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Journal of Bacteriology, March 2007, p. 1856-1865, Vol. 189, No. 5
0021-9193/07/$08.00+0     doi:10.1128/JB.01427-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

CapA, an Autotransporter Protein of Campylobacter jejuni, Mediates Association with Human Epithelial Cells and Colonization of the Chicken Gut{triangledown}

Sami S. A. Ashgar,1,{dagger} Neil J. Oldfield,1,{dagger} Karl G. Wooldridge,1 Michael A. Jones,2,{ddagger} Greg J. Irving,1 David P. J. Turner,1,§ and Dlawer A. A. Ala'Aldeen1,§*

Molecular Bacteriology and Immunology Group, Institute of Infection, Immunity, and Inflammation, School of Molecular Medical Sciences, Queen's Medical Centre, University of Nottingham, Nottingham NG7 2UH,1 Institute for Animal Health, Compton, Berkshire RG20 7NN, United Kingdom2

Received 8 September 2006/ Accepted 8 December 2006


   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Two putative autotransporter proteins, CapA and CapB, were identified in silico from the genome sequence of Campylobacter jejuni NCTC11168. The genes encoding each protein contain homopolymeric tracts, suggestive of phase variation mediated by a slipped-strand mispairing mechanism; in each case the gene sequence contained frameshifts at these positions. The C-terminal two-thirds of the two genes, as well as a portion of the predicted signal peptides, were identical; the remaining N-terminal portions were gene specific. Both genes were cloned and expressed; recombinant polypeptides were purified and used to raise rabbit polyclonal monospecific antisera. Using immunoblotting, expression of the ca.116-kDa CapA protein was demonstrated for in vitro-grown cells of strain NCTC11168, for 4 out of 11 recent human fecal isolates, and for 2 out of 8 sequence-typed strains examined. Expression of CapB was not detected for any of the strains tested. Surface localization of CapA was demonstrated by subcellular fractionation and immunogold electron microscopy. Export of CapA was inhibited by globomycin, reinforcing the bioinformatic prediction that the protein is a lipoprotein. A capA insertion mutant had a significantly reduced capacity for association with and invasion of Caco-2 cells and failed to colonize and persist in chickens, indicating that CapA plays a role in host association and colonization by Campylobacter. In view of this demonstrated role, we propose that CapA stands for Campylobacter adhesion protein A.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Campylobacter jejuni is an important cause of human food-borne gastroenteritis that frequently colonizes poultry and contaminates poultry products (8). Despite the high prevalence and medical and economic importance of C. jejuni infection (16), fundamental aspects of the pathophysiology of colonization and infection remain poorly understood. The mechanisms by which C. jejuni initiates colonization, either in humans or in the avian host, are poorly understood, but the ability to adhere to cells lining the gut is thought to be an important prerequisite for successful colonization and infection. A number of campylobacter adhesins have been proposed, including lipooligosaccharide (38), flagellin (17), PEB1 (43), JlpA (25, 26), the major outer membrane protein (MOMP) (39, 48), P95 (31), and the fibronectin-binding protein CadF (35, 34), but the relative contributions of these adhesins to colonization and disease are unknown.

The autotransporter family of proteins shares a common defining mode of transport to the gram-negative outer membrane or external milieu and represents an extensive and rapidly growing family of virulence-related proteins ubiquitous among gram-negative bacteria (23). Despite sharing a common mode of secretion, autotransporter proteins possess highly diverse N-terminal functional domains (frequently referred to as "passenger" domains). Where functions have been attributed to autotransporter proteins, they have, almost without exception, been implicated in important aspects of the host-pathogen relationship including adhesion, toxigenicity, and intracellular spread (23). Although autotransporter proteins with a role in pathogenesis have been described for Helicobacter pylori (22), they have not previously been reported for the genus Campylobacter. Given the association of these proteins with pathogenesis, we screened the C. jejuni NCTC11168 genome sequence for putative autotransporter proteins. Here we report the identification and characterization of CapA and CapB, and we demonstrate that the former mediates adhesion to human-derived epithelial cells in vitro as well as in vivo colonization of the chicken intestine.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Bacterial strains, growth conditions, and reagents. C. jejuni strain NCTC11168 was kindly provided by J. Ketley (Leicester, United Kingdom). Fresh human fecal isolates of C. jejuni (labeled AVO1 to AVO11) were obtained with minimum laboratory passage (maximum, three subcultures) from the Clinical Microbiology Department, Queen's Medical Centre, Nottingham, United Kingdom (Table 1). Multilocus sequence-typed (MLST) strains were obtained from D. Wareing (Preston, United Kingdom) (Table 1). All Campylobacter isolates were grown under microaerophilic conditions at 37°C on modified CCDA (charcoal-cefoperazone-desoxycholate agar), on blood agar, or in Mueller-Hinton (MH) broth (Oxoid). Escherichia coli strains BL21(DE3)pLysS (Invitrogen), JM109, and JM83 (Promega) were used to express the maltose-binding protein(MBP) fusion proteins. Globomycin was a gift from S. Miyakoshi, Sankyo Co., Ltd., Tokyo, Japan. Culture media were supplemented with 10 µg/ml chloramphenicol or 50 µg/ml kanamycin where appropriate to select for antibiotic-resistant bacteria.


