736 EMBO reports vol. 2 | no. 8 | pp 736–742 | 2001 © 2001 European Molecular Biology Organization
CARD4/Nod1 mediates NF-κB and JNK
activation by invasive Shigella flexneri
Stephen E. Girardin, Régis Tournebize1, Maria Mavris1, Anne-Laure Page1, Xiaoxia Li2,
George R. Stark2, John Bertin3, Peter S. DiStefano3, Moshe Yaniv, Philippe J. Sansonetti1,+
& Dana J. Philpott1,+
Unité des Virus Oncogènes,1Unité de Pathogénie Microbienne Moléculaire and Unité INSERM 389, Institut Pasteur, 28 rue du Dr Roux, Paris, Cédex 15,
75724 France,2The Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, OH and3Millennium Pharmaceuticals Inc., Cambridge, MA, USA
Received April 24, 2001; revised May 31, 2001; accepted June 11, 2001
Epithelial cells are refractory to extracellular lipopoly-
saccharide (LPS), yet when presented inside the cell, it is
capable of initiating an inflammatory response. Using invasive
Shigella flexneri to deliver LPS into the cytosol, we examined
how this factor, once intracellular, activates both NF-κB and
c-Jun N-terminal kinase (JNK). Surprisingly, the mode of acti-
vation is distinct from that induced by toll-like receptors
(TLRs), which mediate LPS responsiveness from the outside-in.
Instead, our findings demonstrate that this response is
mediated by a cytosolic, plant disease resistance-like protein
called CARD4/Nod1. Biochemical studies reveal enhanced
oligomerization of CARD4 upon S. flexneri infection, an event
necessary for NF-κB induction. Dominant-negative versions of
CARD4 block activation of NF-κB and JNK by S. flexneri as
well as microinjected LPS. Finally, we showed that invasive
S. flexneri triggers the formation of a transient complex
involving CARD4, RICK and the IKK complex. This study
demonstrates that in addition to the extracellular LPS sensing
system mediated by TLRs, mammalian cells also possess a
cytoplasmic means of LPS detection via a molecule that is
related to plant disease-resistance proteins.
In contrast to what is known about Toll-like receptors (TLRs) in
mediating responsiveness to bacteria and bacterial products in
cells of the myeloid lineage, the role played by TLRs in pathogen
recognition in epithelial cells remains poorly defined since these
cells are largely unresponsive to extracellular lipopolysaccharide
(LPS) and to non-pathogenic bacteria (Cario et al., 2000; Philpott
et al., 2000). Since most epithelial surfaces are constantly
exposed to extracellular bacteria and bacterial products, lack of
responsiveness of epithelial cells likely prevents the induction of
immune defense mechanisms against the normal microbial
flora. However, invasion of epithelial cells by pathogenic
bacteria initiates inflammatory responses. Infection of epithelial
cells with invasive pathogens, including Shigella flexneri
(Philpott et al., 2000) can induce the innate induction of NF-κB
leading to the production of the pro-inflammatory cytokine, IL-8.
Therefore, epithelial cells can initiate defensive responses to
invasive bacterial pathogens indicating that these cells might
possess an alternate means of pathogen detection.
Entry of S. flexneri into epithelial cells is essential for virulence
and also necessary for the induction of inflammatory responses
as assessed by activation of NF-κB and the production of IL-8
(Philpott et al., 2000). Bacterial internalization allows presentation
of LPS to the intracellular compartment, an event sufficient to
initiate the inflammatory response since microinjection of this
bacterial product directly into epithelial cells induces NF-κB
activation (Philpott et al., 2000). The mechanism by which intra-
cellular LPS activates this response, however, has not yet been
determined. In this study, we sought to identify the mechanism
through which intracellular LPS, delivered by invasive
S. flexneri, activates cell signaling pathways leading to NF-κB
and c-Jun N-terminal kinase (JNK) activation. Here we show that
CARD4/Nod1, a cytosolic protein that resembles a plant disease
resistance protein, is involved in both NF-κB and JNK activation
by intracellular LPS. These findings suggest the existence of an
evolutionarily conserved system of intracellular pathogen recog-
nition and signal transduction in epithelial cells that is
dependent on CARD4.
