Konkel ME, Garvis SG, Tipton SL, et al. Identification and molecular cloning of a gene encoding a fibronectin-binding protein (CadF) from Campylobacter jejuni

Department of Microbiology, Washington State University, Pullman 99164-4233, USA.
Molecular Microbiology (Impact Factor: 4.42). 07/1997; 24(5):953-63. DOI: 10.1046/j.1365-2958.1997.4031771.x
Source: PubMed


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.

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Available from: Steve Garvis, Oct 13, 2014
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    • "Also, Sanchez et al. found that biofilm bacteria grown on abiotic surfaces adhered better to epithelial cells than planktonic, broth grown bacteria (Sanchez et al., 2011b). These two studies, supported by studies in other human pathogens (Konkel et al., 1997; Sulaeman et al., 2012), demonstrate a relationship between epithelial cell adherence and biofilm formation, however, the studies have not investigated the role of this relationship during pneumococcal colonization. "
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    ABSTRACT: Streptococcus pneumoniae (the pneumococcus) is a common colonizer of the human nasopharynx. Despite a low rate of invasive disease, the high prevalence of colonization results in millions of infections and over 1 million deaths per year, mostly in individuals under the age of 5 and the elderly. Colonizing pneumococci form well-organized biofilm communities in the nasopharyngeal environment, but the specific role of biofilms and their interaction with the host during colonization and disease is not yet clear. Pneumococci in biofilms are highly resistant to antimicrobial agents and this phenotype can be recapitulated when pneumococci are grown on respiratory epithelial cells under conditions found in the nasopharyngeal environment. Pneumococcal biofilms display lower levels of virulence in vivo and provide an optimal environment for increased genetic exchange both in vitro and in vivo, with increased natural transformation seen during co-colonization with multiple strains. Biofilms have also been detected on mucosal surfaces during pneumonia and middle ear infection, although the role of these biofilms in the disease process is debated. Recent studies have shown that changes in the nasopharyngeal environment caused by concomitant virus infection, changes in the microflora, inflammation, or other host assaults trigger active release of pneumococci from biofilms. These dispersed bacteria have distinct phenotypic properties and transcriptional profiles different from both biofilm and broth-grown, planktonic bacteria, resulting in a significantly increased virulence in vivo. In this review we discuss the properties of pneumococcal biofilms, the role of biofilm formation during pneumococcal colonization, including their propensity for increased ability to exchange genetic material, as well as mechanisms involved in transition from asymptomatic biofilm colonization to dissemination and disease of otherwise sterile sites. Greater understanding of pneumococcal biofilm format
    Full-text · Article · Jan 2015 · Frontiers in Cellular and Infection Microbiology
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    • "CadF, another outer membrane protein with an apparent molecular weight of 37 kDa, and FlpA have also been shown to connect to fibronectin. These interactions, in turn, result in the activation of integrin receptors to launch a host cell signal cascade leading to restructuring of the actin cytoskeleton mediating the uptake of C. jejuni [11]–[15]. The surface-exposed lipoprotein JlpA is required for efficient adherence of the pathogen to HEp-2 epithelial cells and initiates the activation of NF-κB and p38 MAP kinase. "
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    ABSTRACT: Adherence of Campylobacter jejuni to its particular host cells is mediated by several pathogen proteins. We screened a transposon-based mutant library of C. jejuni in order to identify clones with an invasion deficient phenotype towards Caco2 cells and detected a mutant with the transposon insertion in gene cj0268c. In vitro characterization of a generated non-random mutant, the mutant complemented with an intact copy of cj0268c and parental strain NCTC 11168 confirmed the relevance of Cj0268c in the invasion process, in particular regarding adherence to host cells. Whereas Cj0268c does not impact autoagglutination or motility of C. jejuni, heterologous expression in E. coli strain DH5α enhanced the potential of the complemented E. coli strain to adhere to Caco2 cells significantly and, thus, indicates that Cj0268c does not need to interact with other C. jejuni proteins to develop its adherence-mediating phenotype. Flow cytometric measurements of E. coli expressing Cj0268c indicate a localization of the protein in the periplasmic space with no access of its C-terminus to the bacterial surface. Since a respective knockout mutant possesses clearly reduced resistance to Triton X-100 treatment, Cj0268c contributes to the stability of the bacterial cell wall. Finally, we could show that the presence of cj0268c seems to be ubiquitous in isolates of C. jejuni and does not correlate with specific clonal groups regarding pathogenicity or pathogen metabolism.
    Full-text · Article · Nov 2013 · PLoS ONE
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    • "Campylobacter jejuni is a gram-negative bacterium and the causative agent of campylobacteriosis, a debilitating gastrointestinal illness associated with abdominal cramps, fever and diarrhea.11 C. jejuni encodes two known Fn-binding proteins, an outer membrane protein termed CadF,12 and a recently identified lipoprotein termed FlpA that harbors three FNIII-like domains.13 Needed are studies to dissect the contribution of Fn binding in C. jejuni–host cell interactions. "
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    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.
    Full-text · Article · Oct 2013 · Emerging Microbes and Infections
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