The signaling pathway of Campylobacter jejuni-induced Cdc42 activation: Role of fibronectin, integrin beta1, tyrosine kinases and guanine exchange factor Vav2
- Malgorzata Krause-Gruszczynska1, 2,
- Manja Boehm†1, 2,
- Manfred Rohde†3,
- Nicole Tegtmeyer1,
- Seiichiro Takahashi4,
- Laszlo Buday5,
- Omar A Oyarzabal6 and
- Steffen Backert1, 2Email author
© Krause-Gruszczynska et al; licensee BioMed Central Ltd. 2011
Received: 10 November 2011
Accepted: 28 December 2011
Published: 28 December 2011
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.
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.
Collectively, our findings led us propose that C. jejuni infection triggers a novel fibronectin→integrin-beta1→FAK/Src→EGFR/PDGFR→PI3-kinase→Vav2 signaling cascade, which plays a crucial role for Cdc42 GTPase activity associated with filopodia formation and enhances bacterial invasion.
KeywordsRho 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 Reiter's reactive arthritis as well as the Guillain-Barrè 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 outer-membrane 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-β1, 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 fibronectin-binding 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 F38011ΔcadF, 81-176ΔflaA/B and 81-176ΔflhA 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 Mueller-Hinton (MH) agar amended with 50 μg/ml kanamycin or 30 μg/ml or chloramphenicol at 37°C under microaerobic conditions (generated by CampyGen, Oxoid) for 48 hours.
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-β1-/-) or GD25 cells stably transfected with wt integrin β1A (GD25β1A) or several mutants (GD25β1A-TT788/89AA and GD25β1A-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 Earle's 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 (MβCD, Sigma, 1 mM-10 mM), PF-573228 (Tocris; 10 μM), AG1478 (10 μM) , AG370 (10 μM) , or wortmannin (1 μM) [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 PDGFRβ, dominant-negative PDGFRβ, wt EGFR and dominant-negative 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 manufacturer's 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 antibodies.
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 Cruz).
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 μg/ml gentamicin (Sigma) at 37°C for 2 hours, washed three times with medium, and then incubated with 1 ml of 0.1% (w/v) saponin (Sigma) in PBS at 37°C 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 μg/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 Cdc42-Rac1 interactive binding domain of PAK1 fused to glutathione S-transferase(GST-CRIB), also called GST-CRIB 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 GTPγ-S and GDP, respectively, for 15 min. Cell lysates (treated with bacteria, GTPγ-S, GDP or untreated) were mixed with the PAK-RBD slurry (1 hour, 4°C). 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 Cdc42-GTP using the Lumi-Imager F1 software program (Roche).
Cdc42 activation in infected cells was also determined with the G-LISA™Rac1- 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-LISA's contains a Rac1-GTP-binding 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 Plus, Tecan).
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-β1, Tiam-1, DOCK180 or GAPDH (all Santa Cruz) was performed according to the manufacturer's instructions. As secondary antibodies, horseradish peroxidase-conjugated α-mouse, α-rabbit or α-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 phospho-antibody 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 expression
Recent experiments have indicated that treatment with methyl-beta cyclodextrin (MβCD), 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 MβCD 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-β1, FAK and Src kinases
C. jejuni invasion is inhibited in cells expressing integrin-β1 mutants with defects in fibronectin fibril formation and FAK signaling
C. jejuni induces filopodia formation and invasion in wt cells but not in fibronectin, integrin-β1 and FAK knockout cells
Wild-type but not ΔcadF mutant C. jejuni induces profound FAK, EGFR and PDGFR phosphorylation
Induction of maximal Cdc42-GTP levels requires CadF and is strongly impaired in FAK-/- knockout cells
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 flagellum plays a role in C. jejuni-induced Cdc42 activation, host cells were infected with wt strain 81-176 and its isogenic mutants Δfla A/B lacking the two major flagellar subunits FlaA and FlaB , and ΔflhA, 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 Δfla A/B or ΔflhA 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
Vav2 is required for maximal host cell invasion by C. jejuni
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, 5-triphosphate 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 invasion
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-β1, 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-β1 receptor [59, 60], our current findings indicate a cascade of fibronectin→integrin-β1→FAK/Src-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-β1, 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-β1, 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 host cells.
