Identification of Novel Combinatorial Drug Targets in Mycobacterium tuberculosis

Tuberculosis caused by the Mycobacterium tuberculosis complex (MTBC) remains one of the most important infectious diseases of mankind. Isoniazid (INH) and rifampicin are the main first line drugs used in multidrug treatment of TB but phenotypic tolerance and the development of resistance against these compounds and other TB drugs is a serious and increasing problem. The overall aim of this project is to utilise transposon sequencing (Tnseq) genetic screens of MTBC to identify novel drug targets that function maximally when in combination with INH or rifampicin or increase the sensitivity to these drugs. Drugs directed to these targets could be developed as part of new multidrug therapies. INH is a prodrug which requires activation by the mycobacterial catalase/peroxidase, KatG to forman isonicotinoyl acyl radical, which binds to NAD+/NADH and inhibits enoyl-[acyl-carrier-protein] reductase (InhA)-dependent synthesis of cell wall mycolic acid. A Tnseq screen of BCG in a sub-MIC concentration of INH identified genes whose disruption increased apparent sensitivity to INH. A subset of these genes were functionally related to oxidative stress and were selected for further investigation of individualwild-type strains, mutants and complemented mutants. KatG activity was measured with a catalase assay to test the hypothesis that absence of the oxidative stress gene leads in the production of more KatG which allows for increased activation of the drug INH and increased sensitivity to INH. In a second Tnseq screen, mutants were identified with enhanced sensitivity to rifampicin. The significance of these genes will be investigated further and discussed.