+Corresponding authors. Tel: +33 1 40 61 37 71; Fax: +33 1 45 68 89 53; E-mail: email@example.com or firstname.lastname@example.org
EMBO reports vol. 2 | no. 8 | 2001 737
NF-κB and JNK activation by invasive S. flexneri
RESULTS AND DISCUSSION
Activation of the JNK pathway by invasive
S. flexneri is dependent upon intracellular
presentation of LPS
Our previous work had demonstrated that S. flexneri activated
NF-κB in epithelial cells (including the intestinal cell line Caco-2)
through a pathway dependent upon bacterial entry and the intra-
cellular presentation of LPS. Next, we investigated whether
cellular responses to invasive S. flexneri included the activation
of JNK, a kinase important in the stress response to numerous
stimuli. The major target of JNK, c-Jun, is phosphorylated by this
kinase, which is an event that increases its transcriptional
activity. c-Jun is a component of the transcription factor AP-1,
another important regulator in the inflammatory response
(Foletta et al., 1998). Infection of HeLa epithelial cells with
invasive S. flexneri, but not non-invasive strains, led to the
accumulation of phospho-c-Jun in the nucleus of infected cells
(Figure 1A). This response could be mimicked by the direct
microinjection of LPS (Figure 1B). Furthermore, invasive
S. flexneri led to increased JNK kinase activity compared to
uninfected cells or cells infected with non-invasive strains
(Figure 1C). Therefore, we could demonstrate for the first time
that S. flexneri, as well as intracellular LPS when directly
microinjected into cells, are both potent inducers of the JNK
CARD4/Nod1, a cytosolic protein capable of
mediating LPS responsiveness, self-associates
following S. flexneri infection
Our focus was then to identify the signal transduction pathway
leading from intracellular LPS detection to NF-κB and JNK
activation. We found that invasive S. flexneri activates NF-κB
and JNK via a signal transduction pathway that is distinct from
the TLR/IL-1 pathway (reviewed in O’Neill and Greene, 1998).
Through transient transfection of dominant-negative molecules,
TRAF2 (Song et al., 1997), TRAF6 (Cao et al., 1996) and MyD88
(Muzio et al., 1997) were shown not to play a role in NF-κB
activation by invasive S. flexneri (Figure 2A–C). Moreover,
S. flexneri infection activated NF-κB (Figure 2D) and JNK (data
not shown) in three cell lines deficient in IL-1 signaling
components upstream of TRAF6 (Li et al., 1999), including one
deficient in IRAK. Therefore, an alternate LPS detection system,
independent of the one involving the TLRs, is likely to exist in
mammalian cells in order to respond to intracellular LPS.
Due to the evolutionarily conserved function of TLRs as
pathogen recognition molecules, the possible existence of a
cytosolic TLR-like molecule was pursued. A candidate protein
was found called CARD4 (Bertin et al., 1999) also known as
Nod1 (Inohara et al., 1999). Because of its similarity to plant R
proteins (Figure 3A), its cytosolic location (data not shown) and
its ability to activate NF-κB when overexpressed (Bertin et al.,
1999; Inohara et al., 1999), we hypothesized that CARD4 may
play a role in the detection of intracellular LPS in epithelial cells
infected with invasive pathogens such as S. flexneri. Additionally,
a recent study demonstrated the ability of overexpressed CARD4
to mediate responsiveness to extracellular LPS when co-incubated
with cells for 16 h (Inohara et al., 2001). This approach contrasts
with our experimental model where infection with S. flexneri results
in a cytoplasmic presentation of LPS, followed by a much faster
activation of downstream signaling pathways, within 20 min for
IKK (Philpott et al., 2000) and JNK activation (Figure 1C).
Recently, it was shown that enforced oligomerization of CARD4
via the NBS domain is an event that is sufficient for the induction of
NF-κB (Inohara et al., 2000). We, therefore, investigated the
possibility that S. flexneri infection could induce the activation of
CARD4 by enhancing its self-oligomerization. A full-length myc-
tagged CARD4 was co-expressed with a full-length hemagglutinin
(HA)-tagged CARD4 in order to perform co-immunoprecipitation
experiments following S. flexneri infection. Enhanced self-
association of CARD4 was observed as early as 20 min post-
infection with invasive S. flexneri (Figure 3B). In contrast, infection
with the non-invasive mutant of S. flexneri did not lead to enhanced
oligomerization of CARD4 (data not shown). These findings
provide the first evidence that infection with invasive S. flexneri is a
(patho-) physiological signal inducing CARD4 self-association in
infected epithelial cells.
