MSMEG_1963 and MSMEG_5597 Genes, but Not inhA, Modulate Mycobacterium smegmatis Resistance to Tryptanthrins

The spread of drug-resistant strains of Mycobacterium tuberculosis is a major threat to the global control of tuberculosis (TB), urging the need to constantly develop new anti-TB drugs. Tryptanthrins are convenient compounds for the development of candidate anti-TB drugs due to the easy synthesis, low toxicity, and antimycobacterial activity on both drug-susceptible and drug-resistant M. tuberculosis strains. Enoyl-acyl carrier protein reductase InhA was previously predicted in silico as a possible target for tryptanthrins, while spontaneous tryptanthrin-resistant M. smegmatis mutants were found to have mutations in the MSMEG_1963, MSMEG_4427, and MSMEG_5597 genes. Using the approaches of reverse genetics, we demonstrate that mutations in the MSMEG_1963 and MSMEG_5597 genes lead to a loss of function of their encoded transcriptional repressors and lead to resistance to tryptanthrins. We show that mutations in MSMEG_1963 and MSMEG_5597 lead to overexpression of MSMEG_1964 and MSMEG_5596, respectively, which encode enzymes potentially involved in redox inactivation of tryptanthrins. We also show that InhA is not a biotarget of tryptanthrins, as its overexpression does not affect susceptibility of mycobacteria to tryptanthrins.