Studies are proposed to identify oxidative reactions occurring in phagocytosing leukocytes that are associated with microbicidal action. We have shown that the oxidant, hypochlorous acid (HOC1), inactivates E. coli in reactions that lead to destruction of metabolic energy reserves within the cell and to loss of energy-linked functions such as active transport. To explore the underlying molecular mechanisms, membrane localized energy-linked systems, including the ATP synthetase and selected ion and metabolite transport proteins, will be examined for evidence of oxidative damage. Studies to identify the lethal reactions of hydroxyl radical (OH) or OH-radical-generating systems willl be initiated. Comparison will be made between cell viabilities and loss of specific metabolic functions or biochemical components to identify loci of oxidative attack associated with cellular death. For this purpose differing methods of forming hydroxyl radical will be used that allow its generation in the extracellular medium, at the cell usrface, or within the microbial cell. Following protocols developed for study of inactivation by HOC1, the consequences of OH oxidation upon cellular transport systems, nucleotide concentrations, protonmotive force, and respiratory components (when appropriate) will be determined. For both HOC1 and OH oxidations, various organisms with widely differing metabolic requirements and cellular topographic organization will be studied to determine if particular inactivation mechanisms are """"""""universal"""""""" in character. Organisms exhibiting clearly different mechanisms of oxidative inactivation by HOC1 and OH will be used to identify the toxin produced in neutrophil phagosomes. For these experiments the microbes will be phagocytosed, then immediately recovered to determine the cause of death. These studies should greatly clarify the nature of leukocytic mechanisms comprising host response to infection.
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