiss et al., 2010), protection occurs only after multiple
infections and is of short duration, suggesting that Shigella has
the capacity to dampen the host adaptive immune response.
In contrast, Shigella induces acute inflammation, a hallmark of
the host innate response to infection, which requires TTSS
effector secretion to be elicited. As a consequence of the proin-
flammatory process, massive dendritic cell (DC) and B and T
lymphocyte cell death has been observed in rectal biopsies of
Shigella-infected individuals (Raqib et al., 2002), suggesting
that such an inflammatory response has an impact on the devel-
opment of adaptive immunity. Furthermore, in a model of human
intestinal xenograft, Shigella-infected intestinal epithelial cells
(IECs) exhibit a dramatic decrease in the production of the
chemokine CCL20, resulting in weak recruitment of DCs to the
site of infection (Sperandio et al., 2008). However, during natural
infection, Shigella crosses the intestinal barrier via M cells
located in the lymphoid-associated epithelium where it is likely
to directly encounter DCs and lymphocytes (Sansonetti and
Phalipon, 1999). Moreover, after rupture of the integrity of the
epithelial barrier, Shigella gets access to the lamina propria
where DCs and mainly activated lymphocytes reside.
Whereas some data are available on the effect of Shigella
infection on DCs (Edgeworth et al., 2002; Kim et al., 2008), the
consequences of the interaction between Shigella and lympho-
cytes have not yet been investigated. Hence, we investigated
the outcome of the interaction of Shigella flexneri with primary
human CD4+T lymphocytes and Jurkat cells, a human T cell
line largely used to dissect the molecular mechanisms under-
lying T cell function. We demonstrate that T lymphocytes are
invaded by Shigella and that invasion impairs chemokine-
induced migration, and identify IpgD as the TTSS effector
responsible for the effect. In addition, we provide evidence that
IpgD is injected into T cells in the absence of invasion, putting
forth a model where immune cell functions can be affected
with no need for the bacterium to be intracellular. These results
Cell Host & Microbe 9, 263–272, April 21, 2011 ª2011 Elsevier Inc. 263
start to unravel the possible mechanisms by which TTSS-ex-
pressing pathogens manipulate adaptive immunity.
Shigella Invades Activated Primary Human CD4+T
Lymphocytes and Impairs Their Chemokine-Induced
A possible crosstalk between Shigella and T lymphocytes was
first assessed by analyzing the ability of Shigella to invade
primary human T cells in an unactivated or activated state.
CD4+T lymphocytes purified from PBMCs were incubated at
moi of 10 or 100 bacteria per cell for 30 min with the invasive
Shigella strain M90T or the noninvasive Shigella mutant mxiD
that does not assemble the TTSS needle, and therefore does
not secrete effectors (Allaoui et al., 1993b). In parallel experi-
ments, CD4+T lymphocytes were activated with PMA prior to
infection. To visualize intra- or extracellular bacteria, immunoflu-
orescence analysis with anti-Shigella LPS antibodies on nonper-
meabilized samples (staining only extracellular bacteria) and
GFP-expressing bacteria (total and intracellular bacteria) was
performed. Surprisingly, no intracellular bacteria were detected
in unactivated CD4+T lymphocytes regardless of the moi (data
not shown), while PMA activation rendered CD4+T cells suscep-
tible to invasion by M90T (at both mois) but not by the mutant
mxiD (Figure 1A). About 10% of activated primary CD4+T cells
were invaded at an moi of 10 and 70% at an moi of 100.
Since lymphocyte migration is of utmost importance for T cells
to exert their effector function (Kunkel and Butcher, 2002), we
addressed whether Shigella infection of activated CD4+T
Primary Human T Cells and Impairs Their
Activated primary human CD4+T cells were in-
fected with the Shigella wild-type strain M90T or
the TTSS mutant mxiD. (A) Extra- and intracellular
bacteria were visualized by immunofluorescence
microscopy (IF) of cells infected for 30 min with
GFP-expressing M90T (upper lane) and GFP-ex-
pressing mxiD (lower lane). Total and intracellular
bacteria are seen in green (GFP), extracellular
bacteria labeled prior to cell permeabilization with
cell permeabilization in red. Migration assays
were performed with activated (B) and un-
activated (C) primary human CD4+T cells infected
for 30 min with M90T or mxiD strains at a moi of
10. NI, not infected. Results represent the mean ±
SEM calculated from a minimum of three inde-
pendent experiments. **p < 0.01.
lymphocytes had an effect on their ability
to migrate in response to a chemoattrac-
tant stimulus. An moi of 10 was chosen
for this essay because it resulted in less
than 10% cell death for both noninfected
and infected cells during the time course
of the Transwell chamber migration
assay (data not shown). We observed
a 50% reduction in the ability of activated primary human
CD4+T cells to migrate toward the chemoattractant CXCL12
when infected with M90T (Figure 1B). In contrast, upon infection
with the mxiD mutant, the cell migration index was similar to that
obtained with the noninfected cells (Figure 1B). Migration of un-
activated cells was not affected regardless of the Shigella strain
used for infection (Figure 1C). These results provide evidence
that Shigella invasion of activated primary human CD4+T
lymphocytes is dependent on a functional TTSS and that inva-
sion has a significant impact on one of the critical functional
features of T cells.