∆CARD CARD4 and the LRR domain of CARD4
are dominant-negative inhibitors of NF-κB and
JNK induction by invasive S. flexneri
We then investigated whether CARD4 is involved in NF-κB and
JNK activation following S. flexneri infection. Since the CARD
domain is necessary for NF-κB activation (Bertin et al., 1999;
Inohara et al., 1999), we hypothesized that a CARD4 molecule
lacking this domain may act as a dominant-negative inhibitor of
NF-κB and JNK induction by S. flexneri. Indeed, overexpression
of a ∆CARD CARD4 molecule acted in a dose-dependent manner
to inhibit S. flexneri-induced NF-κB activation (Figure 4A).
Induction of the NF-κB reporter construct by ΤΝFα was much
less affected by overexpression of ∆CARD CARD4 testifying to
the specificity of this response. Furthermore, overexpression of
the ∆CARD CARD4 molecule also blocked JNK induction by
S. flexneri as assessed by an in vitro kinase assay (Figure 4C).
The dominant-negative effect induced by ∆CARD CARD4 over-
expression after S. flexneri infection is likely to be due to either
an interference in the propagation of the signal following
oligomerization of CARD4 through the NBS domain, or titration
of the LPS-induced signaling pathway upstream of CARD4
through the LRR domain. Therefore, we also overexpressed the
LRR domain of CARD4 alone and showed that it inhibited both
NF-κB and JNK activation by S. flexneri (Figure 4B and C), thus
reinforcing the hypothesis that LRR domain overexpression
interferes with upstream signaling pathways initiated by
infection. Whereas the CARD domain appears necessary for the
activation of downstream signaling components, the LRR
domain of CARD4 is likely to be responsible for sensing
intracellular LPS released from invasive S. flexneri. Accordingly,
in vitro studies recently demonstrated the presence of LPS in
CARD4-containing complexes (Inohara et al., 2001). However,
whether LPS directly binds to CARD4 or interacts with this
protein via other intracellular factors has yet to be determined.
We also observed that activation of JNK in cells infected with
S. flexneri was enhanced following overexpression of the full-length
molecule (Figure 4C) implying that low endogenous levels of
this protein may restrict activation.
738 EMBO reports vol. 2 | no. 8 | 2001
S.E. Girardin et al.
Overexpression of the LRR domain of CARD4 also inhibited signal
transduction induced by microinjected LPS (see Supplementary
Table I). Therefore, taken together, these findings implicate
CARD4 as a component of an intracellular LPS detection system
capable of inducing innate immune responses mediated through
the activation of NF-κB and JNK.
Formation of a transient complex containing
CARD4, RICK and the IKK complex
following S. flexneri infection
An induced proximity model for activation of NF-κB by CARD4
demonstrates that oligomerization of CARD4 leads to the
recruitment of RICK, another CARD-containing molecule (also
known as CARDIAK or RIP2; Thome et al., 1998). In turn, RICK
has been shown to recruit the members of the IKK complex
through NEMO, which leads to the activation of NF-κB (Inohara
et al., 2000). We investigated whether infection with the invasive
strain of S. flexneri could also result in the formation of such
complexes. Co-immunoprecipitation studies revealed that, while a
basal interaction between overexpressed CARD4 and RICK could
be detected in uninfected conditions, infection for 20 min with
invasive S. flexneri resulted in an enhanced interaction between
CARD4 and RICK (Figure 5A). These results are in agreement with
our findings that demonstrate oligomerization of CARD4 following
infection (see Figure 3B), since CARD4 self-oligomerization is the
Fig. 1. (A)Shigellaflexneri infectionleads tothephosphorylationofc-Jun.HeLacells were infectedwithwild-type invasive S. flexneriortheplasmid-cured,non-invasive
strain for 2 h. Infected cells were then stained for both phospho-c-Jun using a monoclonal phospho-specific antibody to this protein and LPS to label the infecting
bacteria using a rabbit polyclonal anti-LPS antibody. Stained cells were viewed using conventional fluorescence microscopy. (B) Microinjection of LPS activates
c-Jun phosphorylation. HeLa cells were microinjected with FITC-dextran to identify microinjected cells plus buffer alone or purified Escherichia coli LPS
O111:B4. Cells were then stained for phospho-c-Jun and examined by fluorescence microscopy. (C) Infection with S. flexneri increases JNK kinase activity. HeLa
cells were transfected with Flag-JNK1 for 40 h and then left either uninfected (CTR) or infected for 20 min with invasive (Inv) or non-invasive (NInv) S. flexneri.
