The signaling pathway of Campylobacter jejuni-induced Cdc42 activation: Role of fibronectin, integrin beta1, tyrosine kinases and guanine exchange factor Vav2
Cell Communication and Signaling
The signaling pathway of Campylobacter jejuni- induced Cdc42 activation: Role of fibronectin, integrin beta1, tyrosine kinases and guanine exchange factor Vav2
Malgorzata Krause-Gruszczynska 0
Manja Boehm 0
Nicole Tegtmeyer 0
Omar A Oyarzabal
Steffen Backert 0
0 From the School for Biomedical and Biomolecular Science, University College Dublin , Belfield Campus, Dublin-4 , Ireland
Background: Host cell invasion by the foodborne pathogen Campylobacter jejuni is considered as one of the primary reasons of gut tissue damage, however, mechanisms and key factors involved in this process are widely unclear. It was reported that small Rho GTPases, including Cdc42, are activated and play a role during invasion, but the involved signaling cascades remained unknown. Here we utilised knockout cell lines derived from fibronectin-/-, integrin-beta1-/-, focal adhesion kinase (FAK)-/- and Src/Yes/Fyn-/- deficient mice, and wild-type control cells, to investigate C. jejuni-induced mechanisms leading to Cdc42 activation and bacterial uptake. Results: Using high-resolution scanning electron microscopy, GTPase pulldowns, G-Lisa and gentamicin protection assays we found that each studied host factor is necessary for induction of Cdc42-GTP and efficient invasion. Interestingly, filopodia formation and associated membrane dynamics linked to invasion were only seen during infection of wild-type but not in knockout cells. Infection of cells stably expressing integrin-beta1 variants with well-known defects in fibronectin fibril formation or FAK signaling also exhibited severe deficiencies in Cdc42 activation and bacterial invasion. We further demonstrated that infection of wild-type cells induces increasing amounts of phosphorylated FAK and growth factor receptors (EGFR and PDGFR) during the course of infection, correlating with accumulating Cdc42-GTP levels and C. jejuni invasion over time. In studies using pharmacological inhibitors, silencing RNA (siRNA) and dominant-negative expression constructs, EGFR, PDGFR and PI3-kinase appeared to represent other crucial components upstream of Cdc42 and invasion. siRNA and the use of Vav1/2-/knockout cells further showed that the guanine exchange factor Vav2 is required for Cdc42 activation and maximal bacterial invasion. Overexpression of certain mutant constructs indicated that Vav2 is a linker molecule between Cdc42 and activated EGFR/PDGFR/PI3-kinase. Using C. jejuni mutant strains we further demonstrated that the fibronectin-binding protein CadF and intact flagella are involved in Cdc42-GTP induction, indicating that the bacteria may directly target the fibronectin/integrin complex for inducing signaling leading to its host cell entry. Conclusion: Collectively, our findings led us propose that C. jejuni infection triggers a novel fibronectinintegrinbeta1FAK/SrcEGFR/PDGFRPI3-kinaseVav2 signaling cascade, which plays a crucial role for Cdc42 GTPase activity associated with filopodia formation and enhances bacterial invasion.
Rho family GTPases; Cdc42; EGF receptor; PDGF receptor; Vav2; PI3-kinase; molecular pathogenesis; cellular invasion; signaling; virulence
Food-borne infections with pathogenic bacteria
represent one of the leading causes of morbidity and death in
humans. Estimations by the World Health Organization
WHO suggest that the human population worldwide
suffers from about 4.5 billion incidences of diarrhoea
every year, causing approximately 1.8 million deaths .
Campylobacter has been recognized as the leading cause
of enteric bacterial infection worldwide [2,3]. Two
Campylobacter species, C. jejuni and to less extent C. coli,
are most frequently found in infected persons.
Campylobacter jejuni is a classical zoonotic pathogen, as it is
part of the normal intestinal flora in various birds and
mammals. Because C. jejuni is also present in many
agriculturally important animals, it can contaminate the
final products during food processing . After
ingestion by humans, bacteria remain motile, colonize the
mucus layer in the ileum and colon, interfere with
normal functions in the gastrointestinal tract, and lead to
diseases associated with fever, malaise, abdominal pain
and watery diarrhoea, often containing blood cells [2,3].
In addition, individuals exposed to C. jejuni may develop
late complications, including Reiters reactive arthritis as
well as the Guillain-Barr or Miller-Fisher syndromes
. Increasing amounts of data accumulated in the last
decade suggest that C. jejuni perturbs the normal
absorptive capacity of the human intestine by damaging
epithelial cell functions, either directly by cell invasion
and/or the production of virulence factors, or indirectly
by triggering inflammatory responses [3,6-8].
It has been proposed that invasion of host cells during
infection is a main source of C. jejuni-driven tissue
damage in the intestine. Examination of intestinal
biopsies from infected patients and infection of cultured
human intestinal epithelial cells in vitro indicated that C.
jejuni is capable of invading gut tissue cells [9-11]. In
general, bacterial entry into host cells in vitro may
proceed by microtubule-dependent and/or actin-dependent
pathways [10,12,13]. C. jejuni encodes numerous
outermembrane proteins with proposed roles in bacterial
adhesion such as CadF, FlpA, JlpA and PEB1 [14-17]. For
example, CadF is a well-known bacterial
outer-membrane protein which binds in vitro to fibronectin, an
important extracellular matrix (ECM) protein and
bridging factor to the integrin receptors [15,17-19]. INT-407
intestinal epithelial cells infected with C. jejuni exhibited
membrane ruffling associated with bacterial entry .
Maximal adherence and invasion of INT-407 cells
requires CadF and is accompanied with increased levels
of tyrosine phosphorylation of some yet unknown host
cell proteins [13,21], as well as paxillin, an
integrin-associated scaffold protein . However, the functional
importance of these findings for host cell entry and
which integrin maybe involved in this signaling remained
unclear. CadF and FlpA also seems to be involved in the
activation of the small Rho GTPases Rac1 and Cdc42,
which are required for the cell entry [17,20], but the
exact mechanisms are not yet clear. In addition, mutation
of certain genes in the flagellar export system, deletion of
ciaB (Campylobacter invasion antigen B), waaF and kpsS
genes, led to reduced adhesion and invasion of C. jejuni
in vitro, indicating that their corresponding proteins may
also have functions in host cell invasion [23-28]. It should
be noted, however, that some of these findings are not
reproducible by other research groups. For example, the
role of the described CiaB in invasion as well as the role
of the flagellum as a potential device for the secretion of
virulence factors was called into question . Thus, it is
not clear if the function of the flagellum during invasion
is due to the secretion of bacterial factors into the
medium or bacterial mobility.
Based on pharmacological inhibitor experiments, it was
also reported that multiple host protein kinases, such as
phosphatidylinositol 3-kinase (PI3-K), epidermal growth
factor receptor (EGFR), platelet-derived growth factor
receptor (PDGFR) and heterotrimeric G proteins may also
play a role in epithelial cell invasion by C. jejuni [11,21,30].
Moreover, caveolae structures may also play a role in the
invasion process because expression of dominant-negative
mutants of caveolin-1 significantly decreased C. jejuni
uptake . Once internalized into epithelial cells, C. jejuni
co-localize with microtubules  and survive for
considerable time and consequently may induce cytotoxic responses
in vitro [31-33]. The C. jejuni-containing intracellular
vacuole deviates from the canonical endocytic pathway,
and by inhibition of their entry into lysosomes, the bacteria
may avoid elimination by the host immune system .
