The C. jejuni strains 81–176, 84–25, and F38011 were used in this study. The isogenic F38011ΔcadF, 81–176ΔcadF, 81–176ΔflaA/B, and 81–176ΔflhA mutants were kindly provided by Konkel et al. (1997) and Patricia Guerry (Goon et al., 2006), respectively. All C. jejuni strains were grown on Campylobacter blood-free selective Agar Base (Oxoid) containing Campylobacter growth supplement (Oxoid) or, when appropriate, 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 h.

Human embryonic intestinal epithelial cells (INT-407), obtained from the American Type Culture Collection (ATCC CCL-6), were grown in Eagle’s minimum essential medium (MEM) containing l-glutamine and Earle’s salts (Gibco). Several mouse fibroblast knockout cell lines were cultured in RPMI1640 or DMEM medium, respectively, which were supplemented with 10% fetal calf serum (Gibco). Generation of the floxed FN^+/+ mouse fibroblast cells and FN^−/− knockout cells has been described (Nyberg et al., 2004; Schroeder et al., 2006). The FN^−/− cells were grown in DMEM supplemented with 10% FCS or alternatively, in serum replacement medium for bacterial infection studies (Sigma Aldrich). Monolayers of GD25 mouse fibroblasts (integrin beta1^−/−) or GD25 cells stably transfected with integrin β1A (GD25β1A) were grown in 10% fetal bovine serum (Wennerberg et al., 1996). Mouse knockout cells deficient in (FAK^−/− cells) as well as stable expression of WT FAK in FAK^−/− cells were described previously (Sieg et al., 1999). After reaching a confluency of about 70%, the cells were washed two times with phosphate-buffered saline (PBS), and then starved for 12 h.

For the infection assays, the different cell lines were seeded to give 4 × 10⁵ cells in 12-well tissue culture plates. The culture medium was replaced with fresh MEM without antibiotics 1 h before infection. Bacteria were suspended in (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, 1–10 mM) and PF-573228 (Tocris; 10 μM) were added 30 min prior to infection and kept throughout the duration of the incubation period. All control cells were treated with the 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 with similar results.

The pcDNA3.1-FAK WT and corresponding Y397F, K454R, ΔPR1/ΔPR2, and Y925F mutants were kindly provided by the lab of D. Schlaepfer (Sieg et al., 1999). Transfection of plasmid constructs was performed using GeneJammer transfection reagent according to manufacturer’s instructions (Stratagene). After 48 h, transfected INT-407 cells were infected with C. jejuni strains. All FAK constructs were expressed as C-terminal HA-tag fusion proteins as described (Sieg et al., 1999). The efficiency of transfection was verified both by immunofluorescence microscopy and Western blotting using an α-HA antibody (NEB).

siRNA(s) directed against human DOCK180, Trio and control siRNA containing scrambled sequence were purchased from Santa Cruz. siRNAs targeted against human Rac1 and RhoA were synthesized as follows. Rac1 target sequence (5′-AAAACTTGCCTACTGATCAGT-3′), RhoA target sequence (5′-TACCTTATAGTTACTGTGTAA-3′) were utilized. For down-regulation of Tiam-1 both the siRNA from Santa Cruz and another one obtained from MWG-Biotech [Tiam-1 target sequence (5′-ACAGCTTCAGAAGCCTGAC-3′)], were used simultaneously. Transfection of siRNAs was performed using siRNA Transfection Reagent as recommended by the manufacturer (Santa Cruz).

Infection of eukaryotic cells at a density of 4 × 10⁵ cells per well was carried out as described above. After infection, the cells were washed three times with 1 ml of pre-warmed MEM medium per well to remove non-adherent bacteria. To determine the colony-forming units (CFU) corresponding to intracellular bacteria, the INT-407 cell monolayers were treated with 250 μg/ml gentamicin (Sigma) at 37°C for 2 h, 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 corresponding to 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. All experiments were routinely performed in triplicates. In parallel control experiments, 250 μg/ml gentamicin killed all extracellular bacteria (data not shown).

