We have initiated studies to identify the oxidative cytotoxins of stimulated leukocytes and their cytocidal mechanisms. We have shown that the bactericidal reactions of leukocyte peroxidase- generated hypochlorous acid (HOCl) and chemically similar chloramines cause destruction of metabolic energy reserves within the cell and loss of energy-linked functions such as active transport. The locus of oxidative attack is the bacterial plasma membrane; the metabolic dysfunctions leading to massive net ATP hydrolysis can be traced primarily to selective oxidative inactivation of nutrient transport systems and the proton translocating ATP synthase that links respiration to oxidative phosphorylation. We are applying several analytical methods to identify the molecular sites of attack on the ATP synthase F1 complex that give rise to its inactivation. The common response that we observe by several bacteria with disparate metabolic capabilities and the need for all cells to maintain ATP- synthesizing capabilities suggests that these reactions are capable of accounting for the """"""""universal"""""""" character of HOCl toxicity towards prokaryotes. In the proposed research, we will investigate inactivation of microbial eukaryotes, whose ATP- synthesizing enzymatic systems are mitochondrial, hence nominally protected from extracellularly-generated HOCl. We will initiate parallel studies to identify the lethal reactions of hydroxyl radical and similar active oxygen species (high-valent metal oxo ions) that might be formed in metal-catalyzed reactions involving H2O2. We will compare loss of viability with specific metabolic functions or individual biochemical components to identify the loci of oxidative attack associated with cellular death. For this purpose, we will use differing methods of forming hydroxyl radical to allow its generation in the extracellular medium, at the surface of the bacterial cell, or intracellulary. Following protocols that we have developed for the studies with HOCl, we will examine the consequences of OH and Fenton-type chemical systems upon bacterial transport and respiratory systems, biosynthetic capabilities and selected cytosolic enzymes, nucleotide phosphorylation, and lesions in nucleic acids. Organisms exhibiting cleanly differing mechanisms of oxidative inactivation by HOCl and OH will then be used to probe for the primacy of one or the other within neutrophil phagosomes. These studies will clarify leukocytic mechanisms comprising host cellular response to infection and should yield insights into the nature of chemotactic and inflammatory responses.
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