However, the molecular signaling pathways of early host
cell invasion events and the complex crosstalk between
bacterial and cellular factors are still widely unclear. Here
we identified the signaling cascade of C. jejuni-induced
Cdc42 activation and its role in host cell entry. We utilised
a unique set of mouse knockout cell lines, GTPase
pulldowns, gentamicin protection assays and high-resolution
scanning electron microscopy. Our studies show the
important functions of fibronectin, integrin-b1, several
kinases and the guanine exchange factor Vav2 in the
activation of Cdc42, and the induction of filopodia and
membrane dynamics during C. jejuni infection. Using C. jejuni
mutants strains we also demonstrate that the
fibronectinbinding protein CadF and the flagellum may play roles in
these early invasion-related signaling events.
The C. jejuni strains 81-176, 84-25 and F38011 were
used in this study. The isogenic F38011cadF,
81176flaA/B and 81-176flhA mutants were kindly
provided by Michael Konkel  and Patricia Guerry
. All C. jejuni strains were grown on Campylobacter
blood-free selective Agar Base (Oxoid) containing
Campylobacter growth supplement (Oxoid) or on
MuellerHinton (MH) agar amended with 50 g/ml kanamycin
or 30 g/ml or chloramphenicol at 37C under
microaerobic conditions (generated by CampyGen, Oxoid) for
Knockout fibroblasts and other cell lines
Several mouse fibroblast knockout cell lines were
cultured in RPMI1640 or DMEM medium, supplemented
with 10% fetal calf serum (Gibco). Generation of the
floxed FN+/+ mouse fibroblast cells and FN-/- knockout
cells has been described elsewhere [36,37]. The
FN-/cells were grown in DMEM supplemented with 10%
FCS, or alternatively in serum replacement medium
(Sigma Aldrich). Monolayers of GD25 mouse fibroblasts
(integrin-b1-/-) or GD25 cells stably transfected with wt
integrin b1A (GD25b1A) or several mutants
(GD25b1ATT788/89AA and GD25b1A-Y783/795F) were grown in
10% fetal bovine serum [38-40]. Mouse knockout cells
deficient in focal adhesion kinase (FAK-/- cells) or
fibroblasts derived from c-src-/-, c-yes-/-, and c-fyn-/- triple
knockout mouse embryos (SYF cells) as well as stable
expression of wt FAK in FAK-/- cells or wt c-Src in SYF
cells have been already described [41,42]. Mouse
embryonic fibroblasts from Vav1/2-/- deficient were
prepared as described recently . These cells were grown
on gelatine-coated culture dishes in DMEM containing
10% FCS, non-essential amino acids and sodium
pyruvate . Human embryonic intestinal epithelial cells
(INT-407), obtained from the American Type Culture
Collection (ATCC CCL-6), were grown in MEM
medium containing L-glutamine and Earles salts (Gibco).
After reaching about 70% confluency, the cells were
washed two times with PBS, and then starved for 12
hours before infection.
For the infection experiments, the different cell lines
were seeded to give 4 105 cells in 12-well tissue
culture plates. The culture medium was replaced with fresh
medium without antibiotics 1 hour before infection.
Bacteria were suspended in phosphate-buffered saline
(PBS, pH 7.4), added to the cells at a multiplicity of
infection (MOI) of 100, and co-incubated with host cells
for the indicated periods of time per experiment.
The pharmacological inhibitors methyl-beta cyclodextrin
(MbCD, Sigma, 1 mM-10 mM), PF-573228 (Tocris; 10
M), AG1478 (10 M) , AG370 (10 M) , or
wortmannin (1 M) [21,45] were added 30 min prior to
infection and kept throughout the entire duration of the
experiment. Control cells were treated with the same
amount of corresponding solvent for the same length of
time. We have carefully checked the viability of cells in
every experiment to exclude toxic effects resulting in
loss of host cells from the monolayers. The experiments
were repeated at least three times.
Plasmid DNA Transfection
Eukaryotic expression vectors for human, wt PDGFRb,
dominant-negative PDGFRb, wt EGFR and
dominantnegative EGFR, were kindly provided by Drs. T. Hunter
and G. Gill (University of California, USA). Myc-tagged
wt Vav2 and dominant-negative Vav2 were described
. GFP-fusion proteins of Vav2 include wt, Vav2
Y172/159F, Vav2 R425C, Vav2 W673R and Vav2 G693R
. Transfection of plasmid constructs into host cells
was performed using GeneJammer transfection reagent
according to manufacturers instructions (Stratagene).
After 48 hours, transfected INT-407 cells were infected
with C. jejuni strains for 6 hours. The efficiency of
transfection was verified both by immunofluorescence
microscopy and Western blotting using respective
siRNA directed against human DOCK180, Vav2 and
siRNA containing a scrambled control sequence were
purchased from Santa Cruz. siRNA against human
Cdc42 was synthesised as
5-TTCAGCAATGCAGACAATTAA-3. For down-regulation of Tiam-1, the
siRNAs from Santa Cruz and another one obtained from
MWG-Biotech (5-ACAGCTTCAGAAGCCTG AC-3)
were used simultaneously. Transfection of siRNAs was
performed using siRNA transfection reagent (Santa
Gentamicin protection assay
After infection, eukaryotic cells were washed three times
with 1 ml of pre-warmed MEM medium per well to
remove non-adherent bacteria. To determine the CFU
corresponding to intracellular bacteria, the INT-407 cell
monolayers were treated with 250 g/ml gentamicin
(Sigma) at 37C for 2 hours, washed three times with
medium, and then incubated with 1 ml of 0.1% (w/v)
saponin (Sigma) in PBS at 37C for 15 min. The treated
monolayers were resuspended thoroughly, diluted, and
plated on MH agar. To determine the total CFU of
host-associated bacteria, the infected monolayers were
incubated with 1 ml of 0.1% (w/v) saponin in PBS at 37
C for 15 min without prior treatment with gentamicin.
The resulting suspensions were diluted and plated as
described above. For each strain, the level of bacterial
adhesion and uptake was determined by calculating the
number of CFU. In control experiments, 250 g/ml
gentamicin killed all extracellular bacteria (data not shown).
All experiments were performed in triplicates.
Cdc42-GTP pulldown assay
Cdc42 activation in infected cells was determined with
the Cdc42 activation assay kit (Cytoskeleton, Inc, City,
Country), based on a pulldown assay using the
Cdc42Rac1 interactive binding domain of PAK1 fused to
glutathione S-transferase(GST-CRIB), also called
GSTCRIB pulldown . Briefly, host cells were grown to
70% confluency and serum-starved overnight.
Subsequently, cells were incubated in PBS as a control or
infected with C. jejuni (MOI of 100) in a time course.
Uninfected and infected host cells were washed with
PBS, resuspended in the assay buffer of the kit, and
detached from dishes with a cell scraper. For a positive
and negative control, a portion of the uninfected cell
lysate was mixed with GTPg-S and GDP, respectively,
for 15 min. Cell lysates (treated with bacteria, GTPg-S,
GDP or untreated) were mixed with the PAK-RBD
slurry (1 hour, 4C). Finally, the beads were collected by
centrifugation and washed three times with assay buffer.
