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.
- SourceAvailable from: mediport-online.de[show abstract] [hide abstract]
ABSTRACT: Die akute Campylobacterinfektion verläuft als selbstlimitierende Enteritis. Bakteriämie, Endokarditis, Meningitis, Pankreatitis, septischer Abort und neonatale Sepsis sind selten. Die wichtigste postinfektöse Komplikation ist das Guillain-Barré-Syndrom (GBS). Die unkomplizierte Enteritis wird symptomatisch behandelt. Patienten mit schweren Verläufen oder Immunsuppression erhalten Antibiotika. Resistenzentwicklung gegen Chinolone, seltener gegen Makrolide, ist zu berücksichtigen. Ende 2001 war Campylobacter in Deutschland mit 52.256 Meldungen hinter den Salmonellosen (74.671) und vor den Rotaviren (45.759) der zweithäufigste darmpathogene Erreger. Die fehlende Vermehrung in der Umwelt kompensiert Campylobacter durch eine weite Verbreitung in der Natur und den massiven Befall zahlreicher Wirte. Geflügelfleisch, Rohmilch und Oberflächenwasser sind wichtige alimentäre Risikofaktoren für den Menschen. Die Kontrolle der Campylobacterinfektionen wird erschwert durch eine schnelle Ausbreitung in Schlachtgeflügel während Haltung, Transport und Schlachtprozess. Pasteurisierung der Milch ist eine effektive Intervention, während die Kühlung von Lebensmitteln das Überleben von Campylobacter verlängert. Ansätze zur Prävention einer humanen Infektion sind die Quarantänehaltung des Schlachtgeflügels, Verzicht auf das so genannte “Ausdünnen” der Bestände, Dekontamination der Transportkäfige, das Vorziehen der Schlachtung nicht infizierter Bestände, höchstmögliche Brühtemperaturen der Schlachtkörper, Abkühlung der Körper im Kühlbad, Zusatz antimikrobieller Substanzen zum Prozesswasser und möglicherweise zukünftig eine dekontaminierende Schlussbehandlung. Forschungsbedarf besteht bei der Aufklärung der Pathogenese von Enteritis und GBS. Die zunehmende Resistenzentwicklung gegen antimikrobielle Substanzen erfordert ein interdisziplinäres Überwachungs- und Interventionsprogramm. Enteritis as the characteristic clinical manifestation of Campylobacter infections is rarely complicated by bacteraemia, endocarditis, meningitis, pankreatitis, and septic abortion. The most serious sequelum is the Guillain-Barré syndrome (GBS). Uncomplicated enteritis needs symptomatic treatment only. Severely ill or immunocompromised patients need antimicrobial therapy. In Germany enteropathogenic Campylobacter with 52.256 cases ranked second behind salmonellosis (74.671) in 2001. Campylobacter cannot multiply in the environment. However, this characteristic is compensated by a wide distribution in nature and rapid multiplication in a variety of hosts. Humans are infected mainly over poultry, unpasteurized milk, and untreated drinking water. Control of Campylobacteriosis is difficult due to rapid spread in poultry, during transport, slaughter and processing. Milk pasteurization is an effective intervention, but cool storage of food rather prolongs survival of Campylobacter. Containtment holding of poultry, no intermittend “thinning out” of animals, transport in disinfected crates, slaughtering of Campylobacter-free flocks before Campylobacter-contaminated animals, high temperature scalding, chilling in water, use of decontaminating agents during processing, and probably most effective, decontamination of the final product, are proposed as intervention measures. Besides an interdisciplinary programme for monitoring and controling resistance development, further research should predominately focus on pathogenesis of enteritis and GBS.Bundesgesundheitsblatt - Gesundheitsforschung - Gesundheitsschutz 04/2012; 45(6):497-506. · 0.72 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: Campylobacter jejuni is one of the most important bacterial pathogens causing food-borne illness worldwide. Crossing the intestinal epithelial barrier and host cell entry by C. jejuni is considered the primary reason of damage to the intestinal tissue, but the molecular mechanisms as well as major bacterial and host cell factors involved in this process are still widely unclear. In the present study, we characterized the serine protease HtrA (high-temperature requirement A) of C. jejuni as a secreted virulence factor with important proteolytic functions. Infection studies and in vitro cleavage assays showed that C. jejuni's HtrA triggers shedding of the extracellular E-cadherin NTF domain (90 kDa) of non-polarised INT-407 and polarized MKN-28 epithelial cells, but fibronectin was not cleaved as seen for H. pylori's HtrA. Deletion of the htrA gene in C. jejuni or expression of a protease-deficient S197A point mutant did not lead to loss of flagella or reduced bacterial motility, but led to severe defects in E-cadherin cleavage and transmigration of the bacteria across polarized MKN-28 cell layers. Unlike other highly invasive pathogens, transmigration across polarized cells by wild-type C. jejuni is highly efficient and is achieved within a few minutes of infection. Interestingly, E-cadherin cleavage by C. jejuni occurs in a limited fashion and transmigration required the intact flagella as well as HtrA protease activity, but does not reduce transepithelial electrical resistance (TER) as seen with Salmonella, Shigella, Listeria or Neisseria. These results suggest that HtrA-mediated E-cadherin cleavage is involved in rapid crossing of the epithelial barrier by C. jejuni via a very specific mechanism using the paracellular route to reach basolateral surfaces, but does not cleave the fibronectin receptor which is necessary for cell entry.Gut Pathogens 04/2012; 4(1):3. · 2.74 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: Enteroaggregative Escherichia coli (EAEC) adherence to human intestinal tissue is known to be mediated by aggregative adherence fimbriae (AAF). However, the host cell molecules involved in EAEC adherence remain uncharacterized. In the present study, four key membrane glycoproteins of cultured human intestinal epithelial cells (INT-407) were found to be involved in the interaction with the T8 strain of EAEC. Nine membrane proteins of INT-407 cells were found to interact with EAEC-T8, of which four were identified as fibronectin, epidermal growth factor receptor (EGFR), Thrombospondin-1 (TSP1) and glucose-regulated protein (GRP-94). Our findings were validated by Western blot using antibody against each identified protein. The adherence of EAEC-T8 to INT-407 cells was reduced to c. 26, 29, 37 and 76% in the presence of the antibody against GRP-94, EGFR, fibronectin and TSP-1, respectively. These findings were further substantiated by flow cytometry, where the final mean fluorescence intensity value of the INT-407 cells (c. 1075) resulting from adherent-labelled bacteria was found to be reduced to c. 26, 132, 228 and 597 in the presence of antibody against GRP-94, EGFR, fibronectin and TSP-1. We propose that GRP-94, EGFR, fibronectin and TSP-1 are involved in the aggregative adherence of EAEC-T8 to INT-407 cells.FEMS Immunology & Medical Microbiology 06/2012; 66(2):177-90. · 2.68 Impact Factor