Long-term objectives are to characterize oxidative microbicidal mechanisms within human neutrophils (PMN) with a view towards developing rational therapeutic interventions for infected patients with defects in PMN number or function. Because of the great number and complexity of PMN microbicidal systems, the proposed studies will focus on microbicidal properties of a single PMN- derived enzyme, myeloperoxidase (MPO).
Specific aims are to identify early MPO-induced biochemical changes in target organisms and to evaluate the metabolic consequences of these changes. The goal is to determine which biochemical changes correlate best, and are perhaps responsible for, loss of replicative ability (viability). Preliminary studies have indicated a decrease in E. coli respiration that correlates well with loss of viability (r=0.97), suggesting a relationship between damage to the microbial respiratory chain and microbicidal activity. In the sequence of respiratory electron transport, from nutrients through dehydrogenates, quinones, and terminal oxidase cytochromes to 02, the principal MPO-mediated lesions appear to be in the dehydrogenase group with other components of the chain left substantially intact. Continuing investigations will focus on damage to individual dehydrogenase components: iron-sulfur centers and flavin cofactors. Metabolic consequences of impaired electron transport such as loss of transmembrane pH and electrochemical gradients (proton motive force), loss of active transport, and extensive ATP hydrolysis will be determined. Finally, attempts will be made to rescue organisms from MPO-mediated toxicity by providing growth conditions that bypass the need for products of MPO-oxidized systems. Successful rescue would suggest that damage to the bypassed systems was indeed responsible for the loss of viability observed under usual growth conditions. A further aim is to use the knowledge of MPO-mediated biochemical changes to distinguish MPO from other antimicrobial activity within intact PMNs. PMN microbicidal activity will be compared with MPO-specific changes in microbial structures and correlations will be sought. Specificity for MPO of the PMN-mediated changes will be confirmed using PMNs deficient in MPO (hereditary MPO-deficiency) or in H202 production (chronic granulomatous disease). These studies should identify microbial structures whose modification by PMN results in lethal injury. They may also offer clues for the design of new antimicrobial agents and suggest reasons why certain organisms are able to evade PMN-mediated host defense systems.
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