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  		TABLE 1. C. jejuni strains used in this study

 
Cloning and mutagenesis. A truncated capA gene (corresponding to the Cj0629 open reading frame [ORF]; amino acids [aa] 243 to 1144 of the in silico-reconstructed CapA sequence) encoding a protein with a calculated molecular mass of ca. 102 kDa (compared to the full-length CapA protein of ca. 116 kDa) was amplified by PCR using genomic DNA from C. jejuni NCTC11168 and the Cj0629-specific primers 5'-GGATCCATGGGTGTAAATGTTCGTTC-3' and 5'-GTCGACTTACCAAAGATAATTAAACTGAGC-3'. The resulting PCR product was ligated into pCRT7/NT-TOPO (Invitrogen) according to the manufacturer's instructions. The same PCR product was also ligated into pGEM-T Easy (Promega) and subcloned into pQE30 (QIAGEN) or pMAL-C2x (New England Biolabs). To construct capA and capB isogenic single- and double-mutant strains, the genes were disrupted by insertion of chloramphenicol (62) or kanamycin (54) resistance cassettes into the respective amplified genes. A naturally occurring KpnI site located 979 bp downstream of the putative ATG initiation codon of Cj0628 was used to disrupt this gene. To disrupt capB, a unique KpnI site was introduced by inverse PCR (61) using the capB-specific primers (5'-CGGGGTACCTCAAGCTAAGATCAATAC-3' and 5'-CGGGGTACCTGAGGGCTATAAACAC-3') 191 bp downstream of the putative ATG start codon in Cj1677. The resulting constructs were used to mutate C. jejuni NCTC11168 by natural transformation and allelic exchange as described previously (55). PCR analysis and nucleotide sequencing confirmed the insertion of the appropriate cassette into the correct locus (data not shown). DNA sequencing was carried out using the BigDye cycle sequencing system (ABI Prism; PE Biosystems) in the School of Biomedical Sciences, University of Nottingham.

Expression and purification of recombinant proteins. E. coli JM83 cells containing either pMAL-trCapAB, pMAL-trCapA, or pMAL-trCapB, encoding a region of capA common to both genes (aa 243 to 1144), a capA gene-specific region of capA (aa 38 to 372), or a capB gene-specific region of capB (aa 38 to 349), respectively, were grown in Luria broth to mid-log phase. After induction, the cells were grown for 4 h and harvested by centrifugation. Recombinant fusion proteins were affinity purified using amylase-resin columns (New England Biolabs) and further purified by electroelution from preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels as previously described (19).

Production of a rabbit antiserum against purified recombinant proteins. New Zealand White female rabbits were immunized four times at 2-week intervals with ca. 30 µg of purified MBP-trCapAB, MBP-trCapA, or MBP-trCapB emulsified in Freund's complete (first immunization only) or incomplete adjuvant. The animals were test bled 7 days after the third dose and sacrificed 10 days after the final inoculation. Preimmune sera were obtained prior to immunization. Before use in immunoblotting experiments and immunogold electron microscopy, the sera were preadsorbed overnight at 4°C with a 20% (vol/vol) suspension of 11168capA (for rabbit antiserum against CapAB [R{alpha}CapAB] or R{alpha}CapA) or 11168capB (for R{alpha}CapB) cells grown in MH broth before being cleared by centrifugation and filtration as previously described (51). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting were performed as previously described (1, 33).

Preparation of RNA and RT-PCR. Total cellular RNA was extracted from exponentially growing cultures by using the RNeasy minikit (QIAGEN) according to the recommendations of the manufacturer. RNA preparations were treated with RQ1 RNase-free DNase (Promega) to remove traces of contaminating genomic DNA, after which each preparation was quantified using a NanoDrop microscale spectrophotometer (NanoDrop Technologies). Reverse transcriptase PCR (RT-PCR) was performed using the Omniscript reverse transcriptase kit (QIAGEN) according to the recommendations of the manufacturer. Twenty-microliter reaction mixtures contained 12 µl RNase-free water, 2 µl of 10x RT buffer, 2 µl of 10 mM deoxynucleoside triphosphate mix, 2 µl of 10 µM antisense primer, 1 µl of RNase inhibitor, and 1 µl of Omniscript reverse transcriptase. Antisense primers were 5'-GTCGACTTACCAAAGATAATTAAACTGAGC-3' or 5'-GTCGACGATTACCAAAGATAATTAAACTGAGC-3' for amplification of capA and capB, respectively. Samples were incubated for 60 min at 37°C and then heated at 93°C for 5 min to inactivate the reverse transcriptase. One microliter of the cDNA preparations was used in a subsequent PCR amplification reaction. The 50-µl reaction mixture contained 0.2 µM 5'-GGATCCGCAATAGGAGTGGGTC-3' and 0.2 µM 5'-GTCGACGTTTCCACTTCCATTGG-3' for capA or 0.2 µM 5'-GACGGATCCGGAACTATTAGTGG-3' and 0.2 µM 5'-GTCGACGTTTGTATTGACTCCATAAG-3' for capB, 0.2 µM deoxynucleoside triphosphate mix, 1x PCR buffer (Roche), 0.25 mM MgCl2, and 4 U Biotaq (Roche). The reaction conditions were as follows: 95°C for 2 min, followed by 35 cycles of 94°C for 30 s, 55°C for 30 s, and 68°C for 60 s, and a terminal extension step at 68°C for 5 min using a Techne Progene thermal cycler. PCR products were then analyzed by gel electrophoresis using ethidium bromide staining.

Subcellular fractionation. C. jejuni cells were grown overnight and harvested by centrifugation for 10 min at 11,000 x g. The supernatants were filtered through a 0.2-µm-pore-size Minisart syringe filter (Sartorius) and concentrated approximately 100-fold using a Vivaspin-2 protein concentrator (molecular weight cutoff, 30,000; Vivascience) according to the manufacturer's instructions. The concentrated cell-free supernatant preparations represented the secreted-protein fraction. Cell pellets were resuspended in phosphate-buffered saline (PBS) and disrupted by sonication using an MSE Soniprep 150. After removal of unlysed cells by centrifugation at 250 x g for 2 min, the supernatant was transferred to a new tube and centrifuged at 11,000 x g for 30 min. The supernatant (cytosolic and periplasmic fractions) was transferred to a new tube, and the pellet was resuspended in 25 µl of PBS by vigorous pipetting. Twenty-five microliters of 4% Triton X-100 in PBS was added, and the mixture was incubated at 37°C for 30 min. Triton-insoluble proteins (OMP-enriched fraction) were harvested by centrifugation at 11,000 x g for 30 min. The final supernatant (cytoplasmic membrane protein-enriched fraction) was collected. To determine whether translocation of CapA to the outer membrane was dependent on signal peptidase II, cells were grown at 37°C to mid exponential phase (optical density at 600 nm, 0.5), pelleted by centrifugation at 11,000 x g for 5 min, and resuspended in 10 ml fresh MH broth. The culture was split into two 5-ml tubes, and globomycin (dissolved in methanol) was added to one tube at a final concentration of 300 µg/ml. Tubes were incubated for 5 h at 37°C with shaking, and bacteria were collected by centrifugation at 11,000 x g for 15 min and used to prepare outer membranes as described above. The experiment was repeated independently twice.

