The fibronectin-binding motif within FlpA facilitatesCampylobacter jejuniadherence to host cell and activation of host cell signaling

Abstract

Campylobacter jejuni is a gram-negative, curved and rod-shaped bacterium that causes human gastroenteritis. Acute disease is associated with C. jejuni invasion of the intestinal epithelium. Epithelial cells infected with C. jejuni strains containing mutations in the FlpA and CadF fibronectin (Fn)-binding proteins exhibit reduced invasion of host cells and a C. jejuni CadF FlpA double mutant is impaired in the activation of epidermal growth factor receptor (EGFR) and Rho GTPase Rac1. Although these observations establish a role for Fn-binding proteins during C. jejuni invasion, their mechanistic contributions to invasion-associated signaling are unclear. We examined FlpA, a C. jejuni Fn-binding protein composed of three FNIII-like repeats D1, D2 and D3, to identify the interactions required for cellular adherence on pathogen-induced host cell signaling. We report that FlpA binds the Fn gelatin-binding domain via a motif within the D2 repeat. Epithelial cells infected with a flpA mutant exhibited decreased Rac1 activation and reduced membrane ruffling that coincided with impaired delivery of the secreted Cia proteins and reduced cell association. Phosphorylation of the Erk1/2 kinase, a downstream effector of EGFR signaling, was specifically associated with FlpA-mediated activation of β1-integrin and EGFR signaling. In vivo experiments revealed that FlpA is necessary for C. jejuni disease based on bacterial dissemination to the spleen of IL-10−/− germ-free mice. Thus, a novel Fn-binding motif within FlpA potentiates activation of Erk1/2 signaling via β1-integrin during C. jejuni infection.

Molecular Dissection of the Campylobacter jejuni CadF and FlpA Virulence Proteins in Binding to Host Cell Fibronectin

“…Moreover, the CadF and FlpA proteins, which share only 25.6% similarity at the amino acid level, cannot functionally complement one another as evident in the phenotypic assays performed in this study (e.g., facilitating cell adherence, promoting FN binding, stimulating cell signaling, and enabling effector delivery to host cells). This finding is consistent with the fact that the CadF and FlpA proteins have distinct FN-binding domains [19,20]. Noteworthily, deduced amino acid sequences of the S. aureus FNBPA and FNBPB share a high level (74.2%) of similarity [45], and that complementation of a fnbA fnbB double mutant with either fnbA or fnbB fully restores the FN-binding activity [46].…”

“…Cdc42 and Rac1 are the primary host cell Rho GTPases involved in C. jejuni invasion. We have also reported that Rac1-GTP production is also diminished in human INT 407 cells infected with C. jejuni flpA and cadF flpA double mutant [19]. Given the findings from this study, which demonstrate that CadF and FlpA work together in facilitating cell adherence, promoting FN binding, stimulating cell signaling, and enabling effector delivery to host cells, studies are warranted to determine if disease symptoms arise in animals inoculated with a C. jejuni cadF flpA double mutant.…”

“…The FlpA protein contains a signal peptide sequence and three FN-type III domains. Recombinant CadF and FlpA proteins have been purified and demonstrated to bind to FN in a dose-dependent and saturable fashion [18,19]. CadF mediates binding to FN via a four amino acid motif (Phe-Arg-Leu-Ser) [20], whereas the FN-binding site within FlpA has been localized to a span of nine amino acids (Trp-Arg-Pro-His-Pro-Asp-Phe-Arg-Val) [19].…”

“…Recombinant CadF and FlpA proteins have been purified and demonstrated to bind to FN in a dose-dependent and saturable fashion [18,19]. CadF mediates binding to FN via a four amino acid motif (Phe-Arg-Leu-Ser) [20], whereas the FN-binding site within FlpA has been localized to a span of nine amino acids (Trp-Arg-Pro-His-Pro-Asp-Phe-Arg-Val) [19]. As mentioned above, disruption of either cadF or flpA results in C. jejuni mutants impaired in their ability to bind to cultured epithelial cells [16,18].…”

Conserved core tryptophans of FnII domains are crucial for the membranolytic and chaperone‐like activities of bovine seminal plasma protein PDC‐109

“…Since core tryptophan residues are conserved across all FnII domains as well as their ancestral kringle domains, similar studies could help understand their role in protein-ligand interactions of other proteins containing FnII domains and the significance of such interactions. For example, previous studies have shown that infection by many pathogenic bacteria and viruses involves the interaction of their surface protein with the FnII domain of fibronectin on the host and that such binding is necessary for their life cycle [33][34][35]. Comparison of solution structure of FnII domains of fibronectin and PDC-109 shows similar solvent-exposed orientation of a core tryptophan residue [4,6].…”