Rac1 activation in infected cells was determined with the Rac1 activation assay kit (Cytoskeleton), based on a pull-down assay using the Cdc42–Rac1 interactive binding domain of PAK1 fused to glutathione S-transferase, GST–CRIB (Sander et al., 1998). Briefly, host cells were grown to 70% confluency and serum-starved overnight. Subsequently, cells were incubated in PBS (pH 7.4) 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 for 15 min, respectively. Cell lysates (treated with bacteria, GTPγ-S, GDP or untreated) were mixed with the PAK–RBD slurry (1 h, 4°C). Finally, the beads were collected by centrifugation and washed three times with assay buffer. Activated Rac1 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 quantitated as band intensities representing the relative amount of active Rac1-GTP using the Lumi-Imager F1 software program (Roche).

Rac1 activation in infected cells was also determined by a second approach, 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 immobilized on provided microplates. Bound active Rac1 was detected with a specific antibody and luminescence. The luminescence signal was quantified by using a microplate reader (SpectraFluor Plus, Tecan).

Polyclonal antiserum (α-FlaA and α-MOMP) was raised according to standard protocols (Biogenes GmbH) by immunization of two rabbits with a conserved FlaA-derived peptide (corresponding to amino acids 93–106: KTKATQAAQDGQSL) or a MOMP-derived peptide (amino acids 400–413: NLDQGVNTNESADH), which were conjugated to Limulus polyphemus hemocyanin carrier protein. The resulting antisera were affinity-purified and the specificity against their antigens was confirmed by Western blotting.

Motility phenotypes of strains were tested in MH media containing 0.4% agar. Bacterial cells were harvested from a 36 h culture on conventional agar plates and resuspended in PBS to obtain an optical density at 600 nm of 0.45 (approximately 1 × 10⁹ cfu/ml). The bacteria were incubated for 30 min in the presence or absence of 20 μg/ml α-FlaA antibody or pre-immune serum as control. To ensure that equal amounts of antibody were present on the entire agar surface in the α-FlaA sample, 50 μl of the antibody solutions were plated onto the corresponding plates. Subsequently, 2 μl of a bacterial suspension of 2 × 10⁸ cfu/ml (±antibody) were stabbed into motility agar. Plates were incubated at 37°C under microaerophilic conditions for 24 h, followed by measuring the diameter of the resulting swarms. The results were the mean of at least five separate measurements from three experiments.

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 Rac1 (Upstate), FAK-PY-397 (Biomol), FAK-PY-925 (NEB), FAK, RhoA, fibronectin, β1 integrin, Tiam-1, DOCK180, or GAPDH (all Santa Cruz) was performed according to the manufacturer’s instructions. As a secondary antibody, horseradish peroxidase-conjugated α-mouse, α-rabbit, or α-goat IgG was used (DAKO). Immunoreactive bands were visualized by ECL plus Western Blotting Detection System (Amersham Biosciences). Relative FAK kinase activities were quantitated as band intensities of the FAK-PY397 signals related to its FAK control blot using the Lumi-Imager F1 software program (Roche).

Host cell monolayers grown on coverslips were infected with C. jejuni strains for either 4 or 6 h, then fixed in cacodylate buffer (0.1 M cacodylate, 0.01 M CaCl₂, 0.01 M MgCl₂, 0.09 M sucrose; pH 6.9) 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’s 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 SD.