Electron microscopy and immunogold labeling. C. jejuni cells were grown in 5 ml of MH broth for 2 h at 37°C and prepared for immunogold staining and electron microscopy essentially as previously described (51). Preadsorbed R{alpha}CapA was used as the primary antibody, and preimmune serum from the same rabbit was used as the negative control. The secondary antibody was anti-rabbit immunoglobulin G (IgG) (whole molecule) conjugated to 5-nm gold particles (Sigma). Cells were viewed using a JEOL JEM1010 electron microscope. The numbers of gold particles associated with 25 randomly selected organisms were counted, and the results were tested for statistical significance using a Student t test.

In vitro association and invasion assays. Association and invasion assays were performed essentially as previously described (58). Briefly, Caco-2 cells were grown to confluence in Dulbecco's modified eagle medium (DMEM; Invitrogen) containing 10% fetal calf serum in 24-well tissue culture plates (Costar) at 37°C under an atmosphere of 5% CO2. C. jejuni cells grown overnight in MH broth and resuspended in DMEM were added to wells at 109 CFU per well in 1 ml of DMEM containing 2% fetal calf serum, and plates were incubated at 37°under an atmosphere of 5% CO2 for 2 h. To assess total cell association, monolayers were washed twice with 1 ml PBS per well. To assess invasion, the washed monolayers were further incubated with DMEM containing 100 µg/ml gentamicin for 1 h and then washed twice with 1 ml PBS. In either case, the monolayers were then disrupted by addition of 1 ml 0.5% sodium deoxycholic acid in PBS, and the suspension was homogenized by repeated pipetting. Campylobacter cells were enumerated by serial dilution of the homogenized suspensions on chocolate agar. All assays were repeated three times in duplicate. Statistical significance was measured using the Student t test.

Chicken colonization. Colonization trials were carried out as described by Velayudhan et al. (56). Day-of-hatch specific-pathogen-free Rhode Island Red chicks were obtained from the Poultry Production Unit, Institute for Animal Health, Compton, United Kingdom, and all the birds were inoculated orally with 0.1 ml of Campylobacter-free adult gut flora preparations. Our previous experience has shown that this pretreatment allows greater reproducibility in bacterial colonization experiments. Birds were housed under appropriate specific-pathogen-free conditions until 2 weeks of age, when they were used in colonization trials. Birds were fed on a vegetable-based diet (Special Diet Services, Manea, Cambridgeshire, United Kingdom), which was made freely available to the animals for the duration of the trial. Groups of 20 3-week-old birds with the developed gut flora were inoculated orally with 0.1 ml of culture containing 107 CFU of the desired Campylobacter strain. Cloacae were sampled at weekly intervals with sterile cotton-wool swabs, and fecal excretion was assessed semiquantitatively using a standard method for large groups of birds housed together (49). Cloacal swabs were washed in 2 ml of modified Exeter broth (4); these swabs were then streaked in a standard manner onto Campylobacter blood-free selective agar (Oxoid Ltd., United Kingdom), and colony counts were scored positive (greater than 1 CFU; direct counts) or negative after 2 days of incubation. Swabs were returned to the enrichment medium and incubated under microaerobic conditions at 37°C. After 2 days, the enrichment cultures were checked for Campylobacter by plating on CCDA. These were scored as positive or negative results and are referred to as enrichment counts. At 6 weeks postinfection, the birds were killed humanely, and postmortem bacterial counts were determined from the cecal contents of five birds from each group. Cecal contents were standardized by dilution to 1 g/ml in PBS prior to carrying out decimal serial dilutions. These were plated on Campylobacter blood-free selective agar to determine Campylobacter numbers. Samples of intestinal contents were also placed in modified Exeter enrichment medium for 48 h and then plated out to assess the presence or absence of Campylobacter cells if counts were below the detectable limit.

Southern blotting. Southern blotting was performed using GeneImages random prime labeling and CDP-Star detection modules (Amersham Biosciences) according to the recommendations of the manufacturer. Approximately 50 to 100 µg of genomic DNA was digested overnight with HindIII at room temperature, and samples were separated on a 0.8% agarose gel. To prepare capA- and capB-specific probes, primers 5'-CGGCAATACTCTAGTTATAG-3' and 5'-CATTACCTATGATACCTTG-3' and primers 5'-GGAACATCAAATTCTTTAAC-3' and 5'-GTTCCTGAGTTGTTACCC-3', respectively, were used.

Bioinformatic searches and sequence analysis. To identify novel C. jejuni autotransporter proteins, the amino acid sequences of the known autotransporters AIDA-I (5), Hap (24), Hia (3), IgA1 protease (45), Pic (21), Pet (15), and AspA (51) were used to search the predicted coding sequences of C. jejuni NCTC11168 using the BLAST server available at www.sanger.ac.uk/Projects/C_jejuni/. Protein sequences identified by this search were examined for typical characteristics of autotransporter proteins (23). Protein sequences of the predicted passenger domains (CapA aa 37 to 862 and CapB aa 37 to 839) were used to perform BLASTP searches of the nonredundant protein database (nrdb95) and the Omniome pep database using the WU-BLAST2 servers at http://dove.embl-heidelberg.de/Blast2/ and http://tigrblast.tigr.org/cmr-blast/, respectively. The Neural Network Promoter Prediction (www.fruitfly.org/seq_tools/promoter.html) program was used to examine DNA segments for potential promoter sequences. Ribosome-binding sites were predicted using the Glimmer (version 3.02) server available at http://www.ncbi.nlm.nih.gov/genomes/MICROBES/glimmer_3.cgi, and the output was interrogated using the RBSFinder program (downloaded from ftp://ftp.tigr.org/pub/software/RBSfinder/). The signalP program (www.cbs.dtu.dk/services//SignalP#submission) was used to predict the presence of putative signal peptides, and the Prosite (www.expasy.ch/prosite/) and Pfam (http://pfam.wustl.edu/) databases were used to identify conserved amino acid motifs. Nucleotide sequence data were analyzed using the DNAMAN package (Lynnon Biosoft).


