Tuberculosis remains the leading cause of preventable deaths. Natural spread of multidrug resistant tuberculosis and the potential use of such strains by terrorists make discovery of new targets for antimycobacterial therapy a very critical need. Cell wall lipids constitute a major physical and chemical defensive barrier that helps the pathogen evade the host defenses and antimycobacterial drugs. Therefore synthesis of such unique lipids that are critical for infection can be suitable targets for new antimycobacterial drugs. Multiple methyl branched lipids such as dimycocerosyl phthiocerol (DIM) have been shown to be virulence factors. Elucidation of the biochemical reactions and the nature of the enzymes involved in the biosynthesis of such virulence factors is required for developing novel drags. To this end we propose to 1. Elucidate the functions of lipase genes and their possible role in pathogenesis, a) Express and characterize the catalytic capabilities of lip gene products and determine whether disruption of specific lip genes will result in the absence of a specific class of acyl-lipids or absence of a specific group of esterified fatty acids, and determine whether the mutations affect virulence, b) Directly test whether the expressed lip gene products can release the acyl chains from the expressed synthases in vitro. 2. Elucidate the biological function of tes genes and their possible role in virulence, a) Express tes genes and characterize the catalytic capabilities of their products, b). Disrupt the three tes genes, determine the effects on lipid metabolism by using laC-labeled acetate and """"""""C-labeled propionate as radiotracers and determine the effect of the mutations on virulence of the pathogen in the murine model. 3. Elucidate the function of wes genes, a) Express the wes genes and test whether these gene products are involved in the esterification of the methyl branched acids generated by the large synthases to the hydroxyl groups in the ultimate acceptors, b) Determine the consequences of disrupting the wes genes on lipid metabolism and test the virulence of mutants that show novel biochemical phenotypes. 4. a) Elucidate the role of the novel e subunit in the enzyme that catalyzes the synthesis of methylmalonyl- CoA, the building block for the multiple methyl branched virulence factors, b) Determine whether disruption of accD4 and accD5 affect propionyl-CoA carboxylation, and methylbranched lipid synthesis in M. tuberculosis and its virulence.c) Determine the possible role of succinate as a source for methylmalonyl-CoA. The results of this study are likely to give information and tools for screening chemical libraries to discover new types of drug candidates directed at novel targets in M. tuberculosis and thus help in combating multidrug resistant tuberculosis.
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