Integrin-β1-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-β1 in GD25 cells results in profound assembly defects within the fibrillar ECM meshwork including fibronectin [38, 60, 62]. Cellular pulling forces generated by integrin-β1-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-β1 TT788/789AA mutant is defective in mediating proper cell attachment and is unable to induce fibronectin fibril formation . The conformation of the extracellular integrin-β1 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-β1 mutant. Therefore, we conclude that threonine residues 788-789, which are of critical importance for integrin-β1 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-β1A-Y783/795F cells which are impaired in signaling to FAK due to a defect in β1-dependent autophosphorylation of FAK at tyrosine residue Y-397 . Despite the defect in integrin-β1-mediated FAK activation, FAK was still localized to focal adhesions. This result suggests that besides signaling of integrin-β1 to form correct fibronectin fibril formation, β1-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 pathogens.
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/Src→EGFR/PDGFR→PI3-kinase→Vav2→Cdc42 signaling pathway. However, transfection with both DN-PDGFR 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 internalization.
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 ΔcadF 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 ΔflaA/B or ΔflhA 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-β1, 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 Cdc42-GTP 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-β1 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
Campylobacter adhesin to fibronectin
- C. jejuni :
Campylobacter invasion antigen B
Cdc42-Rac1 interactive binding
domain of kinase PAK1 fused to glutathione S-transferase
colony forming unit
epidermal growth factor receptor
fetal calf serum
field emission scanning electron microscopy
focal adhesion kinase
Fibronectin like protein A
GTPase activating protein
Guanine exchange factor
Jejuni lipoprotein A
- GD25 cells:
integrin β1-/- mouse fibroblasts
- MH agar:
Mueller Hinton agar
multiplicity of infection
platelet-derived growth factor receptor
Periplasmic binding protein 1
- waaF :
heptosyltransferase II gene
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).
- World Health Organization: Global burden of disease (GBD) 2002 estimates. WHO. 2004, Geneva, Switzerland, [http://www.who.int/topics/global_burden_of_disease/en/]Google Scholar
- Nachamkin I, Szymanski CM, Blaser MJ: Campylobacter. Washington, DC: ASM Press 2008.Google Scholar
- Oyarzabal OA, Backert S: Microbial Food Safety: An Introduction. Heidelberg (Germany): Springer Verlag, 2012.View ArticleGoogle Scholar
- Young KT, Davis LM, DiRita VJ: Campylobacter jejuni: molecular biology and pathogenesis. Nat Rev Microbiol. 2007, 5: 665-679. 10.1038/nrmicro1718.View ArticlePubMedGoogle Scholar
- Blaser MJ, Engberg J: Campylobacter. Edited by: Nachamkin I, Szymanski CM, Blaser MJ. 2008, Washington, DC: ASM Press, 99-121.Google Scholar
- Ketley JM: Pathogenesis of enteric infection by Campylobacter. Microbiology. 1997, 143: 5-21. 10.1099/00221287-143-1-5.View ArticlePubMedGoogle Scholar