The Shigella Effector IpgD Impairs Chemokine-Induced
Migration of Activated Primary Human CD4+T
and moesin) proteins, a family of membrane-cytoskeleton cross-
linkers implicated in cell cortex organization, play a crucial role
(Charrin and Alcover, 2006). ERM activation/inactivation state is
dependent on the concentration of phosphatidylinositol 4,5-bi-
sphosphate (PIP2) at the cell membrane (Fehon et al., 2010).
PIP2is also the substrate of the Shigella TTSS effector IpgD.
IpgD is a phosphoinositide 4-phosphatase that generates phos-
phatidylinositol 5-monophosphate (PI5P) from PIP2 (Niebuhr
et al., 2000, 2002). Hence, we hypothesized that the T cell migra-
the enzymatic activity of IpgD within the cells. The cell migration
assay was thus performed with activated primary human CD4+
T cells previously infected with the ipgD mutant and the
Cell Host & Microbe
Inhibition of T Cell Migration by Shigella
264 Cell Host & Microbe 9, 263–272, April 21, 2011 ª2011 Elsevier Inc.
corresponding complemented strain ipgD/pIpgD. ipgD mutant
and M90T infection gave rise to the same percentage of infected
T cells (see Figure S1A available online). However, infection with
the ipgD mutant resulted in a migration index comparable to
that of T cells infected with the mxiD mutant or to the noninfected
ipgDmutationwithipgDresultedina migration phenotype similar
to that of M90T (Figure 2). These results demonstrate that the
virulence effector IpgD is necessary and sufficient to impair the
migration of activated primary CD4+T lymphocytes.
IpgD Inhibits the Migration of Shigella-Infected
We then used Jurkat cells (a human CD4+T cell line) to dissect
the IpgD-mediated molecular mechanism involved in the
impairment of T cell migration observed in the activated
primary human CD4+T cells. We first assessed whether,
upon Shigella infection, Jurkat cells displayed the same pheno-
type as primary human T cells. M90T and mxiD infection at an
moi of 10 resulted in detection of intracellular bacteria only in
Jurkat cells infected with M90T, and bacterial replication was
shown to take place over time (Figure 3A). M90T infection
resulted in ?10% invasion and a 30% reduction in CXCL12-
induced migration when compared to noninfected cells or cells
infected with the mxiD mutant. In addition, IpgD was shown to
account for the impaired cell-migration phenotype (Figure 3B).
These results are all consistent with the invasion and migration
phenotypes observed upon Shigella infection of activated
primary human CD4+T cells.
Interestingly, FACS analysis of M90T-GFP-infected Jurkat
cells from the upper and lower compartments of the Transwell
chamber revealed a population of GFP-high positive cells found
only in the upper compartment (Figure 3C, red circle). IF analysis
demonstrated that the GFP-high positive, nonmigrating Jurkat
cell population was indeed cells heavily invaded by Shigella (Fig-
ure 3C, top panels). In contrast, cells that had migrated into the
lower compartment were GFP-negative noninvaded cells (Fig-
in 100% of Shigella-invaded Jurkat cells, similarly to what was
observed in human primary T cells (Figure S2). No difference in
cell death was observed between the upper and lower wells,
as monitored by PI staining (Figure 3C). In addition, no difference
in Jurkat cell death occurred between M90T and mxiD under
these experimental settings (data not shown).
Figure 2. The Shigella Effector IpgD Accounts for the Reduction of
Migration of Activated Human Primary T Cells
Migration assays were performed as described in Figure 1 with activated
its counterpart complemented strain ipgD/pIpgD. NI, not infected. Results
represent the mean ± SEM calculated from a minimum of three independent
experiments. **p < 0.01.
Figure 3. IpgD Is Responsibleforthe Inhibi-
tion of Jurkat Cell Migration
(A) Counting of intracellular bacteria in Jurkat cells
infected with M90T or mxiD at an moi of 10 after
gentamicin treatment. (B) Migration assay as
previously described in Figures 1 and 2 with
Jurkat cells infected with the strains M90T, mxiD,
ipgD, ipgD/pIpgD, or not infected (NI). (C) FACS
and IF analysis of Jurkat cells infected with GFP-
expressing M90T present in the upper and lower
positive cell population that is only found in the
upper well (nonmigrating cells) corresponds to
heavily invaded cells (red circle). In the lower well,
the cells that have migrated are noninvaded cells
(blue circle). IF pictures show actin in red and
GFP-expressing M90T in green (D). Migration
assay with Jurkat cells expressing IpgD-GFP,
IpgDmut-GFP, GFP alone, or not transfected (N).
Results represent the mean ± SEM calculated
from a minimum of three independent experi-
ments. **p < 0.01. ***p < 0.001.
Cell Host & Microbe
Inhibition of T Cell Migration by Shigella
Cell Host & Microbe 9, 263–272, April 21, 2011 ª2011 Elsevier Inc. 265
Furthermore, migration analysis of Jurkat cells expressing
IpgD-GFP or an inactive IpgD point mutant (IpgDmut-GFP)
demonstrated that inhibition of T cell migration was dependent
on the enzymatic activity of IpgD (Figure 3D). Therefore, both
activated primary human CD4+T lymphocytes and Jurkat cells
are susceptible to Shigella invasion and to the IpgD-mediated
inhibition of their migration.
It is worth mentioning that a battery of Shigella mutants
deleted for the expression of effectors reported to target
different pathways involved in actin cytoskeleton rearrangement
in EC (Tran Van Nhieu and Sansonetti, 1999) have been tested in
the migration assay with Jurkat cells. For all of them, the migra-
tion index of the corresponding infected cells was similar to that
of M90T-infected Jurkat cells (Figure S1B).