Cells were treated for 20 min with 80 J/m2UVC as a positive control. Cells were collected and protein extracts were then used for a JNK kinase assay (see
Methods). Fold activation compared to uninfected cells is presented.
EMBO reports vol. 2 | no. 8 | 2001 739
NF-κB and JNK activation by invasive S. flexneri
critical step for induction of the CARD4–RICK interaction (Inohara
et al., 2000). Interestingly, this increased association was tran-
sient since most of the CARD4–RICK interaction was lost 40 min
after infection. As the oligomerization of CARD4 was not inhib-
ited at this time following infection (see Figure 3B), the observed
downregulation of the CARD4–RICK complex suggests that this
interaction might be modulated at this level through a mecha-
nism yet uncharacterized. This transient interaction between
RICK and CARD4 may be responsible for Shigella-induced acti-
vation of the JNK pathway since we observed that oligomeriza-
tion of RICK is sufficient to activate this pathway (data not
A potential interaction between RICK and the IKK complex
following infection was also investigated. RICK was over-
expressed and antibodies to endogenous IKKα were used to
immunoprecipitate the IKK complex following invasive S. flexneri
infection to investigate whether the IKK complex interacted with
RICK. Similar to the observed interaction between RICK and
CARD4, RICK was observed to transiently associate with IKKα;
complex formation was observed 20 min post-infection, while
Fig. 2. Shigella flexneri induces NF-κB through a signaling pathway distinct
from the TLR/IL-1 pathway. Dominant-negative forms of TRAF2 (A) and
TRAF6 (B) do not inhibit the induction of NF-κB by invasive S. flexneri
(stippled bars), whereas these dominant-negative proteins inhibit NF-κB
activation by TNFα (filled bars) or IL-1 (hatched bars), respectively. HEK293
cells were transfected with vector alone or increasing amounts of DNA
encoding for the dominant-negative forms of TRAF2 (DN-TRAF2) or
TRAF6 (DN-TRAF6) along with a NF-κB luciferase reporter plasmid and
a β-galactosidase plasmid. After 30 h, cells were infected with wild-type
S. flexneri or treated with TNFα (100 ng/ml) or IL-1 (10 ng/ml) for 4 h and
assayed for luciferase activity. (C) Dominant-negative MyD88 (DN-MyD88)
does not inhibit NF-κB activation by invasive S. flexneri (stippled bars) but
affects IL-1- (hatched bars) induced activation of the reporter gene. Increasing
amounts of DNA encoding for dominant-negative MyD88 were transfected
into HEK293 cells and assayed for NF-κB luciferase activity as described
in (A). (D) Shigella flexneri induces NF-κB in HEK293 deficient in IL-1
specific signaling components (see Methods; Li et al., 1999). NF-κB
activation after S. flexneri infection (stippled bars), TNFα (filled bars) or IL-1
(hatched bars) treatment was compared in parental HEK293 cells or three IL-1
signaling mutant cell lines, I1A (IRAK-negative), I2A and I3A. NF-κB
activity was assessed following infection or cytokine treatment by the NF-κB
reporter assay (refer to above) or EMSA. NF-κB reporter assays were
performed in duplicate at least three times and display mean values ± SEM.
Fig. 3. CARD4 oligomerization following S. flexneri infection. (A) Domain
structure of CARD4 compared with plant disease resistance proteins: Tobacco
N protein and Arabidopsis RPS2 protein. (B) Infection with S. flexneri induces
self-association of CARD4. HEK293 cells were transfected with empty vector
or expression vectors encoding either HA-CARD4 or Myc-CARD4 for 24 h and
were left either uninfected or S. flexneri-infected for 20 or 40 min. Cells were
collected and protein extracts (Ext) were subjected to western blotting with
rabbit polyclonal antibodies to Myc or HA to identify the expression levels of
the overexpressed proteins. Another fraction of the protein extracts was used
for immunoprecipitation using a polyclonal anti-HA antibody. Oligomerized
CARD4 was revealed in the immunoprecipitates by western blotting using
antibodies to the Myc-tagged CARD4.