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Identification and sequence analysis of two Campylobacter autotransporter proteins. To identify novel C. jejuni autotransporter proteins, the amino acid sequences of known adhesion-associated and other well-characterized autotransporters were used to search the predicted coding sequences of C. jejuni NCTC11168. Two genes (Cj0628/Cj0629 and Cj1677/Cj1678) identified by this search were found to encode proteins with all of the typical characteristics of autotransporter proteins (23) (Fig. 1). The genes were designated capA (Cj0628/Cj0629) and capB (Cj1677/Cj1678), respectively. In the annotated sequence released by the Sanger Institute (41), each gene was assigned two separate ORFs because of the presence of premature stop codons downstream of homopolymeric tracts. In the case of capA, there is a 5-bp poly(T) tract immediately followed by a 10-bp poly(G) tract between nucleotides 497 and 511. The deletion of a single residue from either tract places the two adjacent coding sequences Cj0628 and Cj0629 within a single ORF. In the case of capB, there is a 7-bp poly(T) tract between nucleotides 578 and 584. Deletion of a single residue from this tract similarly places the adjacent coding sequences Cj1677 and Cj1678 within a single putative gene. Promoter sequences and ribosome-binding sites are predicted to be present upstream of Cj0628 and Cj1677, but not Cj0629 or Cj1678, supporting the hypothesis that the two pairs of ORFs constitute a single gene each.


Figure 1
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  		FIG. 1. Schematic diagram of the predicted protein sequences of CapA (Cj0628/Cj0629) and CapB (Cj1677/Cj1678). Dark shading, signal peptidase II-dependent signal peptides (SPII); light shading, passenger domains; hatched boxes, ß-domains. Predicted signal peptidase II cleavage sites are indicated. Both the Cj0629 and Cj1678 predicted protein sequences end with the motif "YLW," which conforms to the typical autotransporter motif (Y/V/I/F/W)-X-(F/W) (23). Vertical stripes, regions of identity between CapA and CapB. Solid arrows represent the ORFs encoding each protein; homopolymeric tracts resulting in frameshifts are also indicated. Unless otherwise indicated, numbers refer to amino acids.

 
Cj0628 and Cj1677 are both predicted to encode proteins with signal peptidase II domains containing the putative lipid attachment motif 34LAS-37C (28). Although the SignalP algorithm predicts a cleavage site at LASCTHA{downarrow}41T, the presence of the lipid attachment site suggests (by analogy with other lipoproteins) that the actual cleavage site may be at LAS{downarrow}37C (28). It is also noteworthy that 37C is the last of a stretch of 20 residues that are conserved between CapA and CapB. Cleavage at this site would result in the residue at +2 being threonine. In E. coli, lipoproteins are localized to either the inner or the outer membrane depending on the residue at +2. Aspartate at this position, in combination with certain residues at position 3, functions as an inner membrane retention signal (20). The presence of threonine at position 2 would not constitute such a signal, suggesting an outer membrane location for both CapA and CapB (20).

The in silico-reconstructed capA and capB genes encode deduced proteins of 1,144 and 1,121 amino acids and calculated molecular masses of 120 and 118 kDa, respectively. After cleavage of the predicted signal sequences, the molecular masses would be 116 and 114 kDa, respectively. Both proteins are predicted to be weakly acidic, with pIs of 6.8 and 5.7, respectively.

The C-terminal two-thirds of the two protein sequences (extending from 375F in CapA and 349F in CapB) are identical (Fig. 1). By contrast, the N-terminal regions of CapA and CapB are highly divergent except for 20 amino acids immediately preceding the likely signal peptidase II cleavage site. The C-terminal region (residues 862S to 1144W in CapA) contains the predicted autotransporter ß-barrel domain (PROSITE accession no. PS51208) terminating in the "YLW" motif, which conforms to the typical autotransporter motif (Y/V/I/F/W)-X-(F/W) (23). Apart from two cysteine residues in the predicted signal peptide, no other cysteine residues are present in either protein, which is in keeping with an observed low cysteine content of known autotransporter proteins (23).

Analysis of the DNA sequence flanking the capA gene revealed an upstream gene encoding a probable hydrogenase isoenzyme (hypA) and a downstream gene encoding a 321-amino-acid conserved hypothetical protein. The hypA gene is in the same orientation as capA but is separated from it by an 875-bp intergenic sequence containing a 14-bp inverted repeat, suggesting that they are transcriptionally independent. The downstream hypothetical gene is in the opposite orientation to capA, again suggesting that capA is likely to be transcriptionally independent of its neighboring genes. The capB gene lies between murB upstream, encoding a putative acetylenopyruvoylglucosamine reductase, and a downstream gene encoding a 584-amino-acid conserved hypothetical protein. A stem-loop can be found between murB and capB, suggesting that capB and murB are transcriptionally unlinked. Although the gene downstream of capB is predicted to have its own promoter sequence, no potential transcriptional stop signals could be identified between capB and this gene, leaving open the possibility that the two genes are transcriptionally linked.

BLAST searches of available databases with the CapA passenger domain demonstrated similarity with a number of surface proteins, including the AIDA-I autotransporter adhesin (P = 3.8e-6) (5), the surface array protein of Campylobacter fetus (P = 2.0e-5) (6), and the putative autotransporter TapA of Acidithiobacillus ferrooxidans (accession number AJ277640; P = 5.9e-13). A similar search with the passenger domain of CapB demonstrated similarity with a hemagglutinin-related protein of Ralstonia solanacearum (accession number AL646074; P = 3.3e-17) (47), the TapA protein (accession number AJ277640; P = 1.7e-12), and a number of mycobacterial membrane proteins of the PPE (pentapeptide repeat) family (e.g., accession number CAD93639; P = 8.8e-7) (9).