IpgD Is Injected into Jurkat Cells in the Absence
of Shigella Invasion
It was intriguing that 10% invasion resulted in 50% and 30%
reduction in T cell migration for both activated primary human
CD4+T cells and Jurkat cells infected with M90T, respectively.
This suggested that another phenomenon occurred in addition
to invasion that contributed to the inhibition of T cell migration.
Since injection of IpgD has indirectly been shown to occur in
epithelial cells in the absence of invasion (Niebuhr et al., 2002),
we hypothesized that this could also be the case for T cells. To
test this, we adopted a FRET pair-based approach, previously
reported to monitor effector translocation of Gram-negative
bacteria into host cells (Mills et al., 2008). Briefly, to monitor
translocation of IpgD into T cells, cells were preloaded with the
cephalosporin-derived CCF4 probe and infected with Shigella
M90T or mxiD, both expressing IpgD fused to b-lactamase
(M90T-IpgD-bla and mxiD-IpgD-bla). Upon translocation of the
effector fusion protein, the FRET pair CCF4 within the cell is
cleaved by b-lactamase, and the fluorescence emission of the
cells shifts from 535 nm (green) to 450 nm (blue). The threshold
of detection for the FRET pair CCF4 cleavage depends on the
amount of fusion protein delivered to the host cell and the
kinetics of the enzymatic cleavage. To obtain a proper readout,
we used an moi of 500 and a 2 hr incubation period of the
bacteria with the cells after addition of gentamicin to kill the
extracellular bacteria. To assess whether the translocation of
IpgD from extracellular bacteria occurred in the absence of inva-
sion, T cells were preincubated with cytochalasin D, an inhibitor
of Shigella invasion (Mounier et al., 1997). In the presence of
cytochalasin D, cleavage of the FRET pair occurred in about
30% of Jurkat cells infected with M90T-IpgD-bla, while no
cleavage was detected in mxiD-IpgD-bla-infected cells (Fig-
ure 4), demonstrating that IpgD is injected into T cells in the
absence of invasion.
IpgD Decreases the PIP2Pool at the Plasma Membrane
IpgD-mediated hydrolysis of PIP2has been shown to occur in
epithelial cells (Niebuhr et al., 2002) and in Jurkat cells (Guittard
et al., 2009). Since most of the PIP2pool is located at the plasma
membrane,weexpected thattheIpgD-mediated PIP2hydrolysis
in T cells would affect the plasma membrane PIP2pool. To visu-
alize PIP2localization in Jurkat cells, the pleckstrin homology
(PH) domain of PLCd1 was used as a tool for confocal micros-
copy. The PH domain displays high affinity for PIP2, and it is
bound to the plasma membrane when PIP2is present but trans-
locates to the cytosol when PIP2is hydrolyzed (Varnai et al.,
2002). Jurkat cells were transfected to express the PH domain
of PLCd1 coupled to the red fluorescent protein (RFP) (PH-
PLCd1-RFP) together with either wild-type IpgD-GFP or its inac-
tive form (IpgDmut-GFP). Localization of PH-PLCd1-RFP at the
plasma membrane was observed in 92%and 96%of cells trans-
fected with PH-PLCd1-RFP only and in cells cotransfected with
IpgDmut-GFP, respectively. In contrast, the localization of PH-
PLCd1-RFP at the plasma membrane was lost in 81% of cells
cotransfected with IpgD (Figure 5A). Quantitative analysis
revealed a statistically significant decrease of the ratio fluores-
cence intensity at the plasma membrane versus cytosol for
IpgD as compared to IpgDmut, which displayed a ratio similar
to that of the control (Figure 5B). Moreover, the decrease of
PIP2level at the plasma membrane occurred as soon as Jurkat
cells were infected with M90T, but not with the ipgD mutant,
and was sustained for at least 1 hr postinfection (Figure S3).
These results indicate that PIP2is a target of IpgD in Jurkat cells
and that the resulting IpgD-mediated PIP2cleavage modifies the
PIP2pool at the plasma membrane.
IpgD Induces Dephosphorylation of Phospho-ERM
Proteins, Affecting Their Polar Redistribution upon
membrane is crucial for the dynamics of the ERM activation/
inactivation state which is important in the early steps of cell
cortex organization during T cell polarization in response to
a chemokine stimulus (Fehon et al., 2010). We thus investigated
the ratio of nonphosphorylated (inactive) versus phosphorylated
(active) ERMs (phospho-ERMs) in Jurkat cells upon Shigella
infection. Whereas the pool of total ERMs remained unchanged,
a dramatic sustained decrease in the pool of phospho-ERMs
as compared to noninfected cells (Figure 6A). These results
were confirmed in Jurkat cells expressing either IpgD-GFP or
Figure 4. Injection of IpgD into Jurkat Cells in the Absence
FACS analysis of cells loaded with CCF4 and infected with mxiD IpgD-bla (left
panel) or M90T IpgD-bla (right panel) in the presence of Cytochalasin D.
Translocation of the effector fusion protein leads to cleavage of CCF4 and to
an emission switchfrom 535 nmto 450nm.Results representthe mean ±SEM
calculated from three independent experiments.
Cell Host & Microbe
Inhibition of T Cell Migration by Shigella
266 Cell Host & Microbe 9, 263–272, April 21, 2011 ª2011 Elsevier Inc.
IpgDmut-GFP (Figure 6B) and in human primary T cells infected
with M90T versus the ipgD mutant (Figure S4A).
