Browsing by Author "Turan, Tolga"

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    Isolation and characterization of an adhesin protein from the surface of a respiratory pathogen Moraxella catarrhalis
    (2002) Turan, Tolga
    Moraxella catarrhalis is a member of the normal flora of upper respiratory tract. Starting in the early 1980s it gained importance as an important cause of otitis media in children and lower respiratory tract infections in adults with chronic obstructive pulmonary disease. β-lactamase producing strains of M. catarrhalis has been increasing at a very fast rate. In some locations, 100% of the strains are β- lactamase producer. The pathogenesis of infection by this bacterium is not clearly understood which hindered the development of a vaccine. In this study, a surface protein of about 55 kDa was isolated from M. catarrhalis by celite chromatography. It is a heat stable protein and is not affected by 2-mercaptoethanol or dithiothreitol treatment. The immunogenic property of the protein has been determined by immunizing rabbits with M. catarrhalis and detecting the antibody response in serum against 55 kDa protein by Western blotting. In addition, the protein is immunogenic in humans as antibody against 55 kDa protein can be detected in the sputum of patients with M. catarrhalis infection. Moreover, we determined upto 40 amino acids at the N-terminal and also two fragments of the protein. To determine the function of the protein, attachment inhibition assays were performed and it was found that 55 kDa protein competetively inhibits attachment of M. catarrhalis to human pharyngeal epithelial cells (HPEC). Similarly, monoclonal antibody against 55 kDa (mAb) blocks the protein and inhibits the attachment of M. catarrhalis to HPEC. These two lines of evidence show that 55 kDa protein is an adhesin of M. catarrhalis which mediate attachment to HPEC. In addition, immunoflourescence experiments further verified that 55 kDa protein binds to HPEC. To sequence the gene encoding 55 kDa protein, PCR was done using degenerate primers constructed from the Nterminal amino acid sequence. PCR amplification of the possible gene of 55 kDa protein resulted in a 500 bp fragment, but no homology can be obtained with Nterminal amino acid sequence. In addition, a genomic library of M. catarrhalis is prepared and screened with mAb and with a radiolabelled oligonucleotide probe. We isolated several positive clones; therefore in future it might be possible to sequence the gene encoding 55 kDa protein from these clones.