Cloning, mutagenesis, expression, and purification of CapA and CapB. Single-knockout mutants for capA and capB (11168capA and 11168capB, respectively), as well as a double mutant (11168capAcapB) in which both genes were disrupted, were constructed to serve as controls for expression studies and for phenotypic studies. Each gene was disrupted by insertion of either a kanamycin or a chloramphenicol resistance cassette toward the beginning of the structural gene. Successful insertion of the respective antibiotic genes at the intended locations was confirmed by PCR and sequencing (data not shown).

Because capA and capB possess shared as well as gene-specific regions, and in an attempt to avoid possible problems with slipped-strand mispairing at the homopolymeric tracts, we cloned and expressed three separate fragments: the entire downstream ORF of CapA (Cj0629), which corresponds to aa 243 to 1144 of the in silico-reconstructed CapA sequence and contains sequences common to capA and capB; the gene-specific region of capA, which corresponds to aa 38 to 372 of the in silico-reconstructed CapA sequence; and the gene-specific region of capB, which corresponds to aa 38 to 349 of the in silico-reconstructed CapB sequence (Fig. 1). Each fragment was cloned into the pMAL-c2X vector to produce plasmids pMAL-trCapAB, pMAL-trCapA, and pMAL-trCapB, respectively, in order to express MBP fusions of each truncated protein. After induction of E. coli cells harboring pMAL-trCapAB, a recombinant protein (MBP-trCapAB) with a molecular mass of ca. 140 kDa was strongly expressed. This was consistent with the predicted molecular weight of MBP fused to the CapAB common region. The MBP-trCapAB fusion protein was affinity purified using amylase-resin columns and further purified by electroelution from sodium dodecyl sulfate-polyacrylamide gels before being used to raise rabbit antibodies against both CapA and CapB (R{alpha}CapAB). The antiserum recognized proteins with molecular masses of 140 kDa and 115 kDa in lysates of E. coli expressing the recombinant protein; these were assumed to be the full-length recombinant fusion protein and a breakdown product of this protein (Fig. 2, lane 1). Recombinant proteins containing the gene-specific regions of CapA and CapB were expressed and purified in the same way from pMAL-trCapA- and pMAL-trCapB-containing E. coli cells, respectively, and used to generate the CapA-specific antiserum R{alpha}CapA and the CapB-specific antiserum R{alpha}CapB. Both antisera strongly recognized proteins of the expected size in lysates of E. coli expressing the corresponding protein (data not shown).


Figure 2
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  		FIG. 2. Expression of MBP-trCapAB. Lysates of E. coli cells harboring pMAL-trCapAB (lane 1) or pMAL-c2X (lane 2) and induced with isopropyl-ß-D-thiogalactopyranoside (IPTG) were probed in immunoblotting experiments with rabbit antiserum raised against the recombinant fusion protein after purification by gel elution. This antiserum recognized proteins with apparent molecular masses of ca. 140 and ca. 115 kDa. Protein sizes (in kilodaltons) are given on the right. Positions of molecular mass markers are given on the left.

 
CapA but not CapB is expressed in Campylobacter. To determine whether CapA and/or CapB is naturally expressed in C. jejuni, we probed whole-cell extracts of C. jejuni with R{alpha}CapAB in immunoblotting experiments. A single ca. 116-kDa protein (consistent with the expected molecular weight of the mature CapA protein following cleavage of the putative signal peptide) was observed in the wild-type (WT) C. jejuni strain NCTC11168 but not in 11168capA (Fig. 3, lanes 1 and 2). Expression of the ca. 116-kDa protein was also observed in strain 11168capB but not in strain 11168capAcapB (data not shown). When these strains were probed with the R{alpha}CapA antiserum, an identical profile was observed (data not shown). By contrast, no proteins were detected in cell extracts of 11168, 11168capA, 11168capB, or 11168capAcapB when these strains were probed with R{alpha}CapB. These data indicate that while CapA was consistently expressed in strain NCTC11168, there was no detectable expression of CapB under the culture conditions used.


Figure 3
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  		FIG. 3. Detection of CapA protein in C. jejuni. R{alpha}CapAB recognizes a protein of ca. 116 kDa in whole-cell preparations of strain NCTC11168 (lane 1) and in four heterologous clinical isolates (lanes 3 to 6) but not in 11168capA (lane 2). A protein of the same size was also detected in extracts from isolates obtained from chickens experimentally inoculated with strain NCTC11168 at 2 (lane 7), 3 (lane 8), and 4 (lane 9) weeks postinoculation and in postmortem isolates (lane 10). No protein was detected in extracts of isolates from chickens inoculated with strain 11168capA at 1 week (lane 11) or 2 weeks (lane 12) postinoculation.

 
Southern blotting and immunoblotting were used to assess the presence of the capA and capB genes and the expression of the encoded proteins across a panel of strains. Southern blotting revealed that 2/8 MLST human isolates and 8/11 recent clinical isolates contained the capA gene (Table 1). Both capA-containing MLST isolates but only 4/8 capA-containing recent clinical isolates produced a ca. 116-kDa protein corresponding to CapA (Fig. 3). Similar experiments on capB showed that while none of the 8 MLST human isolates and 4 of 11 recent clinical isolates contained the capB gene (Table 1), no immunoreactive proteins were detected when the same panel of isolates was probed with R{alpha}CapB (data not shown).