We then used Jurkat cells expressing either IpgD-GFP or
IpgDmut-GFP to assess phospho-ERMs redistribution upon
chemokine stimulus. The transfected cells were incubated for
45 s with CXCL12 followed by anti-phospho-ERM antibodies
to visualize phospho-ERMs localization. The number of Jurkat
cells displaying recruitment of phospho-ERMs at one pole of
the cell were counted by IF microscopy, and it was observed
in cells expressing GFP as a control or IpgDmut-GFP. In
contrast, phospho-ERMs localization at the cell pole was barely
was similar to that of cells incubated in the absence of CXCL12
(Figures 6C and 6D). Those results were consistent with the
observation that IpgD-mediated dephosphorylation of phos-
pho-ERMs remained unchanged in the presence of CXCL12
both for Shigella-infected Jurkat cells (Figure S4B) and primary
human T cells (Figure S4C). Our data collectively suggest that
inhibition of T cell migration induced by IpgD is the consequence
of the blockage of one of the early steps in the process of cell
migration, i.e., cell polarization in response to a chemoattractant
involving PIP2and ERMs.
Four main messages arise from this study. First, we show that
Shigellainvades activatedhuman CD4+Tcellsin aTTSS-depen-
dent manner. Second, injection of TTSS effectors takes place in
T cells in the absence of invasion. Third, Shigella invasion and
TTSS injection result in the inhibition of chemokine-induced
T cell migration. Fourth, inhibition of T cell migration relies on
the bacterial TTSS effector IpgD that hydrolyses PIP2through
its phosphoinositol 4-phosphatase activity. Our results suggest
that the subsequent reduction of the PIP2pool at the plasma
membrane impairs the dynamics between the inactive (non-
phosphorylated) and active (phosphorylated) forms of the ERM
proteins. This presumably affects the polar cap formation (also
called posteriorization of the plasma membrane), a critical step
for the switch, upon chemokine stimulation, from unpolarized
to occur (Charrin and Alcover, 2006).
Invasion of T cells by Shigella requires a functional TTSS, as
previously reported for other nonphagocytic cells including
IECs (Allaoui et al., 1993b). This suggests that the effectors
and mechanisms triggering actin cytoskeleton rearrangements
for Shigella entry into IECs (Tran Van Nhieu and Sansonetti,
1999) may be similar in T cells. Accordingly, we observed that
the phenotype of the ipaA mutant when incubated with T cells
was similar to the phenotype previously reported with IECs,
i.e., a reduced invasion capacity as compared to wild-type
Shigella (Figure S1A) (Tran Van Nhieu et al., 1997). IpaA is
a TTSS effector that binds vinculin, a key component of focal
adhesions, and stimulates actin depolymerization. By targeting
b1-integrin, IpaA also stimulates the GTPase activity of RhoA,
thereby inducing the loss of actin stress fibers (Demali et al.,
2006). Salmonella typhimurium and Yersinia entercolitica are
two Gram-negative enteroinvasive
a TTSS. S. typhimurium has been reported to invade in vitro
T cell lines, whereas Y. enterocolitica has not (Verjans et al.,
Interestingly, activated but not unactivated primary human
CD4+T cells are invaded by Shigella. We speculate that, as
compared to unactivated T cells, activated cells upregulate the
expression of surface molecules that would favor interactions
with Shigella effectors involved in the entry process. For
instance, the expression of CD44 and a5b1integrins is modu-
lated upon lymphocyte activation (Kinashi, 2007; Ponta et al.,
2003). IpaB, one of the effectors required for Shigella entry into
EC, has been shown to interact with the CD44 receptor located
within the lipid rafts (Skoudy et al., 2000), and cholesterol deple-
acts with a5b1integrins like IpaC and IpaD, two other key players
of the bacterial entry process into EC (Watarai et al., 1996). If
CD4+T cells are invaded by Shigella in vivo, discrimination
between activated and unactivated T cells would result in a pref-
erential targeting of activated T cells in the lamina propria as
opposed to the lymphoid follicules associated to the intestinal
mucosa and their population of naive T cells.
We found that Shigella invasion and presumably injection of
effectors in the absence of invasion into the T cell cytoplasm
results in the inhibition of chemokine-induced migration of T
lymphocytes. T celltrafficking isessential for efficient Tcell func-
tions. The ordered, directional migration of T lymphocytes is
indeedakey processintheir development, immunesurveillance,
and the immune response (Kunkel and Butcher, 2002). Inhibition
of cell migration has been reported for other pathogens. For
Figure 5. IpgD Reduces the PIP2Pool at the Plasma Membrane
(A) The pleckstrin homology (PH) domain of PLCd1 was used to monitor PIP2
localization in Jurkat cells expressing either PLCd1PH-RFP (C for control,
and IpgD-GFP (lower lane).
(B) The ratio of the fluorescence intensity of PLCd1PH-RFP at the PM (average
fluorescence intensity of four defined boxes of 2 3 10 pixels of the plasma
J software. Bars represent mean ± the SEM of two independent experiments
(25 cells scored for each construct). ***p < 0.001.