In our previous studies we have shown that small Rho GTPases such as Rac1 and Cdc42 are activated by C. jejuni, and inhibitors as well the expression of dominant-negative constructs have indicated that active Rac1 maybe one important host factor required for bacterial entry into host cells (Krause-Gruszczynska et al., 2007). Here we aimed to identify the signaling pathway leading to Rac1 activation. First, we confirmed that Rac1 is activated in infected non-phagocytic intestinal epithelial cells (INT-407) using a novel commercial assay, called G-LISA (see Materials and Methods). The results show that wild-type (WT) C. jejuni strains 81–176, 84–25, or F38011 induced the generation of active Rac1-GTP during the course of infection (Figure 1A and data not shown). To confirm that Rac1 is indeed necessary for C. jejuni invasion, we downregulated Rac1 expression by siRNA. Down-regulation of Rac1 expression by about 90% lead to a significant drop in the number of intracellular bacteria as determined by gentamicin protection assays (Figure 1B). As controls, a scrambled siRNA or siRNA against RhoA, another Rho GTPase member, did not suppress C. jejuni invasion (Figures 1B,C). These results indicate that penetration of C. jejuni into cultured cells specifically requires Rac1.

Previous studies have shown that addition of methyl-beta cyclodextrin (MβCD), an agent that sequesters cholesterol, decreased the ability of C. jejuni to enter several cultured epithelial cell lines, suggesting that lipid rafts may be required for efficient host cell invasion (Watson and Galán, 2008). Thus, we investigated if intact lipid rafts may be required for C. jejuni-triggered Rac1 activation. Indeed, addition of MβCD blocked C. jejuni-induced Rac1 activation and internalization into INT-407 cells in a dose-dependent manner (Figure 1D), suggesting that a lipid raft-associated host receptor maybe targeted by C. jejuni to trigger downstream signaling leading to Rac1 activation. Since C. jejuni encodes CadF, an outer membrane protein binding to the extracellular matrix protein fibronectin (Konkel et al., 2001), we proposed that a classical fibronectin → integrin-β1 → FAK pathway could be involved in activating Rac1. To investigate this question, we utilized fibroblast cell lines derived from fibronectin^−/−, integrin-β1^−/− (so called GD25 cells) and FAK^−/− knockout mice (Wennerberg et al., 1996; Sieg et al., 1999; Nyberg et al., 2004), which have the advantage that the cells are completely devoid of expressing the proteins of interest (Figures 2A–C). As controls, we infected in parallel experiments floxed fibronectin^+/+ cells, GD25 cells re-expressing WT integrin-β1A (GD25β1A) and FAK^−/− cells re-expressing WT FAK. Infection studies showed that WT C. jejuni can effectively enter the control cells, while the knockout cells exhibited significant deficiency for bacterial uptake (Figures 2A–C). In addition, transient transfection of FAK^−/− knockout cells with HA-tagged WT FAK restored the capability of C. jejuni to enter these cells, while expression of FAK mutants that were either not capable of autophosphorylation (FAK Y397F), impaired kinase activity (FAK K454R), lacking several proline residues in two PxxP-motifs (FAKΔPR1/2) necessary for association with SH3-containing proteins such as p130CAS or Graf, or lacking the Grb2-binding site (FAK Y925F; Sieg et al., 1999; Hauck et al., 2002), significantly reduced the internalization of C. jejuni (p ≤ 0.001) by about 35–50% (Figure 2D). This indicates an important role of FAK signaling downstream of fibronectin and integrins in facilitating efficient uptake of C. jejuni.

Next we infected the WT fibroblasts and their corresponding fibronectin^−/−, GD25, and FAK^−/− knockout cell lines and analyzed the interaction of C. jejuni with the surface of host cells by high resolution FESEM. First, we confirmed that the WT C. jejuni strains had single bipolar flagella (Figure 3A, white arrows). After infection, the micrographs revealed that the bacteria were able to attach to the host cell surface, followed by cellular invasion which was observed predominantly after 4–6 h of infection (Figure 3B). Interestingly, similar to our earlier observations with infected INT-407 cells (Krause-Gruszczynska et al., 2007), we found that C. jejuni entered the WT fibroblast cells in a very specific fashion, first with its leading flagellar tip followed by the opposite flagellar end (Figure 3B, yellow arrows). Tight engulfment of the bacteria and membrane ruffles (red arrows), filopodia-like structures (blue arrows) as well as the appearance of elongated microspikes (green arrowheads) were also regularly observed. In contrast, infection of fibronectin^−/−, GD25 and FAK^−/− knockout cell lines also revealed bound bacteria at the surface of the cells with short microspikes present, but no membrane ruffles or invading C. jejuni could be detected (Figure 3C). The predominant observation of membrane ruffling in WT cells confirms the typical occurrence of Rac1 GTPase activation followed by dynamic membrane rearrangements during the C. jejuni invasion process, and this depends on the expression of three crucial host cell factors, fibronectin, integrin β1, and FAK.

