Genetic Analysis of Bordetella Pathogenesis

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DESCRIPTION (provided by applicant): An early and often crucial step in the development of infection is adherence of the pathogen to epithelial surfaces of its host. Filamentous hemagglutinin (FHA), expressed by members of the gram negative genus Bordetella, is prototypical of a large family of afimbrial adhesins that are both surface-associated and released into the surrounding milieu. This highly immunogenic protein is a primary component of all acellular pertussis vaccines. In vitro studies using B. pertussis and FHA purified from B. pertussis have identified various binding domains within FHA and suggest that this protein may function as an adhesin when surface-associated (by mediating attachment to ciliated respiratory epithelia) and an immunomodulator when released (by interacting with integrin receptors on the surface of epithelial cells and macrophages). In vivo roles for FHA, however, are poorly understood due to lack of natural-host animal models for studies with the human-adapted pathogen B. pertussis. We have developed genetic tools, a variety of in vitro assays, and highly sensitive animal models for studying the role(s) of FHA and its various binding domains in the context of a natural bacterial-host interaction using B. bronchiseptica and rabbits, rats and mice. Our preliminary data with a B. bronchiseptica strain expressing FHA from B. pertussis indicate that these sensitive assays will allow us to dissect the roles of specific FHA binding domains in adherence and immunomodulation. We propose to 1) construct Bb strains expressing chimeric FHA molecules and to use them to determine how the various FHA binding domains contribute to adherence, colonization and the development of innate and humoral immune responses in vivo, 2) investigate the molecular bases for FHA-mediated host responses using in vitro assays, 3) investigate the mechanism of FHA processing and localization, and 4) investigate the importance of FHA processing and localization in the development of respiratory infection.