Examination of Campylobacter jejuni Putative Adhesins Leads to the Identification of a New Protein, Designated FlpA, Required for Chicken Colonization

Abstract

Campylobacter jejuni colonization of chickens is presumably dependent upon multiple surface-exposed proteins termed adhesins. Putative C. jejuni adhesins include CadF, CapA, JlpA, major outer membrane protein, PEB1, Cj1279c, and Cj1349c. We examined the genetic relatedness of 97 C. jejuni isolates recovered from human, poultry, bovine, porcine, ovine, and canine sources by multilocus sequence typing (MLST) and examined their profile of putative adhesin-encoding genes by dot blot hybridization. To assess the individual contribution of each protein in bacterium-host cell adherence, the C. jejuni genes encoding the putative adhesins were disrupted by insertional mutagenesis. The phenotype of each mutant was judged by performing in vitro cell adherence assays with chicken LMH hepatocellular carcinoma epithelial cells and in vivo colonization assays with broiler chicks. MLST analysis indicated that the C. jejuni isolates utilized in this study were genetically diverse. Dot blot hybridization revealed that the C. jejuni genes encoding the putative adhesins, with the exception of capA, were conserved among the isolates. The C. jejuni CadF, CapA, Cj1279c, and Cj1349c proteins were found to play a significant role in the bacterium's in vitro adherence to chicken epithelial cells, while CadF, PEB1, and Cj1279c were determined to play a significant role in the bacterium's in vivo colonization of broiler chicks. Collectively, the data indicate that Cj1279c is a novel adhesin. Because Cj1279c harbors fibronectin type III domains, we designated the protein FlpA, for fibronectin-like protein A.

Molecular mechanisms and biological role ofCampylobacter jejuniattachment to host cells

“…Purified JlpA inhibited adherence of C. jejuni to HEp-2 cells confirming the role of the former as an adhesin [23]. However, inactivation of jlpA gene did not affect the ability of C. jejuni to bind to chicken LMH cells or to colonise broiler chickens [17], and also did not reduce attachment to human T84 human colonic adenocarcinoma cell [24] suggesting that only certain host cells are able to produce receptors for this adhesin.…”

“…Disruption of the flpA gene impairs bacterial ability to adhere to chicken LMH hepatocellular carcinoma epithelial cells and to human INT 407 cells, and reduces bacterial ability to colonise chickens [17]. In addition, bacterial binding to host cells was inhibited by anti-FlpA specific antiserum in a dose-dependent way [18].…”

“…CapA mutant showed decreased adherence to human Caco-2 cells and also low colonisation efficiency in chicks [25]. In another study, mutation of capA also showed decreased adherence to chicken epithelial cells in vitro, although it did not result in a reduced ability of mutants to colonise chicks (47%) [17].…”

“…Host cell receptors have been identified only for outer membrane proteins CadF and FlpA specific to fibronectin (Fn) [16][17][18][19] and JlpA, specific to heat shock protein 90 [20].…”

“…Fn-binding protein FlpA contains Fn type III domains [17,18]. Disruption of the flpA gene impairs bacterial ability to adhere to chicken LMH hepatocellular carcinoma epithelial cells and to human INT 407 cells, and reduces bacterial ability to colonise chickens [17].…”