The Campylobacter jejuni Cj0268c Protein Is Required for Adhesion and Invasion In Vitro

Adherence of Campylobacter jejuni to its particular host cells is mediated by several pathogen proteins. We screened a transposon-based mutant library of C. jejuni in order to identify clones with an invasion deficient phenotype towards Caco2 cells and detected a mutant with the transposon insertion in gene cj0268c. In vitro characterization of a generated non-random mutant, the mutant complemented with an intact copy of cj0268c and parental strain NCTC 11168 confirmed the relevance of Cj0268c in the invasion process, in particular regarding adherence to host cells. Whereas Cj0268c does not impact autoagglutination or motility of C. jejuni, heterologous expression in E. coli strain DH5α enhanced the potential of the complemented E. coli strain to adhere to Caco2 cells significantly and, thus, indicates that Cj0268c does not need to interact with other C. jejuni proteins to develop its adherence-mediating phenotype. Flow cytometric measurements of E. coli expressing Cj0268c indicate a localization of the protein in the periplasmic space with no access of its C-terminus to the bacterial surface. Since a respective knockout mutant possesses clearly reduced resistance to Triton X-100 treatment, Cj0268c contributes to the stability of the bacterial cell wall. Finally, we could show that the presence of cj0268c seems to be ubiquitous in isolates of C. jejuni and does not correlate with specific clonal groups regarding pathogenicity or pathogen metabolism.


Introduction
Campylobacter jejuni is a Gram-negative spiral shaped bacterium, which is pervasive in mammals and birds. The chicken intestine is the natural reservoir that is frequently colonized by the pathogen. In recent years, C. jejuni has emerged as an abundant reported cause of bacterial diarrhoea in industrialized as well as developing countries with approximately 2.5 million estimated cases per year in the United States and more than 60,000 annual cases in Germany. Contaminated chicken meat, beef and milk products are common sources of transmission and human infection [1][2][3][4]. The progress of the disease can vary from watery to bloody diarrhea including fever and abdominal cramps. In rare cases immunopathological sequelae such as Guillain-Barré syndrome might arise even months or years post infection [5,6].
Adherence of C. jejuni to intestinal epithelial cells is essential for successful infection in human hosts. In the past, in vitro as well as in vivo studies revealed distinct proteins which are important for C. jejuni to adhere to its particular target cells. For instance, PEB1, the periplasmic component of an aspartate/glutamate ABC-transporter, mediates adherence and invasion of human epithelial cells and is important for the intestinal colonization of mice [7][8][9]. Furthermore, the major outer membrane protein MOMP adheres to fibronectin and is involved in binding of C. jejuni to the membrane of INT407 cells [10]. CadF, another outer membrane protein with an apparent molecular weight of 37 kDa, and FlpA have also been shown to connect to fibronectin. These interactions, in turn, result in the activation of integrin receptors to launch a host cell signal cascade leading to restructuring of the actin cytoskeleton mediating the uptake of C. jejuni [11][12][13][14][15]. The surface-exposed lipoprotein JlpA is required for efficient adherence of the pathogen to HEp-2 epithelial cells and initiates the activation of NF-kB and p38 MAP kinase. Consequently, JlpA seems to be involved in the