CapA, an autotransporter protein of Campylobacter jejuni, mediates association with human epithelial cells and colonization of the chicken gut

Abstract

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.

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, β-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.

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-β-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.

FIG. 3.

Detection of CapA protein in C. jejuni. RαCapAB 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.

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 RαCapAB. (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 RαCapAB (B) and RαJlpA (C).

FIG. 5.

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

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).

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.