Activated Cdc42 was then visualized by immunoblotting
as described below. To confirm equal amounts of
protein for each sample, aliquots of the lysates from
different time points were also analyzed by immunoblotting.
The GTPase activities were quantified as band
intensities representing the relative amount of active
Cdc42GTP using the Lumi-Imager F1 software program
Cdc42 activation in infected cells was also determined
with the G-LISARac1- activation assay (Cytoskeleton).
Host cells were grown to 70% confluency in tissue
culture petri dishes and serum depleted overnight. The
cells were infected with C. jejuni for the indicated times
per experiment. Subsequently, cells were washed with
PBS, resuspended in lysis buffer of the kit and harvested
from the dishes with cell scraper. Total protein
concentration in each lysate was determined by protein assay
reagent of the kit. The G-LISAs contains a
Rac1-GTPbinding protein immobilised on provided microplates.
Bound active Cdc42 was detected with a specific
antibody and luminescence. The luminescence signal was
quantified by using a microplate reader (SpectraFluor
SDS-PAGE and immunoblotting
Proteins from transfected and/or infected host cells were
separated on 10-15% polyacrylamide gels and blotted
onto polyvinylidene difluoride (PVDF) membranes
(Immobilon-P; Millipore). Staining with primary
antibodies against FAK-PY-397 (Biomol), EGFR-PY-845,
PDGFR-PY-754 (both NEB), FAK, Cdc42, RhoA,
Fibronectin, integrin-b1, Tiam-1, DOCK180 or GAPDH (all
Santa Cruz) was performed according to the
manufacturers instructions. As secondary antibodies, horseradish
peroxidase-conjugated a-mouse, a-rabbit or a-goat IgG
(DAKO) was used. Immuno-reactive bands were
visualized by ECL plus Western Blotting Detection System
(Amersham Biosciences). Relative FAK, EGFR and
PDGFR kinase activities were quantified as band
intensities of the corresponding activation-specific
phosphoantibody signals related to its non-phospho control blots
using the Lumi-Imager F1 software program (Roche).
The strongest seen phospho-band levels per experiment
were taken as 100% kinase activity.
FESEM (Field Emission Scanning Electron Microscopy)
Host cell monolayers grown on coverslips were infected
with C. jejuni strains for either 4 or 6 hours, then fixed
with cacodylate buffer (0.1 M cacodylate, 0.01 M CaCl2,
0.01 M MgCl2, 0.09 M sucrose; pH6.9) containing 5%
formaldehyde and 2% glutaraldehyde, and subsequently
washed several times with cacodylate buffer. Samples
were dehydrated with a graded series of acetone (10, 30,
50, 70, 90 and 100%) on ice for 15 min for each step.
Samples in the 100% acetone step were allowed to reach
room temperature before another change of 100%
acetone. Samples were then subjected to critical-point
drying with liquid CO2 (CPD030, Bal-Tec, Liechtenstein).
Dried samples were covered with a 10 nm thick gold
film by sputter coating (SCD040, Bal-Tec, Liechtenstein)
before examination in a field emission scanning electron
microscope (Zeiss DSM-982-Gemini) using the Everhart
Thornley SE detector and the inlens detector in a 50:50
ratio at an acceleration voltage of 5 kV.
All data were evaluated using Student t-test with
SigmaStat statistical software (version 2.0). Statistical
significance was defined by P 0.05 (*) and P 0.005 (**). All
error bars shown in figures and those quoted following
the +/- signs represent standard deviation.
Activation of Cdc42 by C. jejuni is time-dependent, and
bacterial invasion requires intact lipid rafts and Cdc42
We have previously shown that small Rho GTPases such
as Cdc42 are activated by C. jejuni. Inhibitors, toxins,
expression of dominant-negative constructs and other
experiments have indicated that active Cdc42 could be
an important host determinant required for bacterial
invasion . In the present study, we identified and
characterized the signaling pathway leading to C.
jejuniinduced Cdc42 activation. First, we confirmed that
Cdc42 is activated in infected non-phagocytic INT-407
intestinal epithelial cells using a novel commercial
GLisa assay and GTPase pulldowns of the GST-CRIB
construct. The results showed that infection with wild-type
(wt) C. jejuni strains 81-176, F38011 or 84-25 induced
the accumulation of active Cdc42-GTP in a time
dependent manner (Figure 1A and data not shown). In order
to confirm that Cdc42 is indeed necessary for the entry
of C. jejuni into host cells, we downregulated Cdc42
expression by siRNA. Downregulation of Cdc42
expression by > 95% lead to a significant drop in the number
of intracellular colony-forming units (CFU), as
quantified in gentamicin protection assays (Figure 1B).
Transfection of a scrambled siRNA as control did not reveal
suppressive effects on C. jejuni invasion (Figure 1B).
These results indicate that invasion of C. jejuni into
cultured host cells requires Cdc42.
Recent experiments have indicated that treatment with
methyl-beta cyclodextrin (MbCD), an agent sequestering
cholesterol in lipid rafts, decreased the ability of C.
jejuni to invade cultured epithelial cell lines . Thus,
we tested if the integrity of lipid rafts may be also
required for C. jejuni-mediated Cdc42 activation.
Indeed, addition of MbCD to INT-407 cells inhibited C.
jejuni-induced Cdc42 activation and bacterial
internalization in a dose-dependent fashion (Figure 1C),
suggesting that one or more lipid raft-associated host cell
receptor(s) maybe activated by the bacteria to induce
signaling resulting in elevated Cdc42-GTP levels and
subsequently bacterial uptake.
C. jejuni invasion and Cdc42 activation require
fibronectin, integrin-b1, FAK and Src kinases
Because C. jejuni encodes the well-known
fibronectinbinding protein CadF on its surface , we suggested
that a classical fibronectinintegrin-b1focal adhesion
kinase (FAK)Src kinase pathway could be involved in
activating Cdc42. To investigate this hypothesis, we used
fibroblast cell lines derived from fibronectin-/-,
integrinb1-/- (so called GD25 cells), FAK-/- and c-src-/-, c-yes-/-,
and c-fyn-/- (SYF) triple knockout mice [36,38,41,42],
which completely lack expression of the respective genes
(Figure 2A-D). As positive control, we infected with wt
C. jejuni under identical conditions floxed fibronectin+/+
cells, GD25 cells re-expressing wt integrin-b1A
(GD25b1A), FAK-/- cells re-expressing wt FAK and SYF
cells re-expressing c-Src. Gentamicin protection assays
showed that, while the knockout cells exhibited
significant deficiencies for bacterial uptake, C. jejuni invaded all
wt control cells very efficiently (Figure 2A-D). In
addition, Cdc42-GTP levels were determined in the same set
of experiments, showing that activation of Cdc42 also
depends on the expression of each of the above genes.
Figure 1 C. jejuni-triggered Cdc42 activation is time-dependent
and requires intact lipid rafts. (A) Quantification of Cdc42 activity
during the course of infection. INT-407 cells were infected with wt
C. jejuni 81-176 for indicated periods of time. The presence of active
Cdc42-GTP was quantified by G-Lisa and GST-CRIB pulldown. One
hundred % of GTPase activity corresponds to the highest amount of
detected Cdc42-GTP level (right lane). Similar quantities of total
Cdc42 and GAPDH were confirmed by Western blotting. (B) Effect
of Cdc42 expression knockdown on C. jejuni invasion. INT-407 cells
were transfected with Cdc42-siRNA as well as a scrambled siRNA as
control. After 48 hours, cells were infected with C. jejuni for 6 hours.