- Wooldridge KG, Ketley JM: Campylobac ter-host cell interactions. Trends Microbiol. 1997, 5: 96-102. 10.1016/S0966-842X(97)01004-4.View ArticlePubMedGoogle Scholar
- Dasti JI, Tareen AM, Lugert R, Zautner AE, Gross U: Campylobacter jejuni: a brief overview on pathogenicity-associated factors and disease-mediating mechanisms. Int J Med Microbiol. 2010, 300: 205-211. 10.1016/j.ijmm.2009.07.002.View ArticlePubMedGoogle Scholar
- van Spreeuwel JP, Duursma GC, Meijer CJ, Bax R, Rosekrans PC, Lindeman J: Campylobacter colitis: histological immunohistochemical and ultrastructural findings. Gut. 1985, 26: 945-951. 10.1136/gut.26.9.945.PubMed CentralView ArticlePubMedGoogle Scholar
- Oelschlaeger TA, Guerry P, Kopecko DJ: Unusual microtubule-dependent endocytosis mechanisms triggered by Campylobacter jejuni and Citrobacter freundii. Proc Natl Acad Sci USA. 1993, 90: 6884-6888. 10.1073/pnas.90.14.6884.PubMed CentralView ArticlePubMedGoogle Scholar
- Wooldridge KG, Williams PH, Ketley JM: Host signal transduction and endocytosis of Campylobacter jejuni. Microb Pathog. 1997, 21: 299-305.View ArticleGoogle Scholar
- Hu L, Kopecko DJ: Campylobacter jejuni 81-176 associates with microtubules and dynein during invasion of human intestinal cells. Infect Immun. 1999, 67: 4171-4182.PubMed CentralPubMedGoogle Scholar
- Biswas D, Niwa H, Itoh K: Infection with Campylobacter jejuni induces tyrosine-phosphorylated proteins into INT-407 cells. Microbiol Immunol. 2004, 48: 221-228.View ArticlePubMedGoogle Scholar
- Pei Z, Burucoa C, Grignon B, Baqar S, Huang XZ, Kopecko DJ, Bourgeois AL, Fauchere JL, Blaser MJ: Mutation in the peb1A locus of Campylobacter jejuni reduces interactions with epithelial cells and intestinal colonization of mice. Infect Immun. 1998, 66: 938-943.PubMed CentralPubMedGoogle Scholar
- Konkel ME, Monteville MR, Rivera-Amill V, Joens LA: The pathogenesis of Campylobacter jejuni-mediated enteritis. Curr Issues Intest Microbiol. 2001, 2: 55-71.PubMedGoogle Scholar
- Poly F, Guerry P: Pathogenesis of Campylobacter. Curr Opin Gastroenterol. 2008, 24: 27-31. 10.1097/MOG.0b013e3282f1dcb1.View ArticlePubMedGoogle Scholar
- Euker TP, Konkel ME: The cooperative action of bacterial fibronectin-binding proteins and secreted proteins promote maximal Campylobacter jejuni invasion of host cells by stimulating membrane ruffling. Cell Microbiol. 2011, doi: 10.1111/j.1462-5822.2011.01714.xGoogle Scholar
- Moser I, Schroeder W, Salnikow J: Campylobacter jejuni major outer membrane protein and a 59-kDa protein are involved in binding to fibronectin and INT 407 cell membranes. FEMS Microbiol Lett. 1997, 157: 233-238. 10.1111/j.1574-6968.1997.tb12778.x.View ArticlePubMedGoogle Scholar
- Konkel ME, Gray SA, Kim BJ, Garvis SG, Yoon JJ: Identification of the enteropathogens Campylobacter jejuni and Campylobacter coli based on the cadF virulence gene and its product. Clin Microbiol. 1999, 37: 510-517.Google Scholar
- Krause-Gruszczynska M, Rohde M, Hartig R, Genth H, Schmidt G, Keo T, Koenig W, Miller WG, Konkel ME, Backert S: Role of small Rho GTPases Rac1 and Cdc42 in host cell invasion of Campylobacter jejuni. Cell Microbiol. 2007, 9: 2431-2444. 10.1111/j.1462-5822.2007.00971.x.View ArticlePubMedGoogle Scholar