To determine whether capA and capB were transcribed, the WT strain NCTC11168 and its mutant derivatives were examined for mRNA transcription using RT-PCR. PCR amplification using primers specific for capA produced products of the expected size when genomic DNA or RNA treated with RT from strain NCTC11168 or 11168capB was used as a template. No capA-specific products were amplified from strain 11168capA or 11168capAcapB. Control amplifications using RNA in which RT was omitted did not yield products from strains NCTC11168 and 11168capB, confirming the absence of contaminating genomic DNA from the RNA preparations (data not shown). Primers specific for capB amplified a product from the genomic DNA from strains NCTC11168 and 11168capA but not from 11168capB or 11168capAcapB. No capB-specific products were amplified from any of the strains either after treatment with RT or in a control reaction in which RT was omitted (data not shown). Thus, expression of capA could be demonstrated for strains NCTC11168 and 11168capB but not for strain 11168capA or 11168capAcapB. We were unable to detect expression of capB in any of the strains tested (data not shown).

CapA is an outer membrane, surface-exposed protein. To determine the subcellular localization of CapA in C. jejuni, cells of C. jejuni strain NCTC11168 or 11168capA were fractionated, and the resulting fractions were probed in immunoblotting experiments with R{alpha}CapAB. The ca. 116-kDa immunoreactive protein, assumed to be mature CapA, was detected predominantly in the outer membrane-enriched fraction, with a relatively small amount in the cytoplasmic membrane-enriched fraction, but was absent from the soluble (pooled cytoplasmic and periplasmic) fraction and from concentrated culture supernatants (secreted-protein fraction) (Fig. 4A). Immunoblotting experiments with antisera against JlpA, a known outer membrane protein of C. jejuni (25), gave an identical profile, confirming that CapA is an outer membrane protein (data not shown).


Figure 4
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  		FIG. 4. Subcellular localization of CapA. (A) Secreted proteins (lanes 1 and 2), outer membrane protein-enriched fractions (lanes 3 and 4), cytoplasmic membrane protein-enriched fractions (lanes 5 and 6), and soluble intracellular proteins (lanes 7 and 8) of either NCTC11168 (odd-numbered lanes) or 11168capA (even-numbered lanes) were separated on a 10% acrylamide gel and probed in immunoblotting experiments with R{alpha}CapAB. (B and C) Samples of outer membrane protein-enriched fractions from C. jejuni NCTC11168 grown in the absence (lane 9) or presence (lane 10) of globomycin (and adjusted to ensure equal protein loading) were also probed with R{alpha}CapAB (B) and R{alpha}JlpA (C).

 
To reinforce the prediction that CapA is a lipoprotein, WT C. jejuni NCTC11168 and 11168capA cells were grown to exponential phase in the presence or absence of globomycin, a specific inhibitor of lipoprotein signal peptidase II (13). Levels of CapA and the known lipoprotein JlpA were significantly reduced in outer membrane-enriched fractions (Fig. 4B and C) and increased in inner membrane fractions (data not shown) of WT cells grown in the presence of globomycin, whereas levels of MOMP in the inner and outer membranes (data not shown) were not affected by globomycin treatment, thus indicating that CapA is likely to be a lipoprotein.

Immunogold labeling and electron microscopy were used to determine whether CapA is accessible on the bacterial cell surface. WT C. jejuni NCTC11168 cells were strongly immunogold labeled (800 to 1,200 gold particles per cell; 25 bacterial cells counted) in contrast to 11168capA cells (115 to 170 gold particles per cell; 25 bacterial cells counted). This difference was statistically significant (P < 0.001), confirming the surface accessibility of CapA (Fig. 5).


Figure 5
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  		FIG. 5. Immunogold electron microscopy of C. jejuni NCTC11168 (A) or 11168capA (B) probed with R{alpha}CapA followed by gold-labeled goat anti-rabbit IgG. WT (but not capA mutant) cells are strongly labeled. Bars, 500 nm.

 
CapA plays a role in association and invasion of human cells in vitro. In view of CapA's similarity to adhesins of other pathogens, it was important to determine whether it had any role in the interaction of C. jejuni with human cells. WT cells of strain NCTC11168, capA and capB mutant derivatives, and a capA capB double mutant were tested for their abilities to associate with and invade monolayers of the human enterocyte-like cell line Caco-2. The mutant strain 11168capA and the double-mutant strain 11168capAcapB both had significantly reduced propensities to associate with Caco-2 cells in vitro, and even greater reductions in the ability to invade these cells were observed (Fig. 6). By contrast, the mutant strain 11168capB associated with and invaded Caco-2 cells at levels similar to those of the WT parent (Fig. 6). Although the invasive capacity of the 11168capAcapB strain appears to be reduced compared to that of the 11168capA strain (Fig. 6B), this difference was not statistically significant (P > 0.05). Furthermore, the observed differences in association and invasion were not the result of impaired growth characteristics, since growth curve assays carried out using liquid cultures showed no significant differences between any of the mutant strains and the WT (data not shown).


Figure 6
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  		FIG. 6. CapA is required for maximal cell association and invasion of Caco-2 cells by C. jejuni strain NCTC11168. Cells of C. jejuni strain NCTC11168, 11168capA, 11168capB, or 11168capAcapB were added to differentiated monolayers of Caco-2 cells and incubated for 2 h. After a wash, monolayers were disrupted with sodium deoxycholate, and viable bacteria were enumerated by plate counting (A). Gentamicin protected bacteria were similarly enumerated after an additional incubation for 1 h in the presence of gentamicin (B). Experiments were performed in quadruplicate. Error bars represent 1 standard deviation. Similar results were obtained in two independent experiments. The reductions in both association and invasion compared to that for the wild type were statistically significant (P < 0.05) for the 11168capA and 11168capAcapB strains. The apparent difference in the invasive capacity of the 11168capAcapB strain compared to the 11168capA strain (B) was not statistically significant (P > 0.05).