Intracellular bacteria were quantified by gentamicin protection
assays. Immunoblotting with a-Cdc42 antibody confirmed
downregulation of the protein. GAPDH expression levels were determined
as control. (C) Effects of MbCD targeting lipid rafts on host cell
internalization of C. jejuni. INT-407 monolayers were pre-incubated
with the indicated concentrations of MbCD for 30 min, followed by
6 hours infection with wt C. jejuni 84-25. Intracellular C. jejuni were
quantified by gentamicin protection assays. The presence of active
Cdc42-GTP was analyzed by CRIB-GST pulldown and quantified. One
hundred % of activity corresponds to the highest amount of
detected Cdc42-GTP level (lane 2). Similar quantities of total Cdc42
and GAPDH were confirmed by Western blotting. (*) P 0.05 and
(**) P 0.005 were considered as statistically significant as
compared to the control.
C. jejuni invasion is inhibited in cells expressing
integrinb1 mutants with defects in fibronectin fibril formation
and FAK signaling
In order to investigate the importance of fibronectin and
integrin-b1 signaling in more detail, we utilized two
well-established mutant cell lines, GD25 knockout cells
stably expressing integrin-b1A TT788/89AA which
exhibit functional FAK signaling but a defect in extracellular
fibronectin fibril formation , and GD25b1A-Y783/
795F cells, which have a pronounced defect in FAK
autophosphorylation at Y-397 . These cells were
infected with C. jejuni followed by gentamicin
protection assays. The number of intracellular C. jejuni was
found to be significantly reduced in the
integrin-b1-deficient GD25 cells and was restored when wt
integrinb1A was stably expressed (Figure 3A). However, the
expression of TT788/89AA or Y783/795F mutants in
GD25 cells did not rescue the capability of C. jejuni to
invade these cells; especially the FAK-signaling deficient
GD25b1A-Y783/795F cells exhibited a highly
pronounced defect for the uptake of bacteria (Figure 3A).
Interestingly, these results strictly correlated with the
parallel Cdc42 activation assays, showing that the same
integrin-b1 mutant cell lines are also widely deficient in
their capability to induce Cdc42-GTP production during
C. jejuni infection (Figure 3B). This data suggests that
integrin-mediated fibronectin fibril formation and FAK
downstream signaling are also required for efficient
Cdc42 activation and C. jejuni uptake.
C. jejuni induces filopodia formation and invasion in wt
cells but not in fibronectin, integrin-b1 and FAK knockout
The above results led us to propose that fibronectin,
integrin-b1 and FAK may form a signaling complex to
induce Cdc42 activity during infection. Thus, we asked
if we could visualize classical Cdc42-triggered filopodia
on cells upon contact with the bacteria. To investigate
this question, we infected wt fibroblasts with wt C.
GAPDH GAPDH GAPDH GAPDH
Figure 2 Importance of fibronectin, integrin-b1, FAK and Src kinases expression on C. jejuni invasion. The following cells lines were
infected with wt C. jejuni 81-176 for 6 hours. (A) Fibronectin-deficient cells (Fn-/-) and corresponding floxed wt cells (Fn+/+), (B)
integrin-b1deficient cells (GD25) and GD25 stably re-expressing wt integrin-b1A (GD25-b1A) cells, (C) FAK-deficient cells (FAK-/-) and FAK-/- cells stably
reexpressing wt FAK and (D) Src kinase-deficient cells (SYF-/-) and SYF-/- cells stably re-expressing wt c-src. Intracellular C. jejuni were quantified by
gentamicin protection assays, and Cdc42 activation by CRIB-GST pulldowns. (**) P 0.005 was considered as statistically significant. Fibronectin,
integrin-b1, FAK and Src expression was analyzed by immunoblotting. GAPDH expression levels were determined as loading control.
(**) P 0.005 was considered as statistically significant. Similar
quantities of total Cdc42 and GAPDH were confirmed by Western
jejuni followed by analysis of host cells by FESEM. The
micrographs showed that C. jejuni profoundly induced
filopodia formation at the periphery and top of infected
host cells (Figure 4A, up to 7 m long, blue arrows),
while only very few of these structures could be seen in
non-infected control wt fibroblasts (Figure 4B). Next, we
infected fibronectin-/-, GD25 and FAK-/-knockout cell
lines and their corresponding wt control cells, followed
by the analysis of the interaction of C. jejuni with the
surface of host cell surface by high-resolution FESEM.
Infection of fibronectin-/-, GD25 and FAK-/-knockout
cell lines revealed the presence of attached bacteria
(yellow arrows) at the surface of the cells with short
microspikes (up to 1 m long, green arrowheads), but no
indication of induced membrane dynamics was seen
(Figure 5A). Filopodia formation or invading C. jejuni
could be detected only rarely in any infected knockout
cell line. In contrast, infection of wt fibroblasts under
the same conditions revealed tight engulfment of the
attached bacteria associated with long filopodia (blue
arrows) and/or ruffles (red arrows) and somewhat
elongated microspikes (green arrowheads) as shown in
Figure 5B. In agreement with our earlier observation in
infected INT-407 cells , we found that C. jejuni
entered the wt fibroblasts in a very specific fashion, first
with its flagellum followed by the bacterial cell with the
opposite flagellar end (Figure 5B, bottom). The
Figure 3 C. jejuni invasion is impaired in cells expressing
integrin-b1 point mutations exhibiting defects in fibronectin
fibril organisation or FAK phosphorylation. (A)
Integrin-b1deficient cells (GD25) and GD25 stably re-expressing mutated
integrin subunit b1A (GD25-b1 ATT788-9AA or GD25-b1 AY783/795F
or wild-type b1A (GD25-b1A) cells were infected with wt C. jejuni
81-176 for 6 hours. Intracellular C. jejuni were quantified by
gentamicin protection assays. (B) The presence of active Cdc42-GTP
was quantified by CRIB-GST pulldowns. One hundred % of activity
corresponds to the highest amount of detected Cdc42-GTP level.
Figure 4 High resolution FESEM of C. jejuni-induced filopodia
formation. Representative sections of wild-type fibroblasts
incubated for 6 hours with wt C. jejuni 81-176 (A) and non-infected
fibroblast control cells that were mock-treated (B) are shown.
Infection revealed the occurrence of membrane protrusion events
with long filopodia at the periphery and on top of cells which were
only sporadically seen in the non-infected control cells (blue
Figure 5 High resolution FESEM of C. jejuni-induced filopodia formation and invasion. (A) Infection of GD25 knockout cells with wt C.
jejuni 81-176 (yellow arrows) for 6 hours revealed bacterial attachment to the cell surface with short microspikes (green arrowheads) present, but
membrane dynamics events or invasion were rarely seen. Similar observations were made with infected fibronectin-/- or FAK-/- cells. (B) Infecting
C. jejuni in wt cells were regularly associated with long filopodia (blue arrows) membrane ruffling (red arrows), as well as elongated microspikes
generation of filopodia in wt cells confirms the typical
occurrence of Cdc42 GTPase activation during infection,
followed by dynamic membrane rearrangements and
host entry, dependent on the expression of fibronectin,
integrin-b1 and FAK.