Cell Host & Microbe
Inhibition of T Cell Migration by Shigella
Cell Host & Microbe 9, 263–272, April 21, 2011 ª2011 Elsevier Inc. 267
instance, the adenylate cyclase toxin CyaA released by
Bordetella pertussis inhibits T cell chemotaxis by interfering with
chemokine receptor signaling through its cyclic AMP (cAMP)-
elevating activity (Paccani et al., 2008). The HIV-1 protein Nef
inhibits T cell chemotaxis in response to the ligand CXCL12
(Choe et al., 2002) by downmodulating LFA-1 expression on
T cells, therefore diminishing adhesion and polarization of
T cells, resulting in decreased migration across the endothelium
(Park and He, 2009). In addition, Nef strongly induces phosphor-
ylation of cofilin, thus inactivating this evolutionarily conserved
actin-depolymerizing factor that promotes cell motility when
unphosphorylated (Stolp et al., 2009). The hepatitis C envelope
2 protein inhibits T cell motility by targeting protein kinase C
signaling upon LFA-1 integrin ligation (Volkov et al., 2006). Our
data reveal another type of virulence effector and mechanism
involved in the impairment of T cell migration.
To date, no TTSS effector has been demonstrated to be
involved in dampening T cell chemotaxis. We provide evidence
that IpgD-mediated inhibition of T cell migration is dependent
on its phosphoinositol 4-phosphatase activity, which leads to
hydrolysis of PIP2with a resulting decrease of its pool at the
plasma membrane. In epithelial cells, IpgD-mediated PIP2
cleavage is responsible for dramatic morphological changes
that lead to a decrease in membrane tether force associated
with membrane blebbing and actin filament remodeling (Niebuhr
et al., 2002). As shown here, IpgD is not required for T cell inva-
sion (Figure S1A). This is fully consistent with the fact that,
although involved in the formation of the fully structured entry
foci, IpgD is not involved in invasion of IECs (Niebuhr et al.,
2002). As previously illustrated with the Yersinia TTSS effectors
(Trosky et al., 2008), our results emphasize the extraordinary
power of such an injection device to deliver a given effector
with a particular enzymatic activity into different cell types,
thereby triggering a diversity of outcomes to modulate the host
As to the mechanism involved, it is known that pathogens
target the phosphoinositides (PIs) network (Bakowski et al.,
2010; Smith et al., 2010). PI metabolism plays a key role in
the regulation of receptor-mediated signal transduction, actin
remodeling, and membrane trafficking in eukaryotic cells
(De Matteis and Godi, 2004). Thus, it is not surprising that
several intracellular bacterial pathogens modulate and exploit
PI levels, directly or indirectly, to ensure their survival and effi-
cient intracellular replication. Like Shigella, Salmonella enterica,
Mycobacterium tuberculosis, and some Escherichia coli secrete
effectors mimicking mammalian phosphatases. For example,
the Salmonella effector SopB shares similarity with mammalian
PI4P and PI5P phosphatases (Marcus et al., 2001; Norris et al.,
1998) mainly in the catalytic domain (Ungewickell et al., 2005).
SopB was found to diminish specifically the cortical PIP2pool,
thus destabilizing cytoskeleton-plasma membrane interactions
(Terebiznik et al., 2002). Viruses also exploit PIs, as exemplified
by the HIV Tat protein that binds with a high affinity to PIP2,
resulting in the perturbation of the PIP2-mediated recruitment
of cellular proteins to the plasma membrane (Rayne et al.,
2010). However, Shigella is the first pathogen to be reported to
provoke a massive and sustained dephosphorylation of phos-
pho-ERMs by means of IpgD-mediated PIP2hydrolysis. Acute
ERM protein inactivation plays a critical physiological role in
lymphocytes. Lymphocyte recirculation from blood into tissues
and then back into blood is crucial for efficient adaptive immune
responses. While in blood, the cytoskeleton of the lymphocyte
assures that it is spherical and relatively rigid, allowing it to
survive the hemodynamic stress of circulation. Regulated
binding to the vascular endothelium and migration into tissue
are triggered by chemokines on the endothelial surface that
activate G protein-coupled receptors (GPCRs) on the lympho-
cyte. One very rapid consequence is global reorganization of
the cytoskeleton into a configuration appropriate for a flexible
migration-capable cell. Because ERMs provide a conformation-
ally regulated connection from the cortical actin cytoskeleton to
the plasma membrane (Niggli and Rossy, 2008), rapid conver-
sion of ERMs from their active to inactive conformations plays
a key role in this process (Charrin and Alcover, 2006). The
Figure 6. IpgD Induces ERM Dephosphory-
lation, Thus Blocking Chemokine-Induced
Phospho-ERM Polarization in Jurkat Cells
phoERM (pERM) antibodies of whole-cell lysates
of Jurkat cells (A) infected with the strains M90T or
ipgD at different time points postinfection or not
infected (NI) or of Jurkat cells (B) expressing
IpgD-GFP, IpgDmut-GFP, or not transfected (N).
Jurkat cells were lysed 18 hr after transfection.
Anti-ERM and anti-actin antibodies were used as
Jurkat cells not stimulated and not transfected
(N), CXCL12-stimulated and not transfected (N+),
CXCL12-stimulated and expressing IpgD-GFP,
CXCL12-stimulated and expressing IpgDmut-
GFP. White arrows show localization of pERM at
one pole of the cell. (D) Quantification of the cells
displaying pERM polarization as observed in (C).