proinflammatory host cell response upon C. jejuni infection [16,17]. Furthermore, CapA, an autotransporter protein of C. jejuni, has been shown to be associated with the adherence and invasion of epithelial cells by the pathogen and plays an important role in the colonization of the chicken gut. Recently, it was demonstrated that the bacterial outer membrane protein Cj0091 mediates adherence of C. jejuni to INT407 cells and contributes to the colonization of chickens as well [18,19]. Taken together, all these adhesion factors contribute significantly to the interaction between host cell and bacterial pathogen to allow the subsequent process of cellular invasion. In addition to these proteins described to be involved in the process of adherence, also lipooligosaccharides (LOS) are important for pathogen-host cell interactions given that C. jejuni strains deficient in genes involved in LOS metabolism (wlaRG, wlaTB and wlaTC) exhibited diminished adherence properties to chicken embryo fibroblasts [20]. Recently, our group generated a C. jejuni mutant deficient in sulphite:cytochrome c oxidoreductase (SOR) which exhibited a down-regulated transcription of genes involved legionaminic acid synthesis and possessed reduced adherence properties to Caco2 cells [21]. Finally, a recently identified C. jejuni type VI secretion system (T6SS) was shown to be involved in cell adhesion. Following functional knockout of the T6SS-genes hcp1 and icmF1, the adherence capacity of the respective mutants to T84 colon epithelial cells was reduced by approximately 50% as compared to the parental strain [22].
In this report we describe the C. jejuni gene cj0268c which has been shown by us and others to be important for the capability of the pathogen to infect host cells [23,24]. This protein with a molecular weight of 40.2 kDa and an isoelectric point of 8.93 possesses a putative transmembrane domain around amino acid 60 and a SPFH domain encompassing the amino acids 64 to 259. Proteins containing the stomatin/prohibitin/flotillin/HflK/C (SPFH) domain can be found in divergent species ranging from bacteria to mammals. The precise function of this domain, however, is still unclear even though mammal proteins containing SPFH domains are frequently found in lipid raft microdomains within several cellular membranes [25][26][27]. In support of this, Hinderhofer et al. [28] could categorize altogether 1090 SPFH domain-containing proteins from 497 different bacterial species encompassing all phyla into 12 subfamilies. However, despite the knowledge acquisition about the evolutionary development of prokaryotic SPFH proteins, the general biological function of this motif is still unclear. Here we demonstrate that Cj0268c is required for adhesion of C. jejuni to different host cells. Heterologous expression revealed its potential to alter the adhesion capacity of E. coli. The exact subcellular localization of Cj0268c was not known yet. Although analysis of the protein sequence using SignalIP 4.1 (Technical University of Denmark) revealed no evidence for the presence of a signal peptide, flow cytometry analysis after immunolabeling of Cj0268c in E. coli indicated the protein to reside in the periplasmic space with no exposure of the C-terminus at the bacterial surface. Furthermore, we determine the relevance of Cj0268c regarding motility, autoagglutination, the resistance of C. jejuni to bile salts and the stability of the bacterial cell to the nonionic surfactant Triton X-100. Finally, we investigate the affiliation of cj0268c to particular clonal groups of C. jejuni.