- Hu L, McDaniel JP, Kopecko DJ: Signal transduction events involved in human epithelial cell invasion by Campylobacter jejuni 81-176. Microb Pathog. 2006, 40: 91-100. 10.1016/j.micpath.2005.11.004.View ArticlePubMedGoogle Scholar
- Monteville MR, Yoon JE, Konkel ME: Maximal adherence and invasion of INT 407 cells by Campylobacter jejuni requires the CadF outer-membrane protein and microfilament reorganization. Microbiology. 2003, 149: 153-165. 10.1099/mic.0.25820-0.View ArticlePubMedGoogle Scholar
- Karlyshev AV, Linton D, Gregson NA, Lastovica AJ, Wren BW: Genetic and biochemical evidence of a Campylobacter jejuni capsular polysaccharide that accounts for Penner serotype specificity. Mol Microbiol. 2000, 35: 529-541.View ArticlePubMedGoogle Scholar
- Kanipes MI, Holder LC, Corcoran AT, Moran AP, Guerry P: A deep-rough mutant of Campylobacter jejuni 81-176 is noninvasive for intestinal epithelial cells. Infect Immun. 2004, 72: 2452-2455. 10.1128/IAI.72.4.2452-2455.2004.PubMed CentralView ArticlePubMedGoogle Scholar
- Konkel ME, Klena JD, Rivera-Amill V, Monteville MR, Biswas D, Raphael B, Mickelson J: Secretion of virulence proteins from Campylobacter jejuni is dependent on a functional flagellar export apparatus. J Bacteriol. 2004, 186: 3296-3003. 10.1128/JB.186.11.3296-3303.2004.PubMed CentralView ArticlePubMedGoogle Scholar
- Guerry P: Campylobacter flagella: not just for motility. Trends Microbiol. 2007, 15: 456-461. 10.1016/j.tim.2007.09.006.View ArticlePubMedGoogle Scholar
- Hu L, Kopecko DJ: Campylobacter. Edited by: Nachamkin I, Szymanski CM, Blaser MJ. 2008, Washington, DC: ASM Press, 297-313.Google Scholar
- Larson CL, Christensen JE, Pacheco SA, Minnich SA, Konkel ME: Campylobacter. Edited by: Nachamkin I, Szymanski CM, Blaser MJ. 2008, Washington, DC: ASM Press, 315-332.Google Scholar
- Novik V, Hofreuter D, Galán JE: Identification of Campylobacter jejuni genes involved in its interaction with epithelial cells. Infect Immun. 2010, 78: 3540-3553. 10.1128/IAI.00109-10.PubMed CentralView ArticlePubMedGoogle Scholar
- Watson RO, Galán JE: Campylobacter jejuni survives within epithelial cells by avoiding delivery to lysosomes. PLoS Pathog. 2008, 4: e14-10.1371/journal.ppat.0040014.PubMed CentralView ArticlePubMedGoogle Scholar
- Konkel ME, Hayes SF, Joens LA, Cieplak W: Characteristics of the internalization and intracellular survival of Campylobacter jejuni in human epithelial cell cultures. Microb Pathog. 1992, 13: 357-370. 10.1016/0882-4010(92)90079-4.View ArticlePubMedGoogle Scholar
- Day WA, Sajecki JL, Pitts TM, Joens LA: Role of catalase in Campylobacter jejuni intracellular survival. Infect Immun. 2000, 68: 6337-6345. 10.1128/IAI.68.11.6337-6345.2000.PubMed CentralView ArticlePubMedGoogle Scholar
- Hofreuter D, Novik V, Galán JE: Metabolic diversity in Campylobacter jejuni enhances specific tissue colonization. Cell Host Microbe. 2008, 4: 425-433. 10.1016/j.chom.2008.10.002.View ArticlePubMedGoogle Scholar
- Konkel ME, Garvis SD, Tipton S, Anderson DE, Cieplak W: Identification and molecular cloning of a gene encoding a fibronectin binding protein (CadF) from Campylobacter jejuni. Mol Microbiol. 1997, 24: 953-963. 10.1046/j.1365-2958.1997.4031771.x.View ArticlePubMedGoogle Scholar