Wild-type but not cadF mutant C. jejuni induces
profound FAK, EGFR and PDGFR phosphorylation
Next, we aimed to investigate if infection activates FAK
autophosphorylation and if this is associated with the
activation of EGFR and PDGFR receptors, which are
also present in membrane lipid rafts. We therefore
infected host cells with wt C. jejuni and an isogenic
cadF deletion mutant in a time course. Protein lysates
from the infected cells were prepared and subjected to
Western blotting using activation-specific
phospho-antibodies for FAK, EGFR and PDGFR (Figure 6A). The
results show that wt C. jejuni significantly induced the
autophosphorylation of FAK at tyrosine residue Y-397
in the active centre, the phosphorylation of EGFR at
Y845 and the phosphorylation of PDGFR at Y-754 over
time (Figure 6A). The data indicated that maximal levels
of kinase activity appeared after 4 hours of infection
(Figure 6B), which correlated with increasing
Cdc42GTP levels over time (Figure 1A) and the invasion
capabilities of wt C. jejuni, as determined by gentamicin
protection assays (Figure 6C). Interestingly, infection
with the cadF mutant, as examined under identical
conditions, revealed that phosphorylation of FAK, EGFR
and PDGFR were widely impaired (Figure 6A, B) and
correlated with the reduced invasiveness of this mutant
(Figure 6C). These observations suggest that CadF
maybe involved in C. jejuni-induced FAK, EGFR and
PDGFR kinase activities, and host cell invasion.
Induction of maximal Cdc42-GTP levels requires CadF and
is strongly impaired in FAK-/- knockout cells
To investigate if FAK is required for C. jejuni-induced
Cdc42 activation, we infected FAK-/- knockout cells and
cells re-expressing FAK under the same conditions with
wt C. jejuni and cadF mutant, followed by CRIB-GST
pulldown assays. While growing levels of activated
Cdc42 were detected in FAK-positive cells over time
with wt C. jejuni, no detectable activation of Cdc42 was
found in FAK-/- cells during the entire course of
infection (Figure 7A). This suggests that FAK is involved in
signaling upstream of Cdc42 activation during invasion
of C. jejuni. Furthermore, significantly reduced
Cdc42GTP levels were observed in both FAK-positive and
FAK-/- cells infected with the cadF mutant (Figure
7A). These findings further support the notion that
CadF could be a significant player in signaling leading
to FAK-mediated activation of Cdc42. However, the
cadF mutant was still able to induce some Cdc42
M-ock+30min+60min+120mi+n240min3-0min6-0min 1-20min2-40mCinj wt
- - - - - + + + + CjDcadF
60 llraub i()n%
Figure 6 Importance of CadF for C. jejuni-induced FAK, EGFR
and PDGFR activation. (A) FAK-positive fibroblasts were infected
with wt C. jejuni F38011 or isogenic F38011cadF for indicated
periods of time. FAK, EGFR or PDGFR activation was analysed by
immunoblotting with indicated antibodies. Total PDGFR expression
levels were determined as loading control. (B) Quantification of FAK,
EGFR and PDGFR kinase phosphorylation during the course of
infection. One hundred % of activity corresponds to the highest
amount of phosphorylation detected per experiment and selected
kinase (lane 5). (C) Intracellular C. jejuni were quantified by
gentamicin protection assays. (*) P 0.005 and (**) P 0.005 were
considered as statistically significant.
GTPase activation in FAK-positive cells, suggesting that
other bacterial factor(s) are also implicated in this
signaling cascade (Figure 7A).
The C. jejuni flagellum is also involved in Cdc42
activation and bacterial invasion
Because CadF is not the sole bacterial factor involved in
C. jejuni-induced Cdc42 activity, we searched for other
bacterial factors involved in this signaling. The C. jejuni
flagellar apparatus has been reported to be a major
pathogenicity determinant [25,26,48]. To test if an intact
Figure 7 Importance of the CadF and flagellar apparatus for C.
jejuni-induced activation of Cdc42 and bacterial invasion. (A)
FAK+/+ and FAK-/- cells were infected with wt C. jejuni F38011 or
isogenic F38011cadF for the indicated periods of time.
Quantification of Cdc42-GTP levels by CRIB-GST pulldown during
the course of infection. One hundred % of activity corresponds to
the highest amount of detected Cdc42-GTP level (lane 4). (B)
FAKpositive cells were infected with the indicated strains in a
timecourse. The presence of bound, active Cdc42-GTP was analyzed in
CRIB-GST pulldown assays followed by Western blotting using
aCdc42 antibody. Similar quantities of individual GTPases at every
time point were confirmed by Western blotting using equivalent
volumes of cell lysates. (C) Quantification of Cdc42-GTP levels during
the course of infection. One hundred % of activity corresponds to
the highest amount of detected Cdc42-GTP level (lane 3). The
amount of intracellular bacteria was quantified by gentamicin
protection assays under the same experimental conditions. (**) P
0.005 were considered as statistically significant as compared to the
flagellum plays a role in C. jejuni-induced Cdc42
activation, host cells were infected with wt strain 81-176 and
its isogenic mutants flaA/B lacking the two major
flagellar subunits FlaA and FlaB , and flhA, a key
element involved in the regulation of flagellar genes and
other pathogenicity factors in C. jejuni . As
expected, activated Cdc42 was detected in FAK-positive
cells between 2-4 hours after infection with wt C. jejuni
(Figure 7B, C). In contrast, no detectable Cdc42
activation and host cell invasion was found in cells infected
with flaA/B or flhA mutants during the entire course
of infection (Figure 7B, C). This indicates that, in
addition to the contribution by CadF as shown above, the
intact C. jejuni flagellum may also play a role in the
activation of Cdc42.
The guanine exchange factor Vav2 is required for C.
jejuni-mediated Cdc42 activation
The following aim was to determine additional signaling
factors downstream of FAK and upstream of Cdc42
activation. Cycling of small Rho GTPases between the
inactive and active forms is commonly stimulated by a class
of proteins called guanine nucleotide exchange factors
(GEFs) and negatively regulated by GTPase activating
proteins (GAPs). GEFs trigger the exchange of GDP for
GTP to generate the active form of a given GTPase,
which is then capable of recognizing downstream targets
[50-52]. To identify which GEF(s) is/are involved in C.
jejuni-induced Cdc42 activation, the expression of
typical GEFs including Vav2, DOCK180 or Tiam-1 was
downregulated using target-specific siRNA, followed by
infection and CRIB-GST pulldowns. While the
downregulation of Vav2 led to the predominant inhibition of
Cdc42-GTP levels (Figure 8A), both downregulation of
Tiam-1 and DOCK180 (Figure 8B, C) or transfection of
non-targeting scrambled siRNA control had no
significant effect on C. jejuni-triggered Cdc42-GTP production
(Figure 8A-C). These findings suggest that Vav2, but not
Tiam-1 or DOCK180, plays a crucial role in C.
jejuniinduced Cdc42 activation.
Vav2 is required for maximal host cell invasion by C.
Next we aimed to consolidate our understanding of the
potential importance and role of Vav2 during infection.