Materials and Methods
Bacterial strains, media and culture conditions C. jejuni strain B2 initially isolated from a patient suffering from gastroenteritis [29,30] and strain NCTC 11168 were grown on Columbia agar supplemented with 5% defibrinated sheep blood at 42uC in microaero-and capnophilic conditions (85% N 2 , 10% CO 2 , 5% O 2 ). If required, appropriate antibiotic concentrations of kanamycin (50 mg ml 21 ) or chloramphenicol (30 mg ml 21 ) were added. Growth experiments were carried out at 42uC in Mueller-Hinton (MH) broth under microaero-and capnophilic conditions. Escherichia coli strain DH5a was grown on Luria bertani (LB) agar or broth at 37uC. When necessary, ampicillin (100 mg ml 21 ) was added. Growth experiments were carried out at 42uC in Mueller-Hinton (MH) broth under the above mentioned conditions for 24 h.

Generation of competent cells and electroporation
10 ml of LB-broth were inoculated with a single E. coli DH5a colony and incubated overnight at 37uC under shaking. Three ml of the overnight culture were grown in 100 ml LB-broth at 37uC to an OD (600 nm) of 0.35-0.45. The culture was transferred into a 50 ml Falcon tube, placed on ice for 10 min and centrifuged for 15 min at 40006g at 4uC. Then the cell pellet was gently resuspended in 30 ml ice cold TFB1 buffer and incubated on ice for 30 min. Cells were pelleted by centrifugation as described above. Then, the cell pellet was carefully dissolved in 2 ml ice-cold TFB2 buffer and incubated on ice for another 30 min. After incubation aliquots of 100 ml were stored at 280uC.
C. jejuni, cells were harvested from Columbia blood agar plates and centrifuged at 5,0006g at 4uC for 10 minutes. After washing of the C. jejuni cells three times in 1 ml ice-cold wash buffer containing 272 mM sucrose and 15% glycerol at 4uC, the pellet was resuspended in 400 ml washing buffer and 100 ml aliquots were used for electroporation, respectively.
For each transformation 0.5 to 3 mg of plasmid DNA were added to an aliquot of competent cells in an ice-cold electroporation cuvette. After incubation of the cuvette containing the mixture of bacteria and DNA, electroporation was performed at 2.5 kV, 25 mF and 200 V using the BTX Electro Cell Manipulator. While adding 500 ml of SOC medium to transform E. coli, in case of C. jejuni, the suspension was transferred onto a non selective Columbia blood agar plate and incubated overnight at 37uC under microaerophilic conditions. Finally, cells were transferred onto a selective plate and incubated at 42uC under microaerophilic conditions for additional 2-3 days. E. coli were plated directly on selective LB agar containing the appropriate antibiotic agent and incubated at 37uC overnight.

Cultivation of cells
Human colon carcinoma Caco2 cells were cultivated in Dulbecco minimal essential medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 1 x non-essential amino acids, 100 U ml 21 penicillin, and 100 mg ml 21 streptomycin. Primary chicken cecal (PCC) cells, kindly provided by Ingrid Hä nel, were isolated as described elsewhere [31] and maintained in Quantum 286 medium for epithelial cells (PAA Laboratories) supplemented with 5% chicken serum and 0.5% chick embryo extract. Both cell lines were incubated in a humidified atmosphere of 95% air and 5% CO 2 at 37uC.

Isolation of nucleic acids
Genomic DNA of C. jejuni was isolated with the QIAamp DNA Mini Kit (Qiagen) according to the instructions of the manufacturer. Plasmid DNA was prepared using the GeneElute Plasmid Miniprep Kit (Sigma) following the manufacturer's protocol.

Insertional knockout of gene cj0268c
Initially, using genomic DNA from strain NCTC 11168 as a template, a 1090 bp DNA fragment representing cj0268c was amplified with primers Cj0268cF and Cj0268cR ( Table 1). The obtained PCR product was XbaI digested and ligated to the XbaI restricted and dephosphorylated plasmid vector pBluescript II KS (Stratagene). For the subsequent knockout, inverse PCR with the primers Cj0268cinvF and Cj0268cinvR using the plasmid described above as a template was carried out. The PCR reaction containing 10 mM Tris-HCl pH 8.3, 50 mM KCl, 1.5 mM MgCl 2 , all four dNTPs (each 0.2 mM) and 10 pmol of each primer was carried out using 1 U PfuUltra High-Fidelity DNA Polymerase (Stratagene) to obtain a blunt end PCR product. Initial incubation at 95uC for 1 min was followed by initial 10 cycles at 95uC for 30 s, 55uC for 30 s and 72uC for 5 min, followed by 25 cycles under the same conditions with the exception of a shifted annealing temperature to 58uC. The resulting PCR product with a length of 3752 bp comprised of the complete cloning vector pBluescript, the 413 59-terminal nucleotides and the 378 39-terminal nucleotides of cj0268c. After gel extraction of the PCR product using the QIAquick PCR Purification Kit (Qiagen), a phosphorylated blunt end kanamycin resistance cassette described previously [21] was ligated with the obtained PCR product using Quick Ligase (New England Biolabs) following the instructions of the manufacturer to obtain plasmid pBcj0268c-kanR.