- Goon S, Ewing CP, Lorenzo M, Pattarini D, Majam G, Guerry P: σ28-regulated nonflagella gene contributes to virulence of Campylobacter jejuni 81-176. Infect Immun. 2006, 74: 769-772. 10.1128/IAI.74.1.769-772.2006.PubMed CentralView ArticlePubMedGoogle Scholar
- Nyberg P, Sakai T, Cho KH, Caparon MG, Fässler R, Björck L: Interactions with fibronectin attenuate the virulence of Streptococcus pyogenes. EMBO J. 2004, 23: 2166-2174. 10.1038/sj.emboj.7600214.PubMed CentralView ArticlePubMedGoogle Scholar
- Schröder A, Schröder B, Roppenser B, Linder S, Sinha B, Fässler R, Aepfelbacher M: Staphylococcus aureus fibronectin binding protein-A induces motile attachment sites and complex actin remodeling in living endothelial cells. Mol Biol Cell. 2006, 17: 5198-5210. 10.1091/mbc.E06-05-0463.PubMed CentralView ArticlePubMedGoogle Scholar
- Wennerberg K, Lohikangas L, Gullberg D, Pfaff M, Johansson S, Fässler R: Beta 1 integrin-dependent and-independent polymerization of fibronectin. J Cell Biol. 1996, 132: 227-238. 10.1083/jcb.132.1.227.View ArticlePubMedGoogle Scholar
- Wennerberg K, Fässler R, Waermegård B, Johansson S: Mutational analysis of the potential phosphorylation sites in the cytoplasmic domain of integrin beta1A. Requirement for threonines 788-789 in receptor activation. J Cell Sci. 1998, 111: 1117-1126.PubMedGoogle Scholar
- Wennerberg K, Armulik A, Sakai T, Karlsson M, Fässler R, Schäfer EM, Mosher DF, Johansson S: The cytoplasmic tyrosines of integrin subunit beta1 are involved in focal adhesion kinase activation. Mol Cell Biol. 2000, 20: 5758-5765. 10.1128/MCB.20.15.5758-5765.2000.PubMed CentralView ArticlePubMedGoogle Scholar
- Sieg DJ, Hauck CR, Schläpfer DD: Required role of focal adhesion kinase (FAK) for integrin-stimulated cell migration. J Cell Sci. 1999, 112: 2677-2691.PubMedGoogle Scholar
- Klinghoffer RA, Sachsenmaier C, Cooper JA, Soriano P: Src family kinases are required for integrin but not PDGFR signal transduction. EMBO J. 1999, 18: 2459-2471. 10.1093/emboj/18.9.2459.PubMed CentralView ArticlePubMedGoogle Scholar
- Schmitter T, Pils S, Sakk V, Frank R, Fischer KD, Hauck CR: The granulocyte receptor carcinoembryonic antigen-related cell adhesion molecule 3 (CEACAM3) directly associates with Vav to promote phagocytosis of human pathogens. J Immunol. 2007, 178: 3797-3805.View ArticlePubMedGoogle Scholar
- Selbach M, Moese S, Hauck CR, Meyer TF, Backert S: Src is the kinase of the Helicobacter pylori CagA protein in vitro and in vivo. J Biol Chem. 2002, 277: 6775-6778. 10.1074/jbc.C100754200.View ArticlePubMedGoogle Scholar
- Biswas D, Itoh K, Sasakawa C: Uptake pathways of clinical and healthy animal isolates of Campylobacter jejuni into INT-407 cells. FEMS Immunol Med Microbiol. 2000, 29: 203-211. 10.1111/j.1574-695X.2000.tb01524.x.View ArticlePubMedGoogle Scholar
- Tamás P, Solti Z, Bauer P, Illés A, Sipeki S, Bauer A, Faragó A, Downward J, Buday L: Mechanism of epidermal growth factor regulation of Vav2, a guanine nucleotide exchange factor for Rac. J Biol Chem. 2003, 278: 5163-5171. 10.1074/jbc.M207555200.View ArticlePubMedGoogle Scholar