First, to identify if Vav2 is also involved in host cell
invasion by C. jejuni, the expression of Vav2 was
suppressed with siRNA, followed by infection and
gentamicin protection assays. The results showed that
downregulation of Vav2 led to a significant drop in the
amount of intracellular bacteria (Figure 9A). In addition,
we investigated if the downregulation of Vav2 may
influence the activity of another small Rho GTPase, Rac1.
Figure 8 Importance of guanine exchange factor Vav2 for C.
jejuni-induced Cdc42 activation. INT-407 cells were transfected for
48 hours with siRNA for Vav2 (A), Tiam-1 (B) or DOCK180 (C) as well
as a scrambled siRNA as control. Immunoblotting with the indicated
antibodies confirmed knockdown of the respective proteins. GAPDH
expression levels were determined as control. Quantification of
Cdc42 GTPase activity after infection with wt C. jejuni 81-176 for 6
hours. The presence of bound, active Cdc42-GTP was analyzed in
CRIB-GST pulldown assays followed by Western blotting using
aCdc42 antibody. One hundred % of activity corresponds to the
highest amount of detected Cdc42-GTP level (lane 1).
Quantification of GTPase activation levels indicated that
while a significant suppressive effect was seen on
Cdc42-GTP, only a slight reduction of Rac1-GTP levels
were observed (Figure 9B). This suggests that Vav2 may
predominantly target Cdc42 in infected INT-407 cells.
Further evidence for an important function of Vav2 in
host cell invasion came from the use of
dominant-negative Vav2. Expression of dominant-negative Myc-tagged
Vav2, but not wt Myc-tagged Vav2, also had some
downregulatory effect on C. jejuni invasion (Figure 9C).
Signaling of Vav2 is functionally linked to growth factor
receptors EGFR and PDGFR
As siRNA-mediated gene silencing or expression of
dominant-negative Vav2 interfered with uptake of C.
jejuni, the impact of Vav2 on C. jejuni host cell entry
was examined in more detail. Vav2 is a substrate of
EGFR/PDGFR kinases and GTPases including Cdc42
can be activated downstream of both receptors through
Vav2 exchange activity [46,53-55]. For this purpose,
INT-407 cells were transiently transfected with wt Vav2
and different Vav2 mutants that were either impaired in
EGFR-dependent phosphorylation of Vav2 (Vav2 Y172/
159F), lacked the primary phosphatidylinositol 3, 4,
5triphosphate binding site (Vav2 R425C) or were not
capable of binding to activated EGFR (Vav2 W673R and
Vav2 G693R) . Gentamicin protection assays
revealed that overexpression of either Vav2 mutant
construct significantly reduced the number of intracellular
C. jejuni bacteria (Figure 9D), further confirming the
importance of Vav2 in bacterial uptake. These findings
also support the view that Vav2, by binding to and
signaling through a C. jejuni-induced EGFR/PDGFR and
PI3-K kinase activation pathway, may contribute Cdc42
activation during infection. Finally, we utilised
Vav1/2-/knockout fibroblasts for infection and gentamicin
protection assays. The determination of total cell-associated
and intracellular C. jejuni bacteria in the same set of
experiments showed that expression of Vav is not only
important for invasion but has also a significant effect
on the binding of C. jejuni to these cells (Figure 9E).
The activities of FAK, EGFR, PDGFR and PI3-K are also
important for C. jejuni-induced Cdc42-GTP levels and
Finally, we wanted to investigate if pharmacological
inhibition of the above described host cell kinases could
confirm the proposed signaling pathway leading to
Cdc42 activation and C. jejuni invasion. For this
purpose, INT-407 cells were pre-treated for 30 min with
AG1478 (EGFR inhibitor), AG370 (PDGFR inhibitor),
wortmannin (PI3-K inhibitor) or PF-573228 (FAK
inhibitor) followed by infection with wt C. jejuni. The
results showed that inhibition of each of these kinases
had a profound suppressive effect on both Cdc42-GTP
levels and bacterial invasion (Figure 10A). To further
corroborate these findings, INT-407 cells were
transiently transfected with wt PDGFR and EGFR constructs,
and their respective dominant-negative forms, followed
by infection with C. jejuni. Gentamicin protection assays
showed that overexpression of either dominant-negative
mutant also significantly reduced the amount of
recovered intracellular C. jejuni, confirming the involvement
of PDGFR and EGFR in uptake of C. jejuni (Figure
10B). These data collectively suggest that we have
identified a novel important pathway of C. jejuni host cell
entry, proceeding by the activation of a
fibronectinintegrin-beta1FAK/SrcEGFR/PDGFRPI3-kinaseVav2Cdc42 signaling cascade.
Invasion of host target cells is a major strategy of a large
group of pathogenic microbes. The entry process
comprises numerous specific steps at the host-pathogen
interface including bacterial binding to one or more
receptors, delivery of signals to the host cell,
Figure 9 Downregulation, elimination or interference with important Vav2 functions reduces the uptake of C. jejuni in host cells. (A)
INT-407 cells were transfected with siRNA against Vav2 or a scrambled siRNA as control. After 48 hours, cells were infected with wt C. jejuni
81176 for 6 hours. Intracellular bacteria were quantified by gentamicin protection assays. (B) The presence of active Rac1-GTP and Cdc42-GTP was
quantified by CRIB-GST pulldowns. One hundred % of activity corresponds to the highest amount of detected GTPase-GTP level. (C) INT-407 cells
were transfected with indicated Myc-tagged or (D) GFP-tagged Vav2 constructs. After 48 hours, cells were infected with wt C. jejuni 81-176 for 6
hours. Intracellular bacteria were quantified by gentamicin protection assays. Expression of the individual Vav2 constructs was verified by Western
blot analysis. GAPDH expression levels were determined as control. (E) Vav2-deficient cells (Vav1/2-/-) or Vav2-expressing control fibroblasts (Vav1/
2+/+) were infected for 6 hours with C. jejuni. Intracellular and cell-associated bacteria were quantified by gentamicin protection assays. (*) P
0.05 and (**) P 0.005 were considered as statistically significant.
Figure 10 Importance of FAK, EGFR, PDGFR and PI3-kinase
activities for C. jejuni-induced activation of Cdc42 and bacterial
invasion. (A) INT-407 monolayers were pre-incubated for 30 min
with the indicated pharmacological inhibitors and infected with C.
jejuni for 6 hours. Intracellular C. jejuni were quantified by
gentamicin protection assays. The presence of active Cdc42-GTP
was quantified by CRIB-GST pulldowns. One hundred % of activity
corresponds to the highest amount of detected Cdc42-GTP level
(lane 2). (B) Effect of overexpression of dominant-negative forms of
PDGFR and EGFR on C. jejuni uptake. 48 hours post transfection
INT407 cells were infected with C. jejuni for 6 hours. Intracellular
bacteria were quantified by gentamicin protection assays.