 
CapA is required for efficient chicken colonization by strain NCTC11168. To examine the role of CapA in chicken colonization, two groups of 20 chickens each were exposed to the WT (but laboratory-adapted) C. jejuni strain NCTC11168 and its capA mutant derivative (11168capA). For the first 2 weeks of the trial, the WT organism was detectable (in most cases only after enrichment) in around two-thirds of the infected birds. From week 3 postinfection, the WT was able to heavily colonize 100% of the chickens and was detectable by direct plating without enrichment, yielding confluent growth on agar media (Fig. 7). In contrast, the 11168capA mutant strain was detected, in small numbers (and usually only after enrichment), in only one-quarter of infected birds for 2 weeks, after which no viable Campylobacter could be detected by direct plating or after enrichment (Fig. 7). Postmortem analysis of birds at 6 weeks postinfection showed that the WT strain colonized at 6.35 x 108 ± 2.3 x 108 CFU/g of cecal contents. We were unable to recover any of the capA mutants from infected birds at this time.


Figure 7
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  		FIG. 7. Chicken colonization experiment comparing the WT C. jejuni strain NCTC11168 (hatched and solid bars) and its isogenic capA mutant (open and shaded bars). Detection by direct culture (solid and shaded bars) or after enrichment (hatched and open bars) is indicated. Each group consisted of 20 birds.

 
Expression of CapA but not CapB after adaptation to the chicken gut. Cell extracts of sequential isolates obtained at 2, 3, 4, and 6 weeks postinoculation were probed with R{alpha}CapAB in immunoblotting experiments. All isolates tested consistently expressed CapA but not CapB (Fig. 3). Identical profiles were obtained when the same extracts were probed with R{alpha}CapA, but no protein expression could be detected when extracts were probed with R{alpha}CapB (data not shown). RT-PCR analysis confirmed capA transcription and a lack of detectable capB transcription in these trial isolates (data not shown). Isolates obtained from chickens infected with the 11168capA mutant strain (obtained at week 2 postinoculation) failed to transcribe or express CapA or CapB (Fig. 3).


   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
This is the first report of the identification and functional characterization of a Campylobacter autotransporter. We identified two genes in silico, here designated capA and capB, encoding potential autotransporter proteins. The former expresses a surface-exposed protein that mediates association with and invasion of human epithelial cells. It also plays an important role in colonization of the chicken gastrointestinal tract (GIT). A role for the related CapB protein could not be demonstrated, since we were unable to detect its transcription or expression in strain NCTC11168 either in vitro, after in vivo passage, or in a range of human isolates.

Detailed bioinformatic analysis indicated that both CapA and CapB possess the characteristic molecular features that are the signature of autotransporter proteins (23). Both proteins were also homologous to a number of known autotransporter proteins, including adhesins of other gram-negative pathogens. Our data show that CapA is transported to the outer membrane and is surface exposed, but it is not released from the cell surface. This is consistent with a role for CapA as a bacterial adhesin. Bioinformatic analysis also predicted that CapA and CapB were lipoproteins. This prediction was reinforced for CapA by inhibiting its translocation to the bacterial outer membrane using the lipoprotein signal peptidase inhibitor globomycin (13). However, we cannot exclude the possibility that indirect effects of globomycin on other lipoproteins, which might be required for the stability and/or export of CapA, may be partly responsible for this effect. Lipidation of autotransporter proteins is relatively unusual and has been confirmed only for the subtilisin autotransporters, SphB1 in Bordetella pertussis (11) and AspA (NalP) in Neisseria meningitidis (51, 52). AlpA, an autotransporter in Helicobacter pylori, also possesses an apparently functional lipoprotein motif, although lipidation of the protein has not been demonstrated (40). Similarly, two members of the putative autotransporter family Pmp in Chlamydia pneumoniae contain signal peptidase II cleavage sites suggestive of lipid modification (18). It is likely that lipidation of CapA is required to anchor this protein to the outer membrane, as has been described for SphB1 (10).

A role for CapA as an adhesin was identified in vitro using the well-established Caco-2 cell model for campylobacter interaction with human enterocytes (58). Since the genome of C. jejuni strain NCTC11168 contains genes encoding several known adhesins, including PEB-1, JlpA, and MOMP, it is perhaps not surprising that ablation of an additional putative adhesin did not dramatically reduce association or cellular invasion in this model. Nevertheless, there was a significant reduction in both parameters for the capA mutant strain compared to its WT parent, indicating that capA has a role in association with human epithelial cells. In view of this demonstrated role, we propose that CapA stands for Campylobacter adhesion protein A. Future studies involving complementation will be required to confirm that this phenotypic effect was not due to an unanticipated effect on another gene.

Successful colonization of the chicken GIT is a multifactorial process. Mutations in genes involved in temperature regulation (7), the heat shock response (63), bile tolerance (36, 46), iron transport (12) and regulation (53), serine catabolism (56), and glycosylation (29, 32) have all been shown to reduce or abolish the ability of C. jejuni to colonize the chicken GIT. Flagella have been shown to be required for passage through the GIT but not for cecal colonization (60). Mutations in the pldA gene (63), which encodes a hemolytic surface phospholipase, and the ciaB gene (63), which encodes a secreted protein required for efficient internalization of C. jejuni into epithelial cells, also lead to a noncolonization phenotype. The ability to adhere to the GIT surface is believed to be an important factor in successful colonization. The fibronectin-binding protein, CadF, was shown to be required for colonization of the ceca of newly hatched chicks by a human clinical isolate of C. jejuni (64). The roles of other Campylobacter adhesins, including MOMP, JlpA, and PEB1, have not been investigated for the chicken, although PEB1 is required for maximal colonization in a mouse model (44). We have demonstrated that CapA plays an important role in the colonization of the chicken gut. Although C. jejuni strain NCTC11168 is laboratory adapted, it is still able to successfully colonize outbred chickens with an intact gut flora. WT cells persisted in high numbers in 100% of infected birds for the entire 6-week experimental period, as has been demonstrated in a separate study (27). The capA mutant, by contrast, was severely impaired in its ability to colonize chickens and was cleared from infected birds within 3 weeks. Since capA is not present in all C. jejuni isolates, it is likely that other adhesins can take its place in other backgrounds. In vitro assays suggested that mutation of capA had no effect on the growth rate. Although unlikely, we cannot exclude the possibility that an in vivo growth rate defect could be responsible for the poor colonization of chickens by the capA mutant.