Cloning of gene cj0268c into pRRC
For functional complementation of the C. jejuni cj0268c-knockout strain, gene cj0268c which was PCR-amplified with primers Cj0268cF and Cj0268cR and XbaI restricted as described above was cloned into likewise digested and dephosphorylated C. jejuni expression vector pRRC [32] to obtain pRRC-cj0268c. In order to provide cj0268c with a His-tag, the gene was amplified with primers Cj0268cF and Cj0268cHis, which are listed in Table 1. PCR was performed in a TRIO-Thermocycler (Biometra) with 10 ng of genomic DNA of C. jejuni as a template. The PCR mixture contained 1 U PfuUltra High-Fidelity DNA Polymerase (Stratagene), 1x inherent reaction buffer, dNTPs (each 0.2 mM) and 10 pmol of each primer. After initial incubation at 95uC for 3 min, 40 cycles at 95uC for 30 s, 55uC for 30 s and 72uC for 2 min were carried out with a final incubation at 72uC for 5 min. Afterwards, the PCR amplicon was XbaI-digested and cloned into pRRC as mentioned above.

Adhesion and invasion assays
Bacterial invasion assays were performed according to the publication of Everest et al. [33]. In brief, Caco2 cells were grown to approximately 80% confluence in a 6 well plate, washed with PBS and inoculated with 400 ml C. jejuni suspension adjusted to an OD (600 nm) of 0.5 which corresponds to a multiplicity of infection (MOI) of 100. To investigate invasion, the C. jejuni suspension was removed after two hours and the cells were washed three times with PBS before further incubation with culture medium supplemented with 100 mg ml 21 gentamicin. For the release of intracellular bacteria, the cells were lysed with 1% Triton X-100 for 10 min and the number of viable bacteria was determined by counting the number of bacteria colony forming units (cfu) grown on Columbia blood agar plates after incubation for 48 h at 42uC under microaerophilic conditions. For investigating bacterial adhesion, 6 well plates containing Caco2 cells and inoculated with C. jejuni or E. coli bacteria were centrifuged at 6006g for 5 min to increase the association of the bacteria with the cells. After incubation for 30 min, the monolayers were washed with PBS, cells were lysed and subsequently, plating of the bacteria was performed as described above. Every experiment was repeated four times.

Motility and autoagglutination assays
Tests for altered motility or autoagglutination of the C. jejuni strains and mutants were carried out as described previously by Tareen

Resistance of C. jejuni to bile salts
Bacteria were harvested from overnight-incubated blood agar plates and adjusted to an OD 600 nm of 0.1 in Muller Hinton broth. Then, the bacterial cultures were supplemented with 0.09, 0.18, 0.37, 0.75, 1.5, and 3% of cholate and deoxycholate, respectively. After incubation for 24 h at 37uC in a microaerophilic atmosphere, OD 600 nm was determined. All tests were carried out in triplicate.

Sensitivity of C. jejuni to Triton X-100
The sensitivity of C. jejuni to Triton X-100 was analyzed using bacterial colonies grown overnight which were harvested from blood agar plates. The bacteria were resuspended in distilled water and adjusted to an OD 600 nm of 0.1. Triton X-100 was added to a final concentration of 1.0%, and after incubation for one hour at room temperature the number of viable bacteria was determined by plating serial dilutions onto blood agar plates and subsequent incubation at 42uC for 48 h in a microaerophilic atmosphere. Every experiment was repeated three times.
Screening of C. jejuni strains for presence of gene cj0268c Genomic DNA samples of 56 C. jejuni strains were analyzed by PCR with 10 pmol of primers 268cGMF and 268cGMR (Table 1) and 1 U Taq-Polymerase (Roche) in a TRIO-Thermocycler (Biometra) under following conditions. After an initial denaturation at 95uC for 1 min, 35 cycles at 95uC for 30 s, 55uC for 30 s and 72uC for 1 min were performed. Afterwards, samples were run on a 2% agarose gel for the detection of the resulting 146 bp PCR amplicon.
Lysis of E. coli DH5a cells and immunoblot analysis E. coli from 5 ml overnight culture were centrifugated and resuspended in 500 ml 1x PBS. After addition of 100 mg lysozyme and NP40 to a final concentration of 1%, lysates were incubated on ice and were casually shaken for 10 min. Following the addition of 22 ml of 5 M NaCl, the lysates were centrifuged for 45 min at 160006g. Then, the supernatants were subject of subsequent immunoblot assays. Aliquots of 20 ml were separated in a 15% SDS-PAGE gel and blotted onto a PVDF membrane (GE Healthcare) by a semidry transport system (Sartorius). After protein transfer, the membrane was blocked for 1 h with 5% milk powder in PBS containing 0.05% Tween 20. Subsequent incubation of the membrane with a 1:3000 dilution of monoclonal mouse anti-His primary antibody (Qiagen) over night at 4uC was followed by labelling of the immune complexes with a 1:3000 dilution of horseradish-peroxidase-conjugated anti mouse secondary antibody (Dianova) for 1 h at room temperature and visualization by ECL chemiluminescence.