Expression of the individual constructs was verified by Western
blotting. GAPDH expression levels were determined as control. (*) P
0.05 and (**) P 0.005 were considered as statistically significant.
programming of intracellular host signaling cascades,
membrane and cytoskeletal dynamics, followed by
engulfment and uptake of the bacterium. These
processes commonly involve the activation of small Rho
family GTPases. Prominent members are the
GTP-binding proteins RhoA, Cdc42 and Rac1, which act as
guanine nucleotide-regulated switches to induce various
responses during the infection process [50,56-58]. Host
cell invasion by the gastrointestinal pathogen C. jejuni
has been reported to cause substantial tissue damage,
but the molecular mechanisms involved remained widely
unknown. We could recently demonstrate that C. jejuni
invasion of INT-407 cells is time-dependent and
associated with increasing activities of small Rho GTPases,
one of which is Cdc42 . The application of
pharmacological inhibitors, GTPase-modifying toxins and
expression of constitutive-active or dominant-negative
Cdc42 plasmids provided evidence that Cdc42 activity
plays a role in host cell invasion of C. jejuni . In the
present report, we aimed to unravel the cascade of
signaling events resulting in C. jejuni-triggered Cdc42
activity. Using knockout cell lines of several host
receptors (fibronectin-/-, GD25 integrin-b1-/-) and kinases
(FAK-/- and SYF), siRNA transfection,
dominant-negative and other expression constructs, G-Lisa, CRIB
pulldowns, gentamicin protection assays and electron
microscopy, we show that C. jejuni exploits a
fibronectinintegrin-b1FAK/SrcEGFR/PDGFRPI3-kinaseVav2 signaling pathway, which is crucial for
activating Cdc42 GTPase function, involved in invasion
of host target cells. Our major findings in this study are
discussed below and have been summarised in a
signaling model (Figure 11).
The use of specific knockout cell lines for C. jejuni
invasion-associated signaling studies has the great advantage
over other cell systems that clear conclusions can be
drawn if the deleted gene of interest is involved in this
process or not. Host cell entry of C. jejuni was largely
reduced in each of the above knockout cell lines,
suggesting that fibronectin, integrin-b1, FAK and Src kinases play
a crucial role in invasion. Since C. jejuni strains express
the conserved major fibronectin-binding protein CadF
[15,17,18,20] and because fibronectin is the natural ligand
for integrin-b1 receptor [59,60], our current findings
indicate a cascade of
fibronectinintegrin-b1FAK/SrcFigure 11 Model for C. jejuni-induced signaling leading to
Cdc42 activation and bacterial invasion. C. jejuni adheres to host
cells via the fibronectin-binding protein CadF, which acts as a
bridge engaging the integrin-b1 receptor. Integrin occupancy and
clustering in lipid rafts leads to recruitment and activation of the
non-receptor tyrosine kinase FAK. Phosphorylation of FAK and Src
triggers a cascade of signals resulting in the formation of protein
complexes leading to activation of other signaling factors as
indicated. Assembly of integrin-dependent signal complexes leads
to phosphorylation and transactivation of PDGFR and EGFR,
followed by stimulation of PI3-K and Vav2. Activated Vav2 then
induces the activation of Cdc42. This signaling potentially causes
localized actin and/or microtubule rearrangements at the site of C.
jejuni entry, resulting in bacterial uptake. In addition to CadF, the C.
jejuni flagellum also appears to play a role in the described signal
cascades. If the flagellum participates by sole bacterial motility, by
translocating bacterial effector proteins or targeting a host receptor
directly is not yet clear and needs to be investigated in future
dependent signaling events occurring during infection. In
line with these observations, we found that Cdc42-GTP
levels triggered by C. jejuni infection were strongly
elevated in cells expressing wt FAK but not in FAK-knockout
cells, and Cdc42-GTP upregulation was verified by two
independent molecular techniques including GST-CRIB
pulldown and G-Lisa. These findings were further
supported by the detection of filopodia formation, membrane
dynamics and engulfment of C. jejuni during infection of
wt control cells, but this was widely impaired in any of the
infected knockout cell lines. These novel data provide a
clear proof that fibronectin, integrin-b1, FAK and Src
kinases are crucial host factors playing significant roles in
C. jejuni-induced Cdc42 activation and filopodia
formation, linked to invasion. Thus, by a strategy engaging
fibronectin, integrin-b1, FAK and Src, the bacteria appear to
hijack the capacity of the integrin receptor complex to
connect with the intracellular cytoskeleton and to create
the necessary pulling forces to trigger C. jejuni entry into
Integrin-b1-dependent fibrillar cell adhesion in healthy
tissues play a crucial role in the organisation of the
ECM because they co-align with proper extracellular
fibronectin fibril structures [60,61]. Genetic elimination
of integrin-b1 in GD25 cells results in profound
assembly defects within the fibrillar ECM meshwork including
fibronectin [38,60,62]. Cellular pulling forces generated
by integrin-b1-mediated linkage to the actin-myosin
network therefore appear to be critical for ECM fibronectin
fibril formation, as force-triggered conformational
changes are essential to expose cryptic oligomerisation
motifs within individual fibronectin proteins [60,63].
Importantly, an integrin-b1 TT788/789AA mutant is
defective in mediating proper cell attachment and is
unable to induce fibronectin fibril formation . The
conformation of the extracellular integrin-b1 domain is
shifted towards an inactive state but the cytoplasmic
part remains functional with respect to activation of
FAK. Interestingly, C. jejuni was widely unable to enter
GD25 cells stably transfected with this integrin-b1
mutant. Therefore, we conclude that threonine residues
788-789, which are of critical importance for integrin-b1
function due to effects on the extracellular conformation
and function of the receptor, play also a crucial role in
proper for fibronectin fibril organisation, important for
efficient C. jejuni host cell entry.
Integrin activation and clustering is tightly associated
with the activation of FAK, and is a strategy of
regulating outside-in signal transduction events leading to
cytoskeletal rearrangements [64,65]. Indeed, the lowest
numbers of intracellular C. jejuni were observed with
GD25-b1A-Y783/795F cells which are impaired in
signaling to FAK due to a defect in b1-dependent
autophosphorylation of FAK at tyrosine residue Y-397 .
Despite the defect in integrin-b1-mediated FAK
activation, FAK was still localized to focal adhesions. This
result suggests that besides signaling of integrin-b1 to
form correct fibronectin fibril formation, b1-dependent
signaling to FAK activation is also required for C.
jejuni-induced Cdc42 signaling and bacterial uptake.
Indeed, FAK autophosphorylation is strongly activated
by C. jejuni and pharmacological inhibition of FAK as
well as infection of FAK-/- cells did not lead to
stimulation of Cdc42 GTPase activity. In addition,
FAK-/mouse embryos in vivo as well as in vitro cultured
FAK-/- cells fail to properly assemble fibronectin fibrils
[60,66]. Therefore, the observed deficiency of
FAK-/cells to internalise C. jejuni is associated with two
phenotypes, inhibited signaling to proper ECM organisation
and downstream signaling leading to GTPase activation.
Thus, fibronectin/integrin-linkages to the dynamic
actin-myosin or microtubuli networks are disrupted in
FAK-deficient cells and necessary pulling forces are not
provided. This setting is similar to that shown for
fibronectin-binding protein-expressing Staphylococcus
aureus, because infected FAK-/- or fibronectin-/- cells were
similarly impaired to internalise these bacteria [37,67].
In addition, the importance of FAK activition has been
reported for other pathogens targeting integrins for
bacterial invasion or other purposes, including Yersinia
pseudotuberculosis [68,69], group B Streptococci 
and Helicobacter pylori [71-73]. Thus, FAK appears to
be a very common target of multiple bacterial
Our observation that FAK activation is required for C.
jejuni-induced Cdc42 activity and host cell entry, led us
to search for other downstream signaling determinants.