Focal adhesion kinase is an intracellular non-receptor tyrosine kinase and important modulator of integrin-dependent focal cell contacts, thereby orchestrating well-known integrin-initiated outside-in signaling events (Sieg et al., 1999; Hauck et al., 2002). FAK localizes with β1-integrins and becomes activated by autophosphorylation at position Y-397, and downstream signaling can be transmitted through subsequent phosphorylation of FAK at Y-925. The next experiment was therefore to examine whether C. jejuni can stimulate the autophosphorylation of FAK. For this purpose, FAK WT cells were infected with WT C. jejuni for different time periods and cell lysates were analyzed with an antibody specific for FAK phosphorylated at position Y-397. Quantification data show that C. jejuni-induced FAK autophosphorylation profoundly, and this correlated with increasing amounts of intracellular bacteria over time (Figure 4A). In addition, C. jejuni-induced the phosphorylation of FAK at Y-925 in a time-dependent manner (Figure 4B). To investigate the potential contribution of the CadF protein for FAK phosphorylation events, the effect of an isogenic ΔcadF mutant was examined under identical conditions. The results show that FAK autophosphorylation was widely impaired in infections with the ΔcadF mutant (Figures 4A,B). These findings suggest that expression of CadF maybe involved in C. jejuni-induced FAK kinase activity.

Next we aimed to investigate if FAK is required for C. jejuni-induced Rac1 activation. For this purpose, FAK^−/− knockout cells and FAK^−/− cells re-expressing FAK were infected under the same conditions as shown in Figures 4A,B, followed by CRIB–GST pull-down assays. While growing levels of activated Rac1 were detected in FAK-positive cells over time, no detectable activation of Rac1 was found in FAK^−/− cells during the entire course of infection (Figure 4C), indicating the involvement of FAK in signaling upstream of Rac1 activation during C. jejuni invasion. Furthermore, significantly reduced Rac1-GTP levels were observed in both FAK-positive and FAK^−/− cells infected with the ΔcadF mutant (Figure 4C). These findings further support the view that cadF plays a role in signaling leading to FAK-mediated activation of Rac1. However, the ΔcadF mutant was still able to induce some GTPase activation in FAK-positive cells suggesting that other bacterial factor(s) are also implicated in this signaling (Figures 4A–C, right lanes).

Our next aim was to determine additional signaling components downstream of FAK and upstream of Rac1 activation during infection with C. jejuni. Cycling of Rho GTPases between the inactive and active forms is positively regulated by guanine nucleotide exchange factors (GEFs) and negatively regulated by GTPase activating proteins (GAPs). GEFs stimulate the exchange of GDP for GTP to generate the active form of a given GTPase, which is then capable of recognizing downstream targets (Schmidt and Hall, 2002; Hsia et al., 2003; Tomar and Schlaepfer, 2009). To identify which GEFs are involved in C. jejuni invasion, the expression of typical GEFs including DOCK180, Tiam-1, or Trio was downregulated using target-specific siRNA. While down-regulation of Tiam-1 and DOCK180 led to the significant reduction in C. jejuni internalization (Figures 5A,B), both down-regulation of Trio (Figure 5C) and transfection with non-targeting scrambled control siRNA sequence had no significant effect on C. jejuni uptake as quantified by gentamicin protection assays (Figures 5A–C). Importantly, the drop in invasion rates correlated with significantly reduced Rac1 GTP levels in CRIB-pull-down assays of Tiam-1 and DOCK180 siRNA-treated cells, but not in Trio siRNA-treated cells (Figures 5D–F). This suggests that two GEFs, Tiam-1 and DOCK180 (but not Trio), play a role in C. jejuni-induced Rac1 activation and invasion.

