IDENTIFICATION AND MOLECULAR-CLONING OF A GENE ENCODING A FIBRONECTIN-BINDING PROTEIN (CADF) FROM CAMPYLOBACTER-JEJUNI

Campylobacter 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. Sca nning electron microscopy of infected INT407 cells suggested that C. j ejuni bound to a component of the extracellular matrix, Binding assays using immobilized extracellular matrix proteins and soluble fibronect in showed specific and saturable binding of fibronectin to C. jejuni. Ligand immunoblot assays using I-125-labelled fibronectin revealed spe cific binding to an outer membrane protein with an apparent molecular mass of 37 kDa. A rabbit antiserum, raised against the gel-purified pr otein, reacted with a 37 kDa protein in all C. jejuni isolates (n=15) as tested by immunoblot analysis. Antibodies present in convalescent s erum from C. jejuni-infected individuals also recognized a 37 kDa prot ein. The gene encoding the immunoreactive 37 kDa protein was cloned an d sequenced. Sequencing of overlapping DNA fragments revealed an open reading frame (ORF) that encodes a protein of 326 amino acids with a c alculated molecular mass of 36 872 Da. The deduced amino acid sequence of the ORF exhibited 52% similarity and 28% identity to the root adhe sin protein from Pseudomonas fluorescens. Isogenic C. jejuni mutants w hich 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 ligand-binding blots. These results indicate that the bindi ng of C. jejuni to fibronectin is mediated by the 37 kDa outer membran e protein which is conserved among C. jejuni isolates.