Many genes in the published sequence of C. jejuni NCTC11168 contain short polymeric tracts of nucleotides that are thought to facilitate high-frequency phase variation (41, 57). The wlaN gene, which encodes a galactosyltransferase, for example, contains a homopolymeric tract that renders the gene phase variable (37). Two identical genes, maf1 and maf4, belonging to a paralogous gene family contain homopolymeric G tracts. Mutations in another member of this family, maf5, were shown to affect flagellum formation and motility, while activation of maf1 through phase variation could partially restore motility to this mutant (30). The presence of homopolymeric tracts at the 5' ends of capA and capB suggests that their expression is also phase variable. In the published genome sequence, both genes contained frameshifts immediately downstream of these polymeric tracts. The presence of predicted promoter sequences, ribosome-binding sites, and signal peptides associated with both upstream ORFs but not their downstream counterparts further supports the hypothesis that each pair of ORFs constituted a single gene that was in an "off" phase at the time of sequencing. In our hands, capA was consistently transcribed and expressed in the isolate of NCTC11168 held in our laboratory. When we examined a number of human isolates, we could detect CapA expression in only 4 of 11 recent low-passage-number isolates and in 2 of 8 MLST isolates. We were able to detect sequences homologous to the capA gene in each of the strains in which CapA expression was demonstrated and in an additional four of the recent isolates.

The capB gene is out of frame in the C. jejuni NCTC11168 sequence released by the Sanger Centre, and we have confirmed that the frameshift is also present in the isolate of NCTC11168 held in our laboratory. Variation in the number of nucleotides in the homopolymeric tract found in capB could, in theory, result in the expression of a full-length molecule, but we were unable to detect expression of capB in strain NCTC11168. To address the hypothesis that capB has been switched off during laboratory adaptation, we examined several human isolates and a series of sequential isolates from our chicken colonization trial, but capB expression was never detected, although transient capB expression in vivo, which is rapidly switched off in vitro, cannot be excluded. Furthermore, we have also been unable to detect capB transcription by RT-PCR in any of our strains. Transcription of the gene should occur independently of any changes in the homopolymeric tract, suggesting that that it is not simply the frameshift that causes the lack of expression. Analysis of the upstream sequences of both capA and capB based on a C. jejuni promoter consensus sequence generated from 21 characterized C. jejuni promoters (59) gave no indication of why capA is transcribed but capB is not (data not shown). We hypothesize that capB either is a pseudogene or is expressed only under certain in vitro or in vivo conditions that we have so far been unable to determine.

The observation that capA is consistently expressed, despite the fact that the frameshift apparent in the published genome sequence was confirmed in the laboratory stock of NCTC11168, is intriguing. It is possible that capA is in frame in a small proportion of cells in a mixed population. Alternatively, transcriptional slippage may facilitate expression of the frameshifted gene. Such a process has been reported in other systems (2). Both possibilities are currently under investigation in our laboratory.

The similarity of the capA and capB genes at the N and C termini raised the possibility of recombination events between the two genes, which could lead to antigenic variation. The presence of two related genes encoding potential adhesins is reminiscent of a pair of genes, babA and babB, in H. pylori (50). BabA is an adhesin that mediates binding to Lewis B blood group antigens on the gastric epithelium. The babA gene was consistently lost or inactivated by phase variation at a CT dinucleotide repeat during experimental infection of macaques (50). In some cases, gene conversion events were observed in which babB replaced the deleted babA gene. We examined this possibility by PCR analysis of DNA extracted from representative chicken isolates obtained 2 and 6 weeks into the trial. In no case was an amplification product detected that would indicate recombination between the two loci, but although we were unable to detect recombination events between capA and capB after chicken colonization, it remains a possibility that under certain circumstances the two genes may recombine to produce new antigenic variants.

Two studies on the gene content of Campylobacter isolates have shown that capA and capB are both present in only a subset of C. jejuni isolates. Pearson et al. (42) showed that the complete capA sequence was present in 6 of 18 strains tested. The C-terminal ORF (Cj0629) was detected in the absence of the N-terminal ORF (Cj0628) in one additional strain. The complete capB sequence was detected in 3 of the 18 strains, the C-terminal ORF (Cj1677) alone in an additional 8 isolates, and the N-terminal ORF (Cj1678) in 1 additional strain. It is interesting that only 2 of the 18 strains resembled C. jejuni NCTC11168 in that they contained the complete capA and capB sequences, whereas 5 of the strains examined contained neither gene. Using a similar microarray-based approach, Dorrell et al. (14) found that 5 of 11 isolates contained the complete capA and capB genes, a further 2 strains contained capA only, and the remainder contained neither gene. The significance of this strain distribution and any correlation with human disease or colonization of animals would require a much larger epidemiological study of the distribution of capA and capB in clinical and veterinary isolates.

CapA is the first autotransporter of C. jejuni to be characterized and has been demonstrated to have an important role in interaction with host cells in vitro and colonization in vivo. The role, if any, of its close homologue, CapB, remains to be determined.


   ACKNOWLEDGMENTS
 
We thank D. Wareing for providing the MLST C. jejuni, S. Miyakoshi for the gift of globomycin, and I. Henderson for useful discussions.


   FOOTNOTES
 
* Corresponding author. Mailing address: Division of Microbiology and Infectious Diseases, A Floor West Block, University Hospital, Nottingham, NG7 2UH, United Kingdom. Phone: 44 (0) 115-823-0748. Fax: 44 (0) 115-823-0759. E-mail: daa{at}nottingham.ac.uk. Back

{triangledown} Published ahead of print on 15 December 2006. Back

{dagger} S.S.A.A. and N.J.O. contributed equally to the present study. Back

{ddagger} Present address: School of Veterinary Medicine and Science, University of Nottingham, Sutton Bonington Campus, Leicestershire LE12 5RD, United Kingdom. Back

§ D.P.J.T. and D.A.A.A. also contributed equally to the present study. Back


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 Introduction
 Materials and Methods
 Results
 Discussion
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