The long-term goal of this project is to improve our understanding of mechanisms of drug action and drug resistance in Mycobacterium tuberculosis and to provide knowledge important for designing new tuberculosis (TB) drugs. Two significant problems seriously compromise our ability to effectively control TB: the emergence of drug-resistant strains, and the lengthy 6 month TB chemotherapy. There is an urgent need to develop new TB drugs that are effective in both aspects. Pyrazinamide (PZA) is a paradoxical front-line TB drug, because while PZA has powerful in vivo sterilizing activity, through killing a population of semi-dormant organisms, and is thus involved in shortening the TB therapy to 6 month; PZA has no activity in vitro against M. tuberculosis under normal culture conditions except under acid pH (5.5). The mode of action of PZA in M. tuberculosis is unknown, and this project proposes to address this issue.
The specific aims of the project are: (1) To determine the effect of pyrazinoic acid (POA), the active form of PZA, on membrane integrity and nutrient transport of M. tuberculosis. (2) To test the potential inhibition of POA on the NAD metabolism in M. tuberculosis. (3) To identify and characterize the efflux genes involved in pumping POA out of the mycobacteria. The health relatedness of this project lies in the possible design of new antituberculous drugs that may further shorten the current 6 month TB chemotherapy based on understanding the mode of action of PZA. The investigators hypothesize that POA as a lipophilic weak acid will partition into the lipid-rich membrane of M. tuberculosis and cause structural and functional damage to the membrane. This hypothesis will be tested in Aim I by EM analysis, by measuring the nutrient transport using radiolabeled amino acids, uracil and thymidine, and by measuring the possible effect of POA on the membrane potential. Because of structural similarity of POA to niacin, they hypothesize that POA may interfere with NAD metabolism in M. tuberculosis by feedback inhibition of NAD biosynthesis and by incorporation as a pseudo-NAD molecule without NAD function. This hypothesis will be tested in Aim 2 by using C 14-labeled precursors of NAD and by tracing C 14-POA in NAD molecule by mass spec and NMR analyses. Because they found the activity of efflux pumps extruding POA in various mycobacteria varies widely and correlates with their susceptibility to PZA, they propose to identify the POA efflux genes by testing putative efflux genes in the Sanger Centre M. tuberculosis genome database and by testing mycobacterial genomic DNA libraries for their ability to confer PZA resistance in M. tuberculosis.
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