We have noted that down-regulation of the individual GEFs Tiam-1 or DOCK180 did not lead to a complete blockade of Rac1 activity and C. jejuni uptake. We therefore proposed that both GEFs may act together in C. jejuni-infected cells. To investigate this question, Tiam-1 and DOCK180 expression was downregulated by siRNA, either alone or simultaneously, followed by G-LISA to determine Rac1 activity. The results show that simultaneous down-regulation of Tiam-1 and DOCK180 led to a profound block of C. jejuni-induced Rac1 activity (Figures 6A,B). A similar strong blockade of Rac1 levels was achieved in infected FAK^−/− cells (Figure 4C) or by infection in the presence of the FAK kinase inhibitor PF-573228 (Figure 6C). As expected, simultaneous down-regulation of Tiam-1 and DOCK180 resulted not only in profound inhibition of Rac1 activity but also profound blockade of C. jejuni invasion (Figure 6D). These data suggest that one important pathway of C. jejuni host cell entry proceeds by activation of a FAK → Tiam-1/DOCK180 → Rac1 signaling cascade.

Since cadF is not the sole bacterial gene involved in C. jejuni-induced GTPase activation, the following aim was to search for other factor(s) playing a role in this signaling. The flagellar apparatus was reported to be one of the most intensively investigated pathogenicity determinant in C. jejuni (Konkel et al., 2004; Guerry, 2007). For this purpose, host cells were infected with WT strain 81–176 and its isogenic mutants ΔflaA/B, lacking the two major flagella subunits FlaA and FlaB (Goon et al., 2006), and ΔflhA, a key element involved in the coordinate regulation of late flagellar genes and other factors in C. jejuni (Carrillo et al., 2004). First, we confirmed the absence of flagella in both mutants (Figure 7A), followed by infection assays. As expected activated Rac1 was detected in FAK-positive cells between 2 and 4 h after infection with WT C. jejuni (Figures 7B,C). In contrast, no detectable Rac1 activation was found in cells infected with ΔflaA/B or ΔflhA mutants during the entire course of infection (Figures 7B,C), indicating an important role of these flagellar genes in activating Rac1 by C. jejuni, in addition to the contribution by cadF as shown above.

There is some controversy in the literature of whether the C. jejuni flagellum-mediated bacterial motility is important for invasion or if the flagellum can secrete invasion-related bacterial factors in the supernatant (Konkel et al., 2004; Novik et al., 2010). To investigate if the flagellar effect on Rac1 is direct or indirect, we were searching for a condition in which the flagellar motility is not affected, but invasion can be impaired. We have generated an α-FlaA antibody against a conserved region at the N-terminus of FlaA, and this antibody recognizes C. jejuni FlaA proteins in Westernblots (Figure 8A). We then pre-incubated WT C. jejuni with the α-FlaA antibody or pre-immune serum as control followed by motility assays in soft agar (see Materials and Methods). Treatment of any of the used C. jejuni strains with the pre-immune serum revealed no significant differences in bacterial motility or invasion as compared to non-treated bacteria (data not shown). The results also showed that while presence of the α-FlaA antibody had a slight but no significant effect on motility of WT C. jejuni or ΔcadF mutant (Figures 8A,B), bacterial invasion was significantly impaired as determined by gentamicin protection assay (Figures 8C,D). This suggests that the flagellum of C. jejuni has a motility-independent activity leading to bacterial entry into host target cells.