Identification and molecular cloning of a gene encoding a fibronectin‐binding protein (CadF) from Campylobacter jejuni

Abstract

SummaryCampylobacter jejuni, a Gram-negative bacterium, is a common cause of gastrointestinal disease. By analogy with other enteric pathogens such as Salmonella and Shigella, the ability of C. jejuni to bind to host cells is thought to be essential in the pathogenesis of enteritis. Scanning electron microscopy of infected INT407 cells suggested that C. jejuni bound to a component of the extracellular matrix. Binding assays using immobilized extracellular matrix proteins and soluble fibronectin showed specific and saturable binding of fibronectin to C. jejuni. Ligand immunoblot assays using 125 I-labelled fibronectin revealed specific binding to an outer membrane protein with an apparent molecular mass of 37 kDa. A rabbit antiserum, raised against the gel-purified protein, reacted with a 37 kDa protein in all C. jejuni isolates (n ¼ 15) as tested by immunoblot analysis. Antibodies present in convalescent serum from C. jejuni-infected individuals also recognized a 37 kDa protein. The gene encoding the immunoreactive 37 kDa protein was cloned and sequenced. Sequencing of overlapping DNA fragments revealed an open reading frame (ORF) that encodes a protein of 326 amino acids with a calculated molecular mass of 36 872 Da. The deduced amino acid sequence of the ORF exhibited 52% similarity and 28% identity to the root adhesin protein from Pseudomonas fluorescens. Isogenic C. jejuni mutants which lack the 37 kDa outer membrane protein, which we have termed CadF, displayed significantly reduced binding to fibronectin. Biotinylated fibronectin bound to a protein with an apparent molecular mass of 37 kDa in the outer membrane protein extracts from wild-type C. jejuni as judged by ligandbinding blots. These results indicate that the binding of C. jejuni to fibronectin is mediated by the 37 kDa outer membrane protein which is conserved among C. jejuni isolates.

Analysis of factors that characterize health impairment in patients with chronic respiratory failure. Quality of Life in Chronic Respiratory Failure Group

“…Furthermore, the highly conserved 50 kDa OMP in the different P. aeruginosa strains that were used demonstrate that this OMP represents a new adhesin for P. aeruginosa, which is different from pilus-adhesin and flagellin-adhesin [34]. A recent study by KONKEL et al [35] reported a significant homology between the amino-acid sequence of a fibronectin-binding protein encoding for the cadF gene of Campylobacter jejuni and the OprF from P. aeruginosa, a 34 kDa aqueous channel of the P. aeruginosa outer membrane. However, the P. aeruginosa OprF has never been described as a fibronectin-binding site, whereas other P. aeruginosa OMP have been recently described for their binding to host extracellular matrix components [27] and to tracheobronchial mucins [21].…”

PCR Detection of Four Virulence-Associated Genes of Campylobacter jejuni Isolates from Thai Broilers and Their Abilities of Adhesion to and Invasion of INT-407 Cells

“…Some genes are involved in Campylobacter adhesion and invasion. Four mutant strains derived from F38011, an excellent colonizer of chicken caeca, dnaJ, cadF, pldA and ciaB are incapable of colonizing the ceca [20,23,43,44]. cadF (Campylobacter adhesin to fibronectin) is an outer membrane protein, which encodes a protein that interacts with a host extracellular matrix protein fibronectin [31], and is required for Campylobacter adherence to and colonization of the host cell surface [20].…”

“…Four mutant strains derived from F38011, an excellent colonizer of chicken caeca, dnaJ, cadF, pldA and ciaB are incapable of colonizing the ceca [20,23,43,44]. cadF (Campylobacter adhesin to fibronectin) is an outer membrane protein, which encodes a protein that interacts with a host extracellular matrix protein fibronectin [31], and is required for Campylobacter adherence to and colonization of the host cell surface [20]. Other genes such as ciaB (Campylobacter invasive antigen B) [21,34] and pldA encoded outer membrane phospholipase A [34] are involved in host cell invasion and are important for caecal colonization [21,43].…”

“…Other genes such as ciaB (Campylobacter invasive antigen B) [21,34] and pldA encoded outer membrane phospholipase A [34] are involved in host cell invasion and are important for caecal colonization [21,43]. The ciaB, pldA and dnaJ (heat shock protein) genes are important to caecal colonization and mutations of these genes severely limit the ability of the mutant strains to colonize the chicken caeca [20,43,44]. The objective of this study was to study the prevalence of C. jejuni in Thai broiler caeca and their ability to adhere and invade and the presence of the 4 genes that are involved in the adherence and invasion of the Campylobacter in the INT-407 cell culture model.…”

Campylobacter jejuni proteins Cj0952c and Cj0951c affect chemotactic behaviour towards formic acid and are important for invasion of host cells

“…Putative adhesion factors have been identified, e.g. the fibronectinbinding proteins CadF and FlpA (Konkel et al, 1997(Konkel et al, , 2010, the autotransporter CapA, and a surface-exposed lipoprotein, JIpA (Jin et al, 2001). In addition, the sialylated lipooligosaccharide outer core of C. jejuni has been demonstrated to be an important factor for the invasion of epithelial cells (Guerry et al, 2000;Louwen et al, 2008).…”