740 EMBO reports vol. 2 | no. 8 | 2001
S.E. Girardin et al.
by 40 min post-infection the complex appeared to be downregulated
(Figure 5B). These results provide the first evidence for the induced
proximity model of NF-κB activation through CARD4–RICK–IKK
complex formation initiated by a physiological stimulus, infection
with S. flexneri.
We have used S. flexneri as a paradigm of an invasive Gram-
negative pathogen to define a signaling pathway that is involved
in the initiation of the inflammatory response following bacterial
infection. CARD4 or other CARD4-like molecules may represent
a common mode of bacterial detection involved in initiating
defensive responses to a number of Gram-negative pathogens
since LPS is a conserved component of this group of bacteria.
Moreover, many of these infections lead to inflammation driven
by the induction of NF-κB and/or AP-1. Further characterization
of the signaling pathways induced by other pathogens may
implicate CARD4 as a common mediator of inflammatory
processes initiated upon infection.
CARD4 is a member of a new family of proteins that possess a
C-terminal LRR and a NBS (Bertin and Distefano, 2000). This
study on CARD4 suggests the possibility that this family of
proteins represents human homologues of plant disease-
resistance proteins. These cytosolic proteins may be involved in
mediating defensive responses to distinct intracellular pathogens
or pathogen products. As it has been shown for the TLRs,
different CARD4-like proteins may exist that are involved in the
recognition of distinct bacterial products. The findings provided
here present the first indication that one of these family
members, CARD4, mediates intracellular pathogen recognition
and signal transduction.
Fig. 4. Constructs expressing either ∆CARD CARD4 or the LRR domain of
CARD4 act as dominant-negative inhibitors in S. flexneri-induced activation
of NF-κB and JNK. Increasing amounts of DNA encoding either (A) ∆CARD
CARD4 or (B) the LRR domain inhibits NF-κB induction by S. flexneri
(stippled bars) in a DNA concentration-dependent manner but only marginally
affects NF-κB activation by TNFα (filled bars) at the higher concentrations of
DNA. Plasmids encoding the truncated forms of CARD4 were transfected into
HEK293 cells along with NF-κB and β-galactosidase reporter plasmids.
Luciferase activity was assayed as described in the Methods. (C) Effect of the
overexpression of full length and truncated forms of CARD4 on JNK
activation. HeLa cells were transfected with Flag-JNK1 and either empty
vector or with expression vectors encoding for CARD4 full length (CARD4-FL),
∆CARD CARD4 or the LRR of CARD4 for 40 h followed by 20 min infection
by invasive (i) or non-invasive (ni) S. flexneri. JNK kinase assays were
performed as described in the Methods. Fold activation compared to the
S. flexneri-induced activation in vector alone expressing cells is presented.
Fig. 5. Interaction between CARD4 and RICK and RICK and IKKα of the
IKK complex folowing S. flexneri infection. (A) Immunoprecipitation using
anti-RICK antibodies demonstrates that RICK and CARD4 interact
transiently following S. flexneri infection. (B) Immunoprecipitation of the
IKK complex using anti-IKKα antibodies showed that RICK (both
endogenous and overexpressed vsv-tagged forms of RICK) associates
transiently with the IKK complex following S. flexneri infection. Experiments
were carried out using HEK293 cells as in Figure 3.
EMBO reports vol. 2 | no. 8 | 2001 741
NF-κB and JNK activation by invasive S. flexneri
Infection of HeLa, HEK293 cells or IL-1 signaling mutant cell
lines. HeLa and HEK293 cells were grown as described
presiously (Philpott et al., 2000). Infection with S. flexneri
activates downstream signaling pathways in both of these cells
lines (Philpott et al., 2000). The three HEK293 cell lines deficient
in IL-1 signaling components have been described previously (Li
et al., 1999). The I1A cell line is deficient in IRAK whereas the
defect in the I2A and I3A cell lines have not been fully charac-
terized, yet the defect likely lies between the IL-1 receptor
complex and IRAK (Li et al., 1999).