- Sander EE, van Delft S, ten Klooster JP, Reid T, van der Kammen RA, Michiels F, Collard JG: Matrix-dependent Tiam1/Rac signalling in epithelial cells promote either cell-cell adhesion or cell migration and is regulated by phosphatidylinositol 3-kinase. J Cell Biol. 1998, 143: 1385-1398. 10.1083/jcb.143.5.1385.PubMed CentralView ArticlePubMedGoogle Scholar
- Konkel ME, Kim BJ, Rivera-Amill V, Garvis SG: Bacterial secreted proteins are required for the internalization of Campylobacter jejuni into cultured mammalian cells. Mol Microbiol. 1999, 32: 691-701. 10.1046/j.1365-2958.1999.01376.x.View ArticlePubMedGoogle Scholar
- Carrillo CD, Taboada E, Nash JH, Lanthier P, Kelly J, Lau PC, Verhulp R, Mykytczuk O, Sy J, Findlay WA, Amoako K, Gomis S, Willson P, Austin JW, Potter A, Babiuk L, Allan B, Szymanski CM: Genome-wide expression analyses of Campylobacter jejuni NCTC11168 reveals coordinate regulation of motility and virulence by flhA. J Biol Chem. 2004, 279: 20327-20338. 10.1074/jbc.M401134200.View ArticlePubMedGoogle Scholar
- Schmidt A, Hall A: Guanine nucleotide exchange factors for Rho GTPases: turning on the switch. Genes Dev. 2002, 16: 1587-1609. 10.1101/gad.1003302.View ArticlePubMedGoogle Scholar
- Hsia DA, Mitra SK, Hauck CR, Streblow DN, Nelson JA, Ilic D, Huang S, Li E, Nemerow GR, Leng J, Spencer KS, Cheresh DA, Schläpfer DD: Differential regulation of cell motility and invasion by FAK. J Cell Biol. 2003, 160: 753-767. 10.1083/jcb.200212114.PubMed CentralView ArticlePubMedGoogle Scholar
- Tomar A, Schläpfer DD: Focal adhesion kinase: switsching between GEFs and GAPs in the regulatory of cell motility. Curr Opin Cell Biol. 2009, 21: 676-683. 10.1016/j.ceb.2009.05.006.PubMed CentralView ArticlePubMedGoogle Scholar
- Liu BP, Burridge K: Vav2 activates Rac1, Cdc42, and RhoA downstream from growth factor receptors but not beta1 integrins. Mol Cell Biol. 2000, 20: 7160-7169. 10.1128/MCB.20.19.7160-7169.2000.PubMed CentralView ArticlePubMedGoogle Scholar
- Marcoux N, Vuori K: EGF receptor mediates adhesion-dependent activation of the Rac GTPase: a role for phosphatidylinositol 3-kinase and Vav2. Oncogene. 2003, 22: 6100-6106. 10.1038/sj.onc.1206712.View ArticlePubMedGoogle Scholar
- Tamás P, Solti Z, Buday L: Membrane-targeting is critical for the phosphorylation of Vav2 by activated EGF receptor. Cell Signal. 2001, 13: 475-481. 10.1016/S0898-6568(01)00172-3.View ArticlePubMedGoogle Scholar
- Tran Van Nhieu G, Caron E, Hall A, Sansonetti PJ: IpaC induces actin polymerization and filopodia formation during Shigella entry into epithelial cells. EMBO J. 1999, 18: 3249-3262. 10.1093/emboj/18.12.3249.PubMed CentralView ArticlePubMedGoogle Scholar
- Boquet P, Lemichez E: Bacterial virulence factors targeting Rho GTPases: parasitism or symbiosis?. Trends Cell Biol. 2003, 13: 238-246. 10.1016/S0962-8924(03)00037-0.View ArticlePubMedGoogle Scholar
- Backert S, König W: Interplay of bacterial toxins with host defense: molecular mechanisms of immunomodulatory signaling. Int J Med Microbiol. 2005, 295: 519-530. 10.1016/j.ijmm.2005.06.011.View ArticlePubMedGoogle Scholar
