Mycobacterium-avium intracellulare is a major opportunistic pathogen of immunosuppressed patients, especially AIDS patients. M. tuberculosis has assumed greater significance with the identification of multi-drug resistant strains. Folate metabolism is well-characterized in a number of other organisms and provides a suitable target for therapeutic intervention. Folate metabolism in mycobacteria, however, has not been characterized. To see if antifols would be potentially active as therapeutic agents for the treatment of MAI infection, we are undertaking studies to characterized folate metabolism in this organism. Studies began by pulsing MAI cultures with both para-aminobenzoic acid and folic acid as well as other reduced folates. In these studies we have been able to show that MAI can synthesize folates de novo. Over the past year we have confirmed studies evaluating the ability of inhibitors of dihydropteroate synthase, as well as dihydrofolate reductase, important enzymes in folate synthesis, to inhibit replication of MAI and M. tuberculosis. Such inhibitors include sulfonamides, dapsone, trimethoprim, and trimetrexate. We have previously demonstrated that some of these drugs can inhibit growth of MAI in liquid culture media at concentrations achievable in patients, although the levels are substantially higher than are needed in vitro to inhibit the target enzyme in crude enzyme preparations. Over the past year we have evaluated the activity of these drugs in cell-culture systems, since Mycobacteria are intracellular pathogens, and efficacy of drugs may be different for free compared to intracellular organisms. The availability of recombinant enzyme would allow the large-scale screening for inhibitors of DHPS that would be active against MAI. We have thus had an MAI library made and have been attempting to clone the DHPS of MAI using immunoscreening as well as DHPS genes from other organisms. The goal of these studies is to identify and improve therapeutic agents for the treatment and prevention of mycobacteria infection.