Wild-type S. flexneri (M90T, serotype 5A) or a plasmid-cured
derivative of the parental strain (BS176) were used to infect cells
at a multiplicity of infection (MOI) of 50. Previous studies
(Philpott et al., 2000) and preliminary evidence had indicated
that this MOI was sufficient to generate a strong NF-κB or
phospho-c-Jun response in 30 min or 1 h, respectively. Infections
were carried out as described previously (Philpott et al., 2000). For
phospho-c-Jun staining, a specific c-Jun antibody recognizing
phosphorylation of c-Jun at serine 63 (Lallemand et al., 1998)
was used followed by anti-mouse secondary antibodies coupled
to Cy3 (Jackson). Staining of LPS using an anti-LPS antibody
(from Dr Armelle Phalipon) followed by FITC-conjugated
secondary antibodies was used to visualize infecting bacteria.
Expression vectors and transient transfections. Vector
constructs expressing dominant-negative MyD88 have been
described previously (Muzio et al., 1997). The LRR domain and
the myc-tagged CARD4 plasmids were constructed by PCR from
the HA-tagged CARD4 plasmid and inserted into pcDNA3
(Invitrogen). The HA-tagged CARD4 and ∆CARD CARD4
plasmids have been described previously (Bertin et al., 1999).
For NF-κB reporter gene assays, HEK293 cells were trans-
fected with FuGene using 300 ng of the NF-κB luciferase
reporter was added along with the indicated amounts of effector
plasmid and a β-galactosidase reporter in order to normalize
transfection efficiencies (Philpott et al., 2000).
Microinjection and fluorescence microscopy. HeLa cells were
plated onto coverslips, transfected and microinjected the
following day with purified Escherichia coli LPS (O111:B4;
Sigma) or bacteria-free supernatants from wild-type S. flexneri as
described previously (Philpott et al., 2000). Staining for NF-κB
and phospho-c-Jun was carried out as described above.
Immunoprecipitation experiments and JNK assays. For
immunoprecipitation experiments, HEK293 cells were plated in
10 cm dishes and transfected with FuGene the following day
using 500 ng of Myc-CARD4, HA-CARD4 and/or VSV-RICK
expression plasmids. Following S. flexneri infection for the
indicated times, immunoprecipitation of HA-CARD4, RICK or
IKKα was performed as previously described (Bertin et al., 1999)
using polyclonal anti-HA (Santa Cruz), polyclonal anti-RICK
(Cayman Chemical, Ann Arbor, MI) or monoclonal IKKα
(Transduction Laboratories). Immunoprecipitates and total protein
extracts were resolved by SDS–PAGE, transferred to nitrocellulose
and western blotting was performed using polyclonal antibodies
against Myc or HA (Santa Cruz Biotechnology).
JNK activity was analyzed by a quantitative JNK kinase assay
(Girardin and Yaniv, 2001). Briefly, HeLa cells were transfected
with 500 ng of Flag-JNK1 expression vector plus 2 µg of empty
vector, HA-tagged CARD full length (FL), ∆CARD CARD4 or LRR
CARD4 vectors. Forty hours post-transfection, cells were left
uninfected or infected with either the plasmid-cured, non-invasive
(ni) strain or wild-type, invasive (i) S. flexneri for 20 min. Flag-JNK1
was immunoprecipitated using antibodies to the Flag epitope.
JNK1 activity of the immunoprecipitates was determined by
dividing the level of c-Jun phosphorylation by the amount of
overexpressed Flag-JNK1 levels for each sample as described
(Girardin and Yaniv, 2001). The level of c-Jun phosphorylation
and Flag-JNK1 expression were determined by densitometry
following STORM and analyses using ImageQuant software
Supplementary data. Supplementary data are available at EMBO
We thank Dr Frédéric Relaix, Institut Pasteur, for providing the
dominant-negative construct of TRAF2, Dr Simon Whiteside,
Institut Pasteur, for dominant-negative TRAF6 and VSV-RICK,
and Dr Marta Muzio, Mario Negri Institute, Milan, for the domi-
nant-negative MyD88 construct. We also thank Dr Dominique
Lallemand for the preparation of anti phospho-c-Jun antibody.
S.E.G. was supported by a Pasteur-Weitzman fellowship from
the Institut Pasteur. P.J.S. is a Howard Hughes International
Research Scholar. D.J.P. was supported by a fellowship from the
Canadian Institutes of Health Research. Research in the Yaniv
and Sansonetti laboratories is supported by grants from the ARC
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