For quantification, 150 cells were counted per
condition and experiment. Results are represen-
tative of three independent experiments. Error bar
represents the standard error of the mean (±SEM).
pERMIF staining on
Cell Host & Microbe
Inhibition of T Cell Migration by Shigella
268 Cell Host & Microbe 9, 263–272, April 21, 2011 ª2011 Elsevier Inc.
importance of PIP2in ERMs function was just highlighted using
a recently devised strategy for inducing rapid hydrolysis of
PIP2. The authors showed that PIP2hydrolysis in itself is suffi-
cient to induce ERMs dephosphorylation, therefore proposing
a key role of PIP2in ERM protein biology, namely hydrolysis-
mediated ERM inactivation. In fact, our results show that
Shigella, via IpgD, triggers this hydrolysis-mediated ERM inacti-
vation. In addition, our data reveal that interfering with PI metab-
olism does not only help pathogens to improve their ability to
enteror surviveintohost cellsbutalsoofferstheopportunity, de-
pending on the targeted host cell type, to modulate the host
immune response by affecting cell migration.
Although we cannot formally exclude the possibility that addi-
tional mechanisms might be involved, our data suggest that
alteration of ERMs dynamics is the key process. First, we did
not detect any difference in the expression levels of the
CXCL12 receptor, CXCR4, in Jurkat cells infected with wild-
type Shigella or the mutant ipgD, suggesting that regulation of
chemokine receptor expression is not involved (data not shown).
Second, no difference in T cell migration was observed between
the ipgB1, ipaA, icsB, ipaC/ipaC351 mutants (Allaoui et al.,1992;
Hachani et al., 2008; Menard et al., 1993; Mounier et al., 2009)
when compared to wild-type Shigella (Figure S1B). Since IpaA,
IpaC, IpgB1, and IcsB target different pathways involved in actin
cytoskeleton rearrangement in EC (Tran Van Nhieu and Sanso-
netti, 1999), these data indicate that those pathways are not
involved in the inhibition of T cell chemotaxis.
In conclusion, besides the indirect manipulation of the T cell-
mediated immunity due to the proinflammatory environment
induced by Shigella, leading to a predominant priming of
Shigella-specific Th17 cells (Sellge et al., 2010), we are eluci-
dating a strategy in which the microorganism has the capacity
to directly interfere with T lymphocytes, and in particular with
their dynamics. Our findings suggest that direct manipulation
can occur via bacterial invasion of T cells or injection of effectors
into T cells without invasion. As for the latter, one may easily
imagine the efficiency of a strategy consisting of injecting effec-
tors as soon as bacterium-cell contacts occur without the need
of invading the cell, a surprising observation in view of the
Shigella intracellular lifestyle. The importance of an ‘‘injection-
only’’-based pathway to ‘‘freeze’’ the host immune response
has been underestimated so far and deserves further investiga-
tion. Whether Shigella via IpgD impairs T cell migration in vivo,
and the consequence in terms of priming of Shigella-specific
immunity, will be further analyzed. Interestingly enough, prelimi-
nary results indicate that upon crossing the intestinal barrier and
reaching the mucosa-associated lymphoid follicles, Shigella can
invade T cells in vivo (Figure S5). We provide insights into the
understanding of the manipulation of T cell-mediated immunity
by Shigella that leads to the poor priming of short-lasting protec-
tive immunity during natural infection in humans.
The following Shigella strains were used: M90T, the Shigella flexneri 5a wild-
type strain (Sansonetti et al., 1982); SF401, the mxiD mutant (M90T-DmxiD);
SF701, the IpgD mutant (M90T-DIpgD); SF709 (M90T-DipgD-pAB17) (Allaoui
et al., 1993a, 1993b); GFP-expressing M90T, and GFP-expressing DmxiD
(Jaumouille et al., 2008). DmxiD and M90T-expressing IpgD fused to b-lacta-
mase were generated by transfecting the strains with a pBAD18 vector
(Guzman et al., 1995), encoding for the chloramphenicol resistence gene,
full-length IpgD, and TEM-1 (accession number AB282997, residues 24–286)
introduced into the multiple cloning site using NheI and XbaI sites (for IpgD)
and XbaI and HindIII (for TEM-1). Bacteria were grown at 37?C on trypticase
soy (TCS) (Becton Dickinson) agar plates containing 0.01% Congo red (Serva).
For cell invasion, a Congo red-positive colony was picked for an overnight
(O/N) culture at 37?C in TCS medium, followed by a subculture in the same
medium to grow the bacteria to logarithmic phase. Bacterial concentrations
were calculated from the optical density of the culture at 600 nm.
Plasmid DNA Purification
E. coli DH5a strains (Invitrogen) containing the eukaryotic expression vectors
were grown O/N in LB medium with corresponding antibiotic at 37?C. The
EndoFree Plasmid Maxi Kit (QIAGEN) was used for the purification of plasmid
DNA, according to the manufacturer’s recommendations. The following
plasmids were used: pKN16, GFP-tagged IpgD- KanR(Niebuhr et al., 2002);
mut-GFP, GFP-tagged IpgD- KanRwith a point mutation of Cys438 changed
into Ser (Niebuhr et al., 2002); GFP, EGFP-KanR(Niebuhr et al., 2002); and
mRFP-PLCd1PH- AmpR, mRFP-tagged PH domain of PLCd1 (kind gift from
Dr. T. Balla) (Varnai and Balla, 1998).
T Cell Lines and Primary T Cells
Jurkat, Clone E6-1 cells (ATCC TIB-152) were used as a human T cell line.