- Giancotti FG, Ruoslahti E: Integrin signaling. Science. 1999, 285: 1028-1032. 10.1126/science.285.5430.1028.View ArticlePubMedGoogle Scholar
- Leiss M, Beckmann K, Girós A, Costell M, Fässler R: The role of integrin binding sites in fibronectin matrix assembly in vivo. Curr Opin Cell Biol. 2008, 20: 502-507. 10.1016/j.ceb.2008.06.001.View ArticlePubMedGoogle Scholar
- Orgel JP, San Antonio JD, Antipova O: Molecular and structural mapping of collagen fibril interactions. Connect Tissue Res. 2011, 52: 2-17. 10.3109/03008207.2010.511353.View ArticlePubMedGoogle Scholar
- Danen EH, Yamada KM: Fibronectin, integrins, and growth control. J Cell Physiol. 2001, 189: 1-13. 10.1002/jcp.1137.View ArticlePubMedGoogle Scholar
- Sechler JL, Rao H, Cumiskey AM, Vega-Colon I, Smith MS, Murata T, Schwarzbauer JE: A novel fibronectin binding site required for fibronectin fibril growth during matrix assembly. J Cell Biol. 2001, 154: 1081-1088. 10.1083/jcb.200102034.PubMed CentralView ArticlePubMedGoogle Scholar
- Tachibana K, Sato T, D'Avirro N, Morimoto C: Direct association of pp125FAK with paxillin, the focal adhesion-targeting mechanism of pp125FAK. J Exp Med. 1995, 182: 1089-1099. 10.1084/jem.182.4.1089.View ArticlePubMedGoogle Scholar
- Miyamoto S, Katz BZ, Lafrenie RM, Yamada KM: Fibronectin and integrins in cell adhesion, signaling, and morphogenesis. Ann NY Acad Sci. 1998, 857: 119-129. 10.1111/j.1749-6632.1998.tb10112.x.View ArticlePubMedGoogle Scholar
- Ilic D, Kovacic B, Johkura K, Schläpfer DD, Tomasevic N, Han Q, Kim JB, Howerton K, Baumbusch C, Ogiwara N: FAK promotes organization of fibronectin matrix and fibrillar adhesion. J Cell Sci. 2004, 117: 177-187. 10.1242/jcs.00845.View ArticlePubMedGoogle Scholar
- Agerer F, Lux S, Michel A, Rohde M, Ohlsen K, Hauck CR: Cellular invasion by Staphylococcus aureus reveals a functional link between focal adhesion kinase and cortactin in integrin-mediated internalisation. J Cell Sci. 2005, 118: 2189-2000. 10.1242/jcs.02328.View ArticlePubMedGoogle Scholar
- Alrutz MA, Isberg RR: Involvement of focal adhesion kinase in invasin-mediated uptake. Proc Natl Acad Sci USA. 1998, 95: 13658-13663. 10.1073/pnas.95.23.13658.PubMed CentralView ArticlePubMedGoogle Scholar
- Eitel J, Heise T, Thiesen U, Dersch P: Cell invasion and IL-8 production pathways inhibited by YadA of Yersinis pseudotuberculosis require common signalling molecules (FAK, c-Src, Ras) and distinct cell factors. Cell Microbiol. 2005, 7: 63-77.View ArticlePubMedGoogle Scholar
- Shin S, Paul-Satyaseela M, Lee JS, Romer LH, Kim KS: Focal adhesion kinase is involved in type III group B streptococcal invasion of human brain microvascular epithelial cells. Microb Pathog. 2006, 41: 168-173. 10.1016/j.micpath.2006.07.003.View ArticlePubMedGoogle Scholar
- Kwok T, Zabler D, Urman S, Rohde M, Hartig R, Wessler S, Misselwitz R, Berger J, Sewald N, König W, Backert S: Helicobacter exploits integrin for type IV secretion and kinase activation. Nature. 2007, 449: 862-866. 10.1038/nature06187.View ArticlePubMedGoogle Scholar
- Tegtmeyer N, Hartig R, Delahay RM, Rohde M, Brandt S, Conradi J, Takahashi S, Smolka AJ, Sewald N, Backert S: A small fibronectin-mimicking protein from bacteria induces cell spreading and focal adhesion formation. J Biol Chem. 2010, 285: 23515-23526. 10.1074/jbc.M109.096214.PubMed CentralView ArticlePubMedGoogle Scholar
