title: Identification and molecular cloning of a gene encoding a fibronectin-binding protein (CadF) from Campylobacter jejuni.
abstract: 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. 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 125I-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 = 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 37kDa 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 36872Da. 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 ligand-binding 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.
authors.affiliation: Department of Microbiology, Washington State University, Pullman 99164-4233, USA. Konkel@mail.wsu.edu
authors.forename: M E
authors.initials: ME
authors.lastname: Konkel
authors.forename: S G
authors.initials: SG
authors.lastname: Garvis
authors.forename: S L
authors.initials: SL
authors.lastname: Tipton
authors.forename: D E
authors.initials: DE
authors.lastname: Anderson
authors.forename: W
authors.initials: W
authors.lastname: Cieplak
mesh_terms: D018829:Adhesins, Bacterial; D000595:Amino Acid Sequence; D000818:Animals; D001425:Bacterial Outer Membrane Proteins / Q000235:genetics* / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D016123:Campylobacter jejuni / Q000235:genetics* / Q000378:metabolism; D002352:Carrier Proteins / Q000235:genetics* / Q000378:metabolism; D002460:Cell Line; D003001:Cloning, Molecular; D004269:DNA, Bacterial; D005109:Extracellular Matrix / Q000378:metabolism; D005798:Genes, Bacterial; D006801:Humans; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D016366:Open Reading Frames; D011817:Rabbits; D017386:Sequence Homology, Amino Acid
chemical_list: D018829:Adhesins, Bacterial; D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; C107872:CadF protein, Campylobacter jejuni; D002352:Carrier Proteins; D004269:DNA, Bacterial; C078635:fibronectin-binding proteins, bacterial