Human, monocyte-depleted, peripheral blood mononuclear cells (PBMCs)
were collected from blood samples. Informed consent was obtained from
donors according to French ethical laws and agreement between Institut Pas-
isolated with the CD4+T cells isolation kit II (Milteny Biotec), according to the
manufacturer’s recommendations. Jurkat T cells, human PBMCs, and purified
CD4+T cells were cultured in RPMI medium (RPMI 1640 GIBCO, Invitrogen)
supplementedwith10%decomplementedfetal bovine serum(FBS) (Biowest),
100 U/ml penicillin (Sigma-Aldrich), and 100 mg/ml streptomycin (Sigma-
Aldrich), at 37?C with 5% CO2. For activation, T cells were incubated for
3 days with 50 ng/ml phorbol 12-myristate 13-acetate (PMA) (Sigma-Aldrich).
The cell concentration was determined by using a Malassez counting
T Cell Infection and Gentamicin Assay
For infection with Shigella, T cells were centrifuged for 10 min at 300 g, resus-
pended in prewarmed RPMI medium without FCS, and seeded in round-
bottomed 96-well plates (TPP) at a concentration of 3 3 105cells in 100 ml
per well. Bacteria were centrifuged for 10 min at 4000 g and resuspended in
RPMI medium without FCS. A bacterial concentration of 6 3 107bacteria/ml
was used for an moi of 10. T cell infection started by adding 50 ml of the bacte-
bated at 37?C in 5% CO2. For the quantification of intracellular bacteria, extra-
cellular bacteria were killed by gentamicin treatment (Sigma-Aldrich) at
concentration of 50 mg/ml 1 hr postinfection. At indicated time points, cells
were lysed with 0.5% sodium desoxycholate and dilutions plated. Enumera-
tion of the bacteria was performed after O/N incubation at 37?C.
Jurkat cells were transfected by electroporation using the Amaxa Cell Line
Nucleofector Kit V (Lonza). The cells were electroporated with 10 mg DNA
encoding IpgD-GFP or IpgDmut-GFP and 5 mg DNA encoding PH-PLCd1-
RFP per 5 3 106cells using the Nucleofector Device electroporator with the
Nucleofector Program X-005 (Lonza). After electroporation, cells were
incubated in RPMI containing 2 mM L-glutamine, 1.5 g/L sodium bicarbonate,
4.5 g/L glucose, 10 mM HEPES, 1.0 mM sodium pyruvate, and 10% FBS for
1 day at 37?C.
For cell death quantification, cell suspensions were incubated for 1 min with
propridium iodide (PI) (dilution 1:1000 of the stock solution) (Sigma-Aldrich)
and FACS analysis performed to detect the PI-positive cell population.
Cell Host & Microbe
Inhibition of T Cell Migration by Shigella
Cell Host & Microbe 9, 263–272, April 21, 2011 ª2011 Elsevier Inc. 269
T cells were grown and infected as described above, using GFP-expressing
bacteria. After 1 hr of infection, T cells were fixed for 15 min with 4% parafor-
maldehyde (PFA) in PBS. Samples were transferred onto glass coverslips pre-
coated with 10 mg/ml poly-L-lysine and centrifuged for 1 min at 300 g. After
PBS washing, cells were incubated for 30 min with rabbit polyclonal serum
specific for Shigella-LPS (collection of the laboratory) (dilution 1:100 in PBS).
The cells were washed with PBS and incubated for 30 min with anti-rabbit-
AlexaFluor 350antibodies diluted 1:100 inPBS (Invitrogen). Cellpermeabiliza-
tion for actin staining was done with 0,1% Triton-X100 for 5 min, followed by
washes with PBS and incubated for 30 min with phalloidine-rhodamine. The
coverslips were mounted using ProLong-mounting medium (Invitrogen).
Immunofluorescence pictures were acquired either by inverted widefield
(Carl Zeiss Inc.) or confocal microscopy (SP5, Leica) under oil immersion.
T cell migration was performed as previously described (Ottoson et al., 2001)
with the addition of gentamicin treatment to kill extracellular bacteria. For
transfected Jurkat cell, no gentamicin was used. The migration index corre-
spondstothepercentage ofmigratedcellsataparticular conditionnormalized
to the percentage of migrated cells in the noninfected controlin the absence of
CXCL12 (negative control). Hence, a migration index of 1 corresponds to that
of the negative control. For the migration assay with transfected Jurkat cells,
only GFP-positive cells were taken into account.
Whole T cell lysates were obtained as previously described (Ottoson et al.,
2001) from 106cells. SDS-PAGE (8%) and immunoblotting were performed
as previously described (Kufer et al., 2006) with anti-phospho-ERM and anti-
ERM Abs (Cell Signal Technology), peroxydase-conjugated goat anti-rabbit
Ab (NordicImmunology) and the Bio-Rad SubstrateECLkit (Bio-Rad) followed
the membrane after immunoblotting, Bio-Rad stripping buffer (Bio-Rad) was
used according to the manufacturer’s instructions.
Transfected Jurkat cells were starved for 1 hr in serum-free RPMI medium.