Permeabilization of E. coli DH5a cells
In order to get access to the bacterial periplasm, E. coli cells were permeabilized. prior to antibody labelling and flow cytometry analysis. 5610 7 bacterial cells of each sample were washed in 1 x PBS and were resuspended in 180 ml 20% sucrose, 50 mM Tris-HCl, pH 8 containing 10 mM EDTA. After 10 min 20 ml lysozyme at a final concentration of 2 mg/ml in 20 mM Tris-HCl, pH 8, 2 mM EDTA, 1.2% NP40 were added, and the samples were incubated at 37uC for 20 min. Finally, cells were washed twice in 20% sucrose, 50 mM Tris-HCl, pH 8.

Antibody labelling of E. coli cells and flow cytometric measurements
Unspecific antibody binding was prevented by incubation of E. coli cells in 100 ml 1 x PBS containing 1% BSA for 30 min at 4uC. Cells were collected by centrifugation at 10.000 g for 2 min and incubated in 100 ml 1 x PBS, 1% BSA containing anti-penta His antibody (Qiagen) at a concentration of 10 mg/ml at 4uC for 30 min. After washing three times with 1 x PBS, 1% BSA, immune complexes were labelled with 50 ml R-phycoerythrinconjugated goat F(ab9) 2 fragment anti-mouse IgG (1:50, Dianova) for 30 min at 4uC. After having been washed, cells were fixed in 1% PFA in PBS. Subsequently, flow cytometric measurements were carried out using a FACSCalibur flow cytometer (Becton Dickinson).

Statistical analysis
The significance of differences (P-value less than 0.01) between mean values was calculated by the Mann-Whitney U test.

