Campylobacter jejuni FlpA Binds Fibronectin and Is Required for Maximal Host Cell Adherence

Campylobacter jejuni is one of the most frequent bacterial causes of food-borne gastrointestinal disease in developed countries. Previous work indicates that the binding of C. jejuni to human intestinal cells is crucial for host colonization and disease. Fibronectin (Fn), a major constituent of the extracellular matrix, is a ∼250-kDa glycoprotein present at regions of cell-to-cell contact in the intestinal epithelium. Fn is composed of three types of repeating units: type I (∼45 amino acids), type II (∼60 amino acids), and type III (∼90 amino acids). The deduced amino acid sequence of C. jejuni flpA (Cj1279c) contains at least three Fn type III domains. Based on the presence of the Fn type III domains, we hypothesized that FlpA contributes to the binding of C. jejuni to human INT 407 epithelial cells and Fn. We assessed the contribution of FlpA in C. jejuni binding to host cells by in vitro adherence assays with a C. jejuni wild-type strain and a C. jejuni flpA mutant and binding of purified FlpA protein to Fn by enzyme-linked immunosorbent assay (ELISA). Adherence assays revealed the binding of the C. jejuni flpA mutant to INT 407 epithelial cells was significantly reduced compared with that for a wild-type strain. In addition, rabbit polyclonal serum generated against FlpA blocked C. jejuni adherence to INT 407 cells in a concentration-dependent manner. Binding of FlpA to Fn was found to be dose dependent and saturable by ELISA, demonstrating the specificity of the interaction. Based on these data, we conclude that FlpA mediates C. jejuni attachment to host epithelial cells via Fn binding.Members of the genus Campylobacter are gram-negative, asaccharolytic, motile bacteria, which grow optimally in the laboratory at temperatures between 37 and 42°C under microaerophilic conditions. Although members of Campylobacter spp. were initially recognized to cause disease in sheep and cattle, Campylobacter jejuni was not recognized as a human pathogen until much later (25). Infection of humans with C. jejuni is characterized by a rapid onset of fever, abdominal cramps, and diarrhea. C. jejuni is now recognized as one of the leading bacterial causes of gastroenteritis in the world. In spite of the incidence of campylobacteriosis, relatively few C. jejuni virulence genes have been characterized, and our understanding of the virulence properties of C. jejuni is limited compared with that of other enteric pathogens, including Salmonella, Shigella, and Yersinia spp.The ability of C. jejuni to cause disease is a complex, multifactorial process. Virulence factors that contribute to the pathogenesis of C. jejuni are associated with motility, host (target) cell adherence, host cell invasion, protein secretion, alteration of host cell signaling pathways, induction of host cell death, evasion of host immune defenses, iron acquisition, and drug/detergent resistance (14, 18). The binding of C. jejuni to specific host cell ligands is hypothesized to play a fundamental role in host colonization and disease progression, since it prevents the organism''s clearance from the intestine by peristalsis and fluid flow. Fauchere et al. (5) reported that C. jejuni isolates recovered from individuals with fever and diarrhea adhered to cultured cells in greater numbers than isolates recovered from asymptomatic individuals. While there is no evidence indicating that C. jejuni produces fimbriae that assist in host colonization (7), a number of constitutively synthesized proteins have been proposed to act as adhesins. Bacterial adhesins are surface-exposed macromolecules that facilitate an organism''s binding to the host cell receptors. Known and putative C. jejuni adhesins include CadF, CapA, FlpA, and PorA (MOMP) (6).An emerging theme among pathogenic microorganisms is their ability to utilize host cell molecules during the infectious process to facilitate their binding and entry into host cells (27). More specifically, many bacterial pathogens have been found to bind to fibronectin (Fn), which in turn modifies host cell signaling pathways to the pathogen''s advantage. Fn exists as a dimer of nearly identical 250-kDa subunits that are linked by a pair of disulfide bonds near their C termini. Each Fn monomer is composed of three types of repeating units: type I (∼45 amino acids), type II (∼60 amino acids), and type III (∼90 amino acids) (22). In total, each monomer contains 12 type I repeats, two type II repeats, and 15 to 17 type III repeats. Fn participates in many cellular interactions, including tissue repair, embryogenesis, blood clotting, and cell migration/adhesion. Plasma Fn, which is synthesized by hepatocytes, is soluble (22). In contrast, Fn involved in host cell-extracellular matrix (ECM) interaction, which is synthesized by chondrocytes, fibroblasts, endothelial cells, macrophages, and certain epithelial cells, is present in an insoluble form (22). Fn serves as an adhesion molecule that anchors cells to ECM components, including collagen and other proteoglycan substrates.The bacterial proteins that bind to ECM components have been termed microbial surface components recognizing adhesive matrix molecules (MSCRAMMs) (23). The C. jejuni CadF protein is a member of the MSCRAMM family and one of the most extensively characterized C. jejuni virulence determinants (10-12, 15, 16, 19-21, 24, 28). CadF mediates the binding of C. jejuni to Fn, promotes bacterium-host cell interactions, and facilitates the organism''s colonization of chickens (10, 11, 15, 16, 20, 21, 28). In addition to CadF, we recently reported that a mutation in Cj1279c resulted in a C. jejuni mutant that poorly colonized broiler chickens compared with a C. jejuni wild-type strain. The product encoded by the Cj1279c gene was termed Fibronectin-like protein A (FlpA) because the protein harbors Fn type III domains (6). The goal of this study was to characterize the binding properties of FlpA and to determine if this protein is a member of the MSCRAMM family. Here we provide experimental evidence that C. jejuni FlpA is surface exposed, promotes the bacterium''s attachment to host epithelial cells, and has Fn binding activity. Assays were also performed to determine if CadF and FlpA act cooperatively to promote binding of C. jejuni to host cells and Fn. We submit that the identification of a second MSCRAMM in C. jejuni highlights the importance of Fn binding in host colonization and disease.