Anaerobic bacteria are frequently recovered from clinical infections throughout the body. Increased numbers of Bacteroides species, as part of a polymicrobial flora, are most often associated with the presence of chronic, purulent infections. The ability of Bacteroides species to induce chronicity to mixed infection has been demonstrated but not understood although it has been suggested that their effect may be on host defense cells, primarily the polymorphonuclear leukocyte (PMN). Progressive destructive periodontal lesions contain increased numbers of Porphyromonas gingivalis (formerly Bacteroides gingivalis) and we hypothesize that this microorganism exerts its effect on disease progression by affecting PMN function in the periodontal environment. We have demonstrated that some parent strains of P. gingivalis affect PMN function whereas others do not. The PMN-modulating activity located in the outer membrane and may not be exposed in heavily encapsulated strains. However, strains release numerous membrane blebs into the extracellular environment and our initial studies have shown that previously """"""""PMN-unreactive"""""""" parent strains release """"""""PMN- reactive"""""""" membrane blebs. Since bleb release is increased at times of physiologic stress and may coincide with, and be a important indicator of, bursts of periodontal destruction, the specific aims of this proposal are directed towards (1) characterizing the physiological and biochemical effects on human peripheral blood PMN P. gingivalis blebs and outer membrane proteins, (2) identifying the presence of PMN-modulating activity in the blebs and outer membranes of previously characterized """"""""PMN-unreactive"""""""" strains, and (3) isolating and characterizing the responsible outer membrane components of P. gingivalis. We will characterize the PMN response from initial membrane depolarization through mobilization of intracellular second messengers, to activation of the respiratory burst, degranulation and PMN locomotion. These assays will be performed in response to chemotactic and non- chemotactic stimulation and the modulation of the responses by membrane blebs and outer membrane proteins will be evaluated. PMN-modulating activity will be examined and compared for blebs and outer membrane components of a variety of previously characterized """"""""pmn-reactive"""""""" and """"""""PMN-unreactive"""""""" parent strains. PMN-modulating molecules will be isolated using Zwittergent detergent extraction of the bacterial surface components followed by chromatographic and electrophoretic separation and purification using PMN assays as a test for biological activity.
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