The anaerobic organism, Bacteroides fragilis is exceptionally resistant to the toxic effects of oxygen and other oxidative stress. This resistance can be attributed to induction of an oxidative stress response (OSR) and this response will be studied to document its role in the pathogenesis of Bacteroides fragilis infection. It is expected that new mechanisms of free radical protection and novel antioxidant defense strategies will be uncovered. This idea is supported by the fact that B. fragilis diverged very early from other eubacteria and has independently evolved the ability to survive extended exposure to oxygen. Further, since this is an anaerobe, it is likely to have stringent requirements for oxygen protection that have resulted in the development of unique highly protective antioxidants. The long term goals of this research are to understand the basic physiological and genetic processes responsible for tolerance of B. fragilis to oxidative stress and to determine how these contribute to virulence. The objectives for this proposal are: 1) Define and characterize the OSR at the protein and genetic levels. Initially, attention will focus on a subset of the OSR, resistance to H2O2 and peroxides (HPR). A set of HPR genes will be cloned and analyzed, and these data will be used to establish a catalog of HPR proteins expressed during the course of oxidative stress. 2) Regulation of the HPR regulon will be studied initially using a model system employing the catalase gene, katB. This will focus on transcriptional regulation and the identification of oxidative stress regulatory molecules. The system will be expanded to include the HPR genes as they become available. 3) Determine the effect of specific mutations in HPR-controlled genes on survival to oxidative stress. Mutants in one or more of these genes will be constructed by allelic exchange and tested for their ability to survive various forms of oxidative stress. Mutants will be subjected to a variety of biochemical tests to determine their mechanism of protection e.g. DNA repair or macromolecule protection. 4) The regulator responsible for control of the HPR regulon will be cloned and its role in the overall OSR will be determined.
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