Neisseria gonorrhoeae (GC) is one of the most common sexually transmitted pathogens in the United States, Infection with GC usually remains localized to the lower genital tract, although certain strains are capable of dissemination. During infection of the urogenital tract with GC, bacteria encounter macrophages in the endometrium and other tissues. Here, surface antigens on the gonococcus trigger the local and systemic humoral immune response that results in the release of cytokines, prostaglandin, and other inflammatory mediators. Although previous efforts have focused on defining immunologic responses to protein antigens, studies would suggest that when phagocytes encounter Gram- negative bacteria, the subsequent cytokine response is due to the interactions of bacterial lipopolysaccharides (LPS) with macrophage receptors. In addition, the activation and fixation of complement by GC plays a major role in bacterial interactions with both professional and non-professional phagocytes. The goal of this proposal is to analyze the nature of the interaction between GC and the mammalian receptors likely to be activated in response to bacterial invasion. Efforts to characterize mammalian receptors for GC will focus on the leukocyte CD11/CD18 integrins. This family of adhesion receptors includes CD11b/CD18 (CR3) and CD11c/CD18 (CR4) which are phagocytic receptors for iC3b and opsonized bacteria, as well as signaling receptors for LPS. The focus of this proposal is to identify the molecular mechanisms involved in phagocytosis and LPS signal transduction using CR3- and CR4-transfected fibroblast cell lines. Because native phagocytes express multiple binding proteins on their surface, they have not been a useful tool for the study of a single receptor class. Furthermore, unlike monocytic lines, transfected fibroblast lines can be genetically manipulated in order to identify genes involved in phagocytosis and LPS signaling.
Four Specific Aim are proposed. First, we will define the individual roles of the complement receptors CR3 and CR4 in phagocytosis and cell activation by GC and gonococcal LOS. The role of the complement receptors will be compared to that of CD14, a well- characterized LPS receptor on macrophages. Second, using differential display we will identify and characterize phagocytosis inducible genes from established wild type (phagocytosing) and cytoplasmic deletion mutant (non-phagocytosing) CR3-transfected CHO line. Finally, we will use somatic cell mutagenesis techniques to generate phagocytosis mutants in the CR3- transfected CHO line. Complementation analysis can then be used to identify genes essential for phagocytosis in human monocytes.
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