The overall objective of the proposed research is to study the role of gonococcal outer membrane components in the entrance and viability of Neisseria gonorrhoeae in two populations of human cells intimately involved in the pathogenesis of gonorrhea: polymorphonuclear leukocytes (PMN) and urogenital epithelial cells.
Four specific aims are proposed to accomplish this objective: 1) Investigate the relative roles of pili, which are anti-phagocytic, and gonococcal outer membrane protein P.II, some which are pro-phagocytic, in the phagocytosis of gonococci by human PMN and mononuclear phagocytes. Well defined gonococcal variants possessing various combinations of pili and P.II types will be used and an attempt will be made to isolate and characterize a putative leukocyte receptor for P.II. Purified radiolabeled P.II will be used to study kinetics of receptor-ligand interactions, to block gonococcus-leukocyte interactions, and to probe for specific leukocyte membrane receptor by using heterobi-functional cross-linking reagents, membrane solubilization, immuno-precipitation with antibodies to P.II, and polyacrylamide gel eletrophoresis. 2) Treat gonococci with human PMN granule contents and investigate the biochemical or physiologic alterations sustained by gonococci that could account, in part or in full, for gonococcal death. Gonococcal inner and outer membrane function and integrity will be investigated, including permeability, metabolite and ion transport, electron transport, cell division, and nucleic acid synthesis. 3) Treat gonococci with human PMN granule contents and investigate the structural alterations sustained by gonococci that could account, in part or in full, for gonococcal death. Gonococcal inner and outer membrane composition and structure will be investigated, including protein, peptidoglycan, phospholipid, and lipopolysaccharide synthesis and turnover. 4) Investigate the relative roles of pili and P.II in the internalization of gonococci by human urogenital epithelial cells. Intracellular viability will also be assessed. We propose that P.II induces internalization of gonococci into urogenital epithelial cells, inside which they continue to grow. Primary cultures of human endometrial and cervical epithelia will be used in conjunction with gonococcal variants possessing various combinations of pili and P.II. These studies will better define the role of gonococcal membrane components in the pathogenesis of gonorrhea at urogenital mucosal surfaces.
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