Molecular characterisation of antibiotic tolerance in Mycobacterium tuberculosis (360G-Wellcome-207487_A_17_Z)

Tuberculosis, caused by Mycobacterium tuberculosis still causes 1.8 million deaths per year and takes months to years to treat. Long treatment times are due to subpopulation(s) of bacteria that, although genetically susceptible, are not killed effectively by antibiotics – termed antibiotic tolerance. Understanding mechanisms of antibiotic tolerance is the key to shortening treatment times for tuberculosis. We have recently shown that mycobacteria use the essential amidotransferase GatCAB to regulate rates of specific errors in gene translation and that mistranslation is both necessary and sufficient for tolerance to rifampicin – the most important anti-tuberculous drug. However, the precise molecular mechanisms by which GatCAB and the mycobacterial translation machinery control fidelity are not understood. We have also recently identified further mechanisms which contribute to rifampicin tolerance. We now propose to determine the mechanism by which GatCAB modulates mistranslation rates. We also propose three complementary forward genetic screens to a) comprehensively identify the pathways that regulate mycobacterial translational fidelity; b) employ bacterial genome-wide association studies to identify mutations that influence rifampicin tolerance in clinical isolates, and c) use saturating transposon insertion mutagenesis and deep sequencing to identify mycobacterial genes that cause differential rifampicin susceptibility in a murine model of antibiotic treatment.