Knockout of cj0268c in C. jejuni reference strain NCTC 11168 and functional complementation
In a previous report we detected gene cj0268c to contribute to the invasion of host cells in C. jejuni strain B2 [24]. Since strain B2 is highly invasive in human colon epithelial cells but fails to infect chicken cells or to colonize birds we wanted to confirm the invasion deficient phenotype in the C. jejuni reference strain NCTC 11168 [34]. This strain is capable of causing gastroenteritis in men as well as settling in livestock and, for this, enables us to test whether cj0268c has a defined function for colonization or infection of a particular host. After introduction of plasmid pBcj0268c-kanR to obtain NCTC 11168::cj0268c we restored the parental phenotype by transformation of the mutant strain with pRRC-cj0268c to obtain NCTC 11168::cj0268c-comp-cj0268c. A description of the genetic arrangement of cj0268c in strain NCTC 11168, the cj0268c knockout mutant, the complemented strain and the location of the primers is shown in Figure 1a.
In order to independently confirm the invasion deficient phenotype of C. jejuni strain B2Dcj0268c in reference strain NCTC 11168, we repeated the gentamicin protection assays on Caco2 cells with the wild type strain, the cj0268c-knockout mutant and the corresponding complemented strain. Thereby, the phenotype of strain B2Dcj0268c could be approved and, hence, the invasiondeficient phenotype demonstrated to be due to the functional loss of cj0268c in both, the C. jejuni strains B2 as well as in NCTC 11168 [24]. If the number of recovered NCTC 11168 wild type colonies is defined as 100%, we detected a mean value of cj0268c-mutant colonies of only 62% (p,0.0007). The percentage of obtained colonies from the complemented strain was 93% which was not significantly different from values of the parental NCTC 11168 strain (Fig. 2a). To address, whether the invasion-deficient phenotype due to the loss of cj0268c was restricted to human cells, we performed gentamicin protection assays with primary chicken cecal cells infected with strains NCTC11168, NCTC11168::cj0268c, and NCTC 11168::cj0268c-comp-cj0268c. In support of our data obtained with Caco2 cells, the invasion capacity of the cj0268c-mutant strain was significantly reduced compared to parental NCTC 11168 strain, but was completely restored in the complemented mutant. If the invasion capacity of wild type strain NCTC 11168 was defined as 100%, the percentage of colonies obtained from mutant strain NCTC 11168::cj0268c was only 62% (62.36, P,0.0007) but was reconstituted to 96% (64.96) by the intact gene in the complemented strain NCTC 11168::cj0268c-comp-cj0268c compared to NCTC 11168 (Fig. 2b).

Adherence
Given that cj0268c encodes a putative transmembrane protein, we next tested whether this protein is involved in the pathogenhost cell adherence process. Applying adhesion assays with wild type strain NCTC 11168, the cj0268c-mutant and the complemented mutant on Caco2 cells, we could decisively detect an adherence-deficient phenotype of NCTC 11168::cj0268c compared to NCTC 11168 which could be restored to wild type level in the cj0268c-complemented strain. When defining the number of colonies recovered from the parental strain NCTC 11168 as 100%, the mean value of corresponding colonies for the mutant NCTC 11168::cj0268c was 60.9% (65.18, P,0.0039) whereas the recovery rate of colonies for the complemented strain was 96.4% (61.77) which represents the wild type adhesion level (Fig. 3a).
To further phenotypically characterize the cj0268c-deficient mutant, we performed assays to test for altered motility or an affected capacity to autoagglutinate compared to the wild type strain and the complemented mutant. As shown in Figures 3b and 3c the mutant strain exhibited the same motility and autoagglu- tination properties as compared to NCTC 11168 wildtype or NCTC 11168::cj0268c-comp-cj0268c and, thereby, excluding a role of cj0268c regarding these characteristics.

E. coli strain DH5a expressing Cj0268c possesses an increased adherence to Caco2 cells
After cloning of cj0268c including a C-terminal His-tag in pRRC, we were able to detect recombinant Cj0268c expression also in E. coli DH5a even though in this vector the chloramphenicol resistance-mediating gene, as well as cj0268c is under control of the C. jejuni 16S promoter. Performing Western-Blot analysis with corresponding E. coli DH5a lysates and a monoclonal antibody against the His-tag, we determined a specific protein band of an approximate size of 41 kDa which represents the expected size of Cj0268c (Fig. 4a). To investigate if Cj0268c expressed in the heterologous context of E. coli intensifies its capability to interact with eukaryotic cells, we repeated the adherence assays with Caco2 cells. By defining the number of recovered E. coli DH5a colonies representing the parental strain as 100%, the relative recovery rate of live bacterial colonies from the Cj0268c-expressing E. coli was 201.7% (66.44). Hence, we could confirm the adherence-mediating phenotype of Cj0268c, indicating that Cj0268c does not necessarily need to interact with other  C. jejuni proteins, but by itself enhances adhesion properties in a heterologous host (Fig. 4b).

