The goal of this project is to better understand the structure and function of a gonococcal membrane protein implicated in pathogenesis. Protein I (P.I) is a major outer membrane protein, and has been implicated in susceptibility to killing by serum and by polymorphonuclear neutrophils, and in ability to invade host cells. This application proposes to use molecular genetic techniques including transposon mutagenesis of cloned genes, and construction of strains carrying alternative forms of P.I. To provide its role in pathogenesis. Isogenic strains will be constructed containing either P.IA or P.IB or chimeric P.IA/P.IB hybrids, by transformation of competent gonococci with cloned P.I structural genes closely linked to a gene from chloramphenicol acetyl transferase. Attempts will be made to isolate P.I-mutants as well. These isogenic strains will be used in studies of serum killing, neutrophil susceptibility, and invasion of HL60 and primary human endometrial epithelial cell cultures, with or without addition of specific monoclonal and polyclonal sera directed at portions of these proteins. Epitopes on P.I will be mapped by analysis of the structure of chimeric P.IA/P.IB proteins, and by construction of recombinant fusion proteins. Previously describe genes for serum resistance and antibiotic resistance that are linked to the genes for P.I will be isolated by """"""""chromosome walking"""""""" from the cloned P.I genes. These studies will expand significantly our knowledge of the roles played in pathogenesis of P.I and will contribute towards rational vaccine development.
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