MOLECULAR AND FUNCTIONAL STUDIES OF H INFLUENZAE PILI

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DESCRIPTION (Adapted from the applicant's abstract): Haemophilus influenzae (Hi) are significant cause of respiratory tract infections, including sinusitis, otitis media, bronchitis, and pneumonia. The first step in the development of Hi infections is colonization of nasopharyngeal tissues, which depends on the ability of Hi to adhere to nasopharyngeal cells. To this end, Hi have evolved pili that bind to ganglioside-containing receptors on epithelial cells and human red cells. Hi pili are complicated structures composed of at least three proteins, including HifA (the major pilus subunit), HifD (whose function is poorly defined), and HifE which contains the actual epithelial cell adhesin. The long-term goal of this project is to identify strategies to interfere with Hi adherence and, thus, prevent Hi infections. This goal will be met through understanding the underlying mechanisms by which the HifE adhesin of Hi mediates adherence to human tissue. Specific Aim I is designed to identify the receptor binding domain(s) of HifE through three approaches: 1. To analyze the amino acid sequences of HifE from Hi respiratory and invasive strains for conserved regions; 2. To identify epitopes defined by monoclonal antibodies that interfere with pilus mediated Hi binding to erythrocytes and epithelial cells; 3. To construct mutations in candidate receptor binding domain(s) by alanine-replacement and to test the function of the mutants in adherence assays. Specific Aim II is designed to compare the receptor binding domain(s) of Hi strains (biotype IV and biogroup aegyptius strains) that occupy unique niches within humans and may possess distinctive receptor binding domains. Specific Aim III is designed to identify tissue specificity of the receptor binding domain(s), by testing in competitive inhibition assays the binding of receptor binding domain mutants to a variety of human epithelial cells and by determining the role of the pilus stalk in binding specificity. Specific Aim IV is designed to identify the immunogenic potential of the receptor binding domain(s). Rabbit antisera will be raised to the domains and binding of the antibodies to HifE assessed by whole cell dot blot assays, Western blot assays, and immunoelectron microscopy. The receptor binding domain antibodies will be tested for their ability to interfere with the adherence of piliated Hi to human erythrocytes and respiratory cells.