Localization of Cj0268c
In order to determine whether Cj0268c is localized at the bacterial surface we carried out flow cytometric measurements with non-permeabilized E. coli cells. After incubation of E. coli expressing a His-tagged version of Cj0268c with a monoclonal anti-His primary antibody followed by labelling with R-Phycoerythrin-conjugated secondary antibody, we could not detect any particular staining of the bacterial cell surface compared to E. coli bacteria that harboured plasmid pRRC without gene cj0268c (Fig. 5). Hence, at least the His-tagged C-terminus of Cj0268c is not localized at the bacterial surface. In contrast, when we permeabilized the bacterial cell wall with EDTA and lysozyme to allow access of the antibodies to the proteins of the periplasm, we could clearly detect immunolabelling of the E. coli population expressing Cj0268c. Whereas anti-His labelling was also obtained after labelling of permeabilized E. coli which had been transformed with the empty vector, the mean fluorescence intensity of antibody-labelled cells was considerably higher for Cj0268cexpressing cells as compared to controls (824.6 FLI units as compared to 484.9; Fig. 5). Control staining with the secondary antibody only of both Cj0268c-positive and negative E. coli confirmed the specificity of the FACS analysis. Together, these results indicate that the protein Cj0268c resides in the periplasmic space.

Resistance to bile salts and Triton X-100
Next we determined whether the presence of Cj0268c strengthens the resistance against bile salts and detergents. Strains NCTC11168, NCTC11168::cj0268c, and NCTC 11168::cj0268ccomp-cj0268c were incubated with different concentrations of cholate and deoxycholate ranging from 0.09 to 3%. Subsequent measurements of the optical density of the cultures revealed no differences between wildtype, cj0268c-mutant and its complement-ed mutant. Given that we were not able to detect any bacterial growth in the presence of bile salt concentrations exceeding 0.75%, further experiments were carried out only with cholate and deoxycholate concentrations up to 0.75%. However, the measurements of the optical densities in these experiments did not reveal any significant differences neither comparing the respective bacterial strains nor with respect to different bile salt concentrations tested (data not shown). Thus, these data suggest that Cj0268c does not exert a functional correlation with e.g. efflux pump systems such as CmeABC, for instance.
To find out whether Cj0268c has any influence on the stability of the bacterial cell wall, we incubated the cj0268c-deficient strain, its complemented version and NCTC 11168 wildtype strain in the presence of the nonionic surfactant Triton X-100 which is commonly used as a detergent to permeabilize cellular membranes. Thereby, the cj0268c mutant strain was much more sensitive to Triton X-100 as compared to wild type and complemented mutant. When we incubated the respective strains in a final concentration of 1% Triton X-100 for 1 h, subsequent plating onto Columbia blood agar yielded significantly fewer CFU of the cj0268c-mutant strain as compared to wild type strain NCTC 11168 and the cj0268c-complemented version. When defining the number of wild type colonies obtained after plating of the fourth dilution as 100%, the relative abundance of cj0268cmutant CFU yielded only a mean of 21.3% (Fig. 6), indicating that Cj0268c significantly contributes to the bacterial cell stability.
The cj0268c gene is ubiquitous in the C. jejuni population In recent studies multilocus sequence typing (MLST) analysis of defined genetic markers allowed the classification of different C. jejuni clonal groups. In addition, the combination of these genetic markers correlated, at least to some degree, with a corresponding animal source [35]. Moreover, after expansion of the MLST analysis by the inclusion of further gene markers, an association with a higher prevalence of campylobacterioses in humans or livestock adaption could be assigned regarding to the distribution of these markers [36]. We therefore studied next whether gene cj0268c fits into one of these clonal groups and, furthermore, could be associated with the C. jejuni settlement of particular animal groups or the severity of human campylobacteriosis. Altogether 56 isolates out of the 266 isolates used in the studies mentioned above from all clonal groups were screened for the presence of gene cj0268c by PCR. We were able to detect gene cj0268c in all isolates of C. jejuni indicating that cj0268c is not related to a distinct clonal group but rather seems to be ubiquitous in C. jejuni (not shown). Thus, a contribution of cj0268c to the C. jejuni settlement of specific animal groups or the clinical course of campylobacteriosis is unlikely.