Cells (5 3 105in 500 ml) cells were then transferred into 12-well plates contain-
ing poly-L-lysin-precoated coverslips. Cells were allowed to settle down for
20 min at 37?C. CXCL12 (Preprotech) (200 ng/ml final concentration) was
added for 45 s at 37?C, 5% CO2. Cells were fixed by adding ice-cold 4%
PFA for 15 min. After PBS washings, samples were blocked for 10 min with
PBS-BSA (1 mg/ml) and then incubated for 1 hr with mouse anti-human-
CD28 (Biolegend) (diluted 1:100 in PBS-BSA). After PBS washings, samples
were incubated with anti-mouse-Cy3 (Jackson Medicorp Inc) Ab (diluted
1:100 in PBS-BSA). Cells were permeabilized for 5 min with 0.1% Triton
X-100, blocked for 10 min with PBS-BSA, and rabbit anti-Phospho-ERM Ab
(Cell Signal Technology) added for 1 hr (diluted 1:100 in PBS-BSA). Anti-
rabbit-Alexa Fluor 647 (Invitrogen) diluted 1:100 in PBS-BSA was added for
1 hr. Coverslips were mounted on glass slides using ProLong mounting
Previous to the injection experiments, the bacterial strains M90T and mxiD
IpgD-bla were tested for secretion of the fusion protein as previously
described (Mounier et al., 1997) and the b-lactamase enzymatic activity tested
by cleavage of Nitrocefin. For this, bacterial cytosolic extracts were obtained
by shaking bacteria with glass beads (<106 mm from Sigma) for 10 min at
30 s?1at 4?C. Samples were then centrifuged at 13,500 rpm for 5 min at
4?C. Twenty microliters of extracts were incubated with 100 ml Nitrocefin
(0.1 mM) for 30 min at room temperature and the absorbance was measured
at 486 nm. For the infection experiments, the optimal cytochalasin D concen-
micin assay described above. Jurkat T cells were centrifuged for 10 min at
300 g, resuspended in EM buffer (120 mM NaCl, 7 mM KCl, 1.8 mM CaCl2,
B (according to the manufacturer’s recommendation, Invitrogen), 2 mM
Probenecid, 5 mg/ml cytochalasin D, and 1 mM CCF4 (Invitrogen) and seeded
in round-bottomed 96-well plates (TPP) at a concentration of 3 3 105cells in
100 ml per well. Loading was carried out for 1 hr at RT in the dark. Subse-
quently, cells were centrifuged and resuspended in 100 ml RPMI containing
2 mM Probenecid and 5 mg/ml Cytochalasin D. Infection was performed as
described above with the M90T IpgD-bla and mxiD IpgD-bla strains at an
moi of 500, and 100 mg/ml gentamicin was added after 20 min. After an addi-
tional incubation of 2 hr, cells were washed with PBS and transferred to flow
cytometry tubes for analysis. Data acquisition was carried out with a CyAn
ADP flow cytometer (DakoCytomation). Live, single cells were gated, and
the fluorescence intensities of cleaved and uncleaved CCF4 were detected
with the 405 nm excitation laser and 450 nm and 535 nm emission filters.
Analysis was performed using the public domain Image J64 program (version
1.42q NIH) as previously described (Kong et al., 2006). Briefly, the fluores-
cence intensity was measured in cells as the average pixel fluorescence inten-
sity within an area of defined size drawn over four distinct areas of the plasma
membrane (2 3 10 pixels) or the average of one box in the cytosol (11 3 11
pixels). Ratios of plasma membrane to cytosolic pixel fluorescence intensity
were determined and subjected to statistical analysis.
The t test was used to compare two groups, and p values < 0.05 were consid-
ered statistically significant. Significant statistically differences were indicated
by asterisks: *p < 0.05; **p < 0.01; ***p < 0.001. The error bars represent the
standard error of the mean (SEM).
Supplemental Information includes five figures and can be found with this
article at doi:10.1016/j.chom.2011.03.010.
We warmly thank C. Parsot (Unite ´ de Pathoge ´nie Microbienne Mole ´culaire,
Institut Pasteur) for very helpful discussion, M.I. Thoulouze and A. Alcover
(Unite ´ de Biologie Cellulaire des Lymphocytes, Institut Pasteur) for sharing
with us their expertise in T cells, G. Chicanne and B. Payrastre (INSERM
U858, I2MR, CHU Rangueil) for their expertise in PI metabolism, and M. Arpin
(Institut Curie, Paris, France) for her advice on the ERM proteins. From the G5
Dynamique des Interactions Ho ˆte-Pathoge `nes, Institut Pasteur, L. Audry and
A.Bobard werehelpfulinprovidingtools fortheinjectionassay,andJ.Enninga
for some experimental design, discussion, and critical reading of the manu-
script. T. Balla (National Institute of Child Health and Human Development,
National Institutes of Health [NIH]) kindly sent PLCd1PH-RFP construct. We
also thank Benoit Marteyn for his very efficient contribution in performing
Shigellainfectionintherabbitilealmodeland processing samplesforimmuno-
histochemistry. We are also grateful to colleagues from the IMAGOPOLE
platform of the Institut Pasteur. C.K. was supported by fellowships from the
European Consortium PATHOGENOMICS, the Institut Pasteur Transversal
Research Program n?251, and the Howard Hughes Medical Institute
(HHMI). E.F. holds a fellowship from the European Initiative for basic research
in Microbiology and Infectious Diseases (EIMID Program). K.N. is a fellow from
the Pasteur-Paris University International Doctoral Program. A. Puhar was
supported first by EMBO long-term fellowship and is presently a Marie Curie
fellow. W.S.-P. is funded by the Pasteur Foundation and the Philips Founda-
tion. P.J.S. is a HHMI Foreign Scholar. The research leading to these results
has received funding from the Institut Pasteur Transversal Research Program
(PTR n?251) and from the European Community’s PEOPLE Seventh Frame-
work Program under grant agreement EIMID IAPP - PIAP-GA-2008-217768.
Received: October 31, 2010
Revised: February 2, 2011
Accepted: March 18, 2011
Published: April 20, 2011
Cell Host & Microbe
Inhibition of T Cell Migration by Shigella
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