Using siRNA knockdown, we tested the importance of a
few well-known GEFs, including Tiam-1, DOCK180 or
Vav2, for the production of Cdc42-GTP levels in
infected cells. Interestingly, Vav2 (but not Tiam-1 or
DOCK180) was required for C. jejuni-induced Cdc42
activation. The importance of Vav2 was then confirmed
by the expression of dominant-negative constructs and
the use of Vav1/2 knockout cells in infection assays.
Bacterial adhesion was also reduced in Vav1/2 knockout
cells, which can be explained by reduced GTPase
activation as compared to wt cells. This is in agreement with
reports showing that Vav2 is also involved in the uptake
of other pathogens including Yersinia and Chlamydia
[74,75]. Moreover, in our studies the expression of
various point mutations in Vav2 linked the signaling
directly to growth factor receptors and PI3-K. The
application of selective inhibitors during C. jejuni infection
showed then that the kinase activities of EGFR, PDGFR
and PI3-K are also required for Cdc42 activation. This
was also confirmed by the expression of
dominant-negative versions of EGFR or PDGFR, which exhibited
suppressive effects on C. jejuni uptake. Extensive research
on the regulation of growth factor receptor activation
and signaling by integrin-mediated cell adhesion
indicates that these two classes of receptors work
cooperatively. Several studies showed that integrin ligation
allows for the maximal activation of EGFR or PDGFR,
thereby producing robust intracellular signals including
small Rho GTPase activation [76,77]. These observations
are in well agreement with our findings, suggesting that
C. jejuni activates, via fibronectin and integrins, a FAK/
signaling pathway. However, transfection with both
DNPDGFR and DN-EGFR constructs resulted in no
additive reduction of C. jejuni invasion. These latter finding
suggests that besides EGFR and PDGFR other signaling
pathway(s) are also implicated in C. jejuni
Our previous study indicated that C. jejuni
pathogenicity factors such as cytolethal distending toxin CDT,
plasmid pVir, the adhesin PEB1 or certain capsular
genes are not required for C. jejuni-induced Cdc42
activation . We found here that an isogenic cadF
mutant less efficiently induced activation of Cdc42 as
compared to wt C. jejuni, suggesting that the
fibronectin-binding protein CadF, probably in concert with FlpA
, could be involved in GTPase activation as shown
here for Cdc42. It appears that CadF does not only act
as a canonical adhesin for bacterial attachment to
fibronectin, but could also stimulate integrins as well as
FAK, EGFR and PDGFR kinase activity, which
subsequently may activate Vav2 and Cdc42, important for
maximal C. jejuni invasion. Since flaA/B or flhA
knockout mutants lacking the flagella induced only very
little Cdc42-GTP levels, another C. jejuni determinant
playing a role in Cdc42 activation is the flagellar
apparatus. The flagellum appears to be a major colonization
determinant of Campylobacter, shown to be essential for
successful infection of several animal models [78-80]. In
addition, FlaA/B proteins play a profound role in C.
jejuni invasion of epithelial cells [16,81-83]. However,
the possible impact of flagellar proteins in host cell
entry is controversial in the literature. One hypothesis is
that the flagella, like their evolutionary related type-III
secretion system counterparts, can secrete
invasion-associated factors such as CiaB and others into the culture
supernatant [15,17,25,48]. The other hypothesis is that
flagella-mediated bacterial motility is the driving force
to permit host cell entry, but deletion of ciaB has no
impact . Thus, it is still not clear if the flagellum,
unlike its well-known function in bacterial motility, may
transport bacterial effectors into the medium or into the
host cell. Alternatively, the flagellum itself may target a
host cell receptor directly to trigger Cdc42 signaling
involved in invasion, which should be investigated in
future studies [Figure 11].
In summary, we provide here several lines of evidence
for a novel invasion-related signaling pathway of C.
jejuni involving fibronectin, integrin-b1, FAK, Src,
EGFR, PDGFR, PI3-K, Vav2 and Cdc42 using three
different strains including the fully-sequenced model
isolate 81-176. Based on our electron microscopic
observations and the use of C. jejuni mutants in
signaling studies, we propose that the flagellum by providing
bacterial motility may bring the CadF adhesin in the
right position, but may also have other effects, in order
to trigger host cell signaling leading to elevated
Cdc42GTP levels and invasion (Figure 11). Interestingly, it
appears that the Cdc42-pathway discovered here is not
the sole pathway involved in C. jejuni invasion. Our
observations support the view that another signaling
cascade involves the small Rho GTPase member Rac1
, which is activated by a pathway involving the same
upstream components (fibronectin, integrin-b1 and
FAK) but two other GEFs, DOCK180 and Tiam-1 ,
which are obviously not involved in C. jejuni-induced
Cdc42 activation as shown here. These findings suggest
that C. jejuni targets two major Rho GTPases by two
independent downstream signal transduction pathways
and therefore provide novel aspects to our knowledge
on the mechanism of C. jejuni host cell entry. In future
studies it will be important to investigate the precise
mechanism of how active Cdc42 regulates microtubule
dynamics and/or actin rearrangements involved in
providing the necessary pulling forces crucial for the
bacterial invasion process.
List of abbreviations used
CadF: Campylobacter adhesin to fibronectin; C. jejuni: Campylobacter jejuni;
CiaB: Campylobacter invasion antigen B; CRIB: Cdc42-Rac1 interactive
binding; GST-CRIB: domain of kinase PAK1 fused to glutathione S-transferase;
CFU: colony forming unit; ECM: extracellular matrix; EGFR: epidermal growth
factor receptor; FCS: fetal calf serum; FESEM: field emission scanning electron
microscopy; FAK: focal adhesion kinase; FlpA: Fibronectin like protein A; GAP:
GTPase activating protein; GEF: Guanine exchange factor; JlpA: Jejuni
lipoprotein A; kpsS: capsular gene; MCD: methyl-beta cyclodextrin; GD25
cells: integrin 1-/- mouse fibroblasts; MH agar: Mueller Hinton agar; MOI:
multiplicity of infection; PDGFR: platelet-derived growth factor receptor;
PEB1: Periplasmic binding protein 1; PI3-K: phosphatidylinositol 3-kinase;
siRNA: silencing RNA; PVDF: polyvinylidenedifluoride; waaF:
heptosyltransferase II gene; wt: wild-type.
We thank Ina Schleicher for excellent technical assistance, and Drs. Patricia
Guerry (Fayetteville State University, USA), Michael Konkel (Pullman
University, USA) and Martin Blaser (New York University, USA) for providing
C. jejuni wt strains and mutants, respectively. We are also very grateful to
Drs. David Schlaepfer (University of California, USA) for providing FAK-/- cells,
Christof R. Hauck (University Konstanz, Germany) for providing Vav1/2-/- cells,
Staffan Johannsson (Uppsala University, Sweden) for the GD25 cell lines and
Phil Soriano (FHCRC, Seattle, USA) for the SYF cells. The work of S.B. is
supported through a SFI grant (UCD 09/IN.1/B2609).
MKG, MB, MR and NT performed and designed the experiments. ST, LB and
OO provided crucial materials and advise for the experiments. SB, the senior/
corresponding author, supervised the experiments and wrote the
manuscript with the help of LB and OO. All co-authors read and approved
the final manuscript.
The authors declare that they have no competing interests.
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