The overall objective of this work is the development of novel chemotherapeutics and chemotherapeutic targets for the treatment of diseases of Mycobacterial origin including tuberculosis, leprosy, and M. avium-intracellulare (MAC) infections of AIDS patients. Pathogenic strains of mycobacteria uniformly modify their major mycolic acids by cyclopropanation while non-pathogenic strains do not. We have used this distinction to clone two genes from a pathogenic species (M. tuberculosis (MTB)) into a non-pathogenic species (M. smegmatis(MSMEG)) which cyclopropanate mycolic acids. cma1 has been shown to function to cyclopropanate the distal cis double bond in the longer meromycolate branch, while cma2 cyclopropanates the proximal double bond. By coexpressing both genes, we have been able to entirely recreate the normal MTB major alpha mycolic acid in MSMEG as well as create two novel mycolates which are hybrids of the MTB and MSMEG structural types by individual expression. Aside from three mycobacterial genes (including a homolog of cma2 we have identified from M. leprae) these enzymes are homologous to the only known enzyme with a related function, the E. coli cyclopropane fatty acid synthase. Using these constructs we have shown that cyclopropanation of cell-wall lipids protects MSMEG from oxidative killing, suggesting a potential role in pathogenesis. In addition we have shown by physical measurements that cyclopropanation changes the fluidity and structure of the mycobacterial cell wall and are using the recombinant organisms to allow a definition of the cell wall structure. Surrounding DNA sequences encodes proteins which are clearly involved in mycolic acid biosynthesis and are revealing new clues about the biosynthesis, regulation, and functions of mycolic acids. Since isoniazid, which directly affects mycolic acid biosynthesis, is an effective chemotherapeutic, each of these genes independently represents a potential drug target. We are actively pursuing the development of cell-free activity assays and the syntheses of compounds designed to inhibit these steps as potential chemotherapeutics for the treatment of diseases of mycobacterial etiology.
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