Discussion
Screening of our transposon-generated mutant library of C. jejuni strain B2 revealed altogether seven genes that mediate a diminished invasion capacity towards Caco2 cells as shown by gentamicin protection assays [24]. Gene cj0268c which was further characterized here has been shown to be involved in host cell invasion earlier [23]. In order to investigate this gene regarding its biological function in further detail, we inactivated cj0268c in C. jejuni reference strain NCTC 11168. This strain, in contrast to strain B2, is able to infect both, human as well as chicken cells. After generation of a corresponding cj0268c-complemented NCTC 11168 strain, we could verify this gene to belong to a number of factors which are important for the adhesion to host cells by the pathogen. Furthermore, we confirmed this role of The numbers of colonies of the respective fourth dilution plated onto blood agar are shown. The cj0268c knockout mutant shows a clearly diminished resistance to Triton X-100. When defining the number of wild type colonies as 100%, the mean value of colonies recovered from the corresponding cj0268c-mutant was 21.3% (69.02, P,0.0039). After complementation of the mutant with an intact copy of cj0268c the percentage of colonies obtained increased to 91.3%. doi:10.1371/journal.pone.0081069.g006 Cj0268c by heterologous expression of cj0268c in E. coli strain DH5a. This result confirmed that Cj0268c possesses an adhesion mediating function alone and does not have to interact with other proteins of C. jejuni. On the other hand, an alteration of surface properties of E. coli after heterologous expression of Cj0268c leading to an indirect effect cannot be ruled out. Since we obtained equal CFUs of the E.coli populations independent of the expression of Cj0268c after incubation for 10 min in the presence of 1% Triton X-100, we could exclude alterations in bacterial stability caused by Cj0268c as the reason for different CFU numbers. However, since the adherence of C. jejuni to human and chicken cells depended exclusively on the presence or absence of Cj0268c irrespective of the specific host cell species, we determined Cj0268c as a protein for the mediation of adherence in general.
The chicken intestine represents a natural habitat for C. jejuni colonization and, hence, resistance to bile salts is essential for the pathogen's survival in such a hostile milieu. One of the resistance mechanisms exerted by C. jejuni employs the multidrug efflux pump system CmeABC, consisting of an outer membrane protein CmeC, a drug transporter CmeB, localized in the inner membrane, and periplasmic CmeB to connect CmaA and CmeC [37][38][39]. Since Cj0268c is a predicted transmembrane protein, interaction with CmeB to stabilize the CmeABC complex for instance was conceivable. However, after incubation of parental strain NCTC 11168 and its corresponding cj0268-mutant, the resistance to bile salts like cholate and deoxycholate exerted by the pathogen strains was virtually identical. Triton X-100 incubation of C. jejuni strains with mutated genes leading to an incomplete lipooligosaccharide metabolism had an inconsistent outcome reported so far. Whereas a mutation in the heptosyltransferase gene waaF (cj1148) decreased the stability of the C. jejuni cell wall, the resistance to Triton X-100 of the galactosysltransferase cj1136deficient mutant was not altered compared to the parental strain [40,41]. Nevertheless in the case of Cj0268c, incubation of the parental strain, the mutant and the complemented mutant with 1% Triton X-100 revealed that Cj0268c is important for maintaining integrity of the bacterial cell wall. Finally, although we could clearly demonstrate Cj0268c to possess an adherencemediating function and, therefore, contributes to the invasion process, the corresponding gene seems to be ubiquitous and not to belong to distinct clonal C. jejuni groups which could be related to pathogenicity according to earlier findings of [34,35].
The in vitro results presented here need to be further complemented by investigating the biological impact of the respective mutant strains in a suitable murine model mimicking human campylobacteriosis.