- Tegtmeyer N, Wittelsberger R, Hartig R, Wessler S, Martinez-Quiles , Backert S: Serine phosphorylation of cortactin controls focal adhesion kinase activity and cell scattering induced by Helicobacter pylori. Cell Host Microbe. 2011, 9: 520-531. 10.1016/j.chom.2011.05.007.View ArticlePubMedGoogle Scholar
- Lane BJ, Mutchler C, Al Khodor S, Grieshaber SS, Carabeo RA: Chlamydial entry involves TARP binding of guanine nucleotide exchange factors. PLoS Pathog. 2008, 4: e1000014-10.1371/journal.ppat.1000014.PubMed CentralView ArticlePubMedGoogle Scholar
- McGee K, Holmfeldt P, Fällman M: Microtubule-dependent regulation of Rho GTPases during internalisation of Yersinia pseudotuberculosis. FEBS Lett. 2003, 533: 35-41.View ArticlePubMedGoogle Scholar
- Cabodi S, Moro L, Bergatto E, Boeri Erba E, Di Stefano P, Turco E, Tarone G, Defilippi P: Integrin regulation of epidermal growth factor (EGF) receptor and of EGF-dependent responses. Biochem Soc Trans. 2004, 32: 438-442. 10.1042/BST0320438.View ArticlePubMedGoogle Scholar
- Alexi X, Berditchevski F, Odintsova E: The effect of cell-ECM adhesion on signalling via the ErbB family of growth factor receptors. Biochem Soc Trans. 2011, 39: 568-573. 10.1042/BST0390568.View ArticlePubMedGoogle Scholar
- Morooka T, Umeda A, Amako K: Motility as an intestinal colonization factor for Campylobacter jejuni. Gen Microbiol. 1985, 131: 1973-1980.View ArticleGoogle Scholar
- Wassenaar TM, van der Zeijst BAM, Ayling R, Newell DG: Colonization of chicks by motility mutants of Campylobacter jejuni demonstrates the importance of flagellin A expression. J Gen Microbiol. 1993, 139: 1171-1175.View ArticlePubMedGoogle Scholar
- Hendrixson DR, DiRita VJ: Identification of Campylobacter jejuni genes involved in commensal colonization of the chick gastrointestinal tract. Mol Microbiol. 2004, 52: 471-484. 10.1111/j.1365-2958.2004.03988.x.View ArticlePubMedGoogle Scholar
- Wassenaar TM, Bleumink-Pluym NMC, van der Zeijst BAM: Inactivation of Campylobacter jejuni flagellin genes by homologous recombination demonstrates that flaA but not flaB is required for invasion. EMBO J. 1991, 10: 2055-2061.PubMed CentralPubMedGoogle Scholar
- Grant CCR, Konkel ME, Cieplak W, Tompkins LS: Role of flagella in adherence, internalization, and translocation of Campylobacter jejuni in nonpolarized and polarized epithelial cell cultures. Infect Immun. 1993, 61: 1764-1771.PubMed CentralPubMedGoogle Scholar
- Yao R, Burr DH, Doig P, Trust TJ, Niu H, Guerry P: Isolation of motile and non-motile insertional mutants of Campylobacter jejuni: the role of motility in adherence and invasion of eukaryotic cells. Mol Microbiol. 1994, 14: 883-893. 10.1111/j.1365-2958.1994.tb01324.x.View ArticlePubMedGoogle Scholar
- Boehm M, Krause-Gruszczynska M, Rohde M, Tegtmeyer N, Takahashi S, Oyarzabal OA, Backert S: Major host factors involved in epithelial cell invasion of Campylobacter jejuni: Role of fibronectin, integrin beta1, FAK, Tiam-1 and DOCK180 in activating Rho GTPase Rac1. Front Cell Infect Microbiol. 2011.Google Scholar
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