Whole-genome sequencing and comparative genomic analysis of mycobacterium smegmatis mutants resistant to imidazo[1,2-b][1,2,4,5]tetrazines, antituberculosis drug candidates

Abstract

The spread of multidrug and extensively drug-resistant Mycobacterium tuberculosis urges the development of novel antituberculosis drugs. Previously, we studied the compounds representing the class of substituted imidazo[1,2-b][1,2,4,5] tetrazines capable of inhibiting serine/threonine protein kinases (STPK) in the original M. smegmatis aphVIII+ test-system. To unveil the mechanism of action of drug candidates, it is necessary to search for mutations in the mycobacterial genome that confer resistance to these compounds. The aim of our work was to find and describe such mutations in M. smegmatis strains. We carried out the whole-genome sequencing of 9 mutants resistant to 3 imidazo[1,2-b][1,2,4,5]tetrazines. Seven of 9 mutant strains were found to have the Y52H mutation in the highly conserved mycobacterial gene MSMEG_1601 encoding a protein with an unknown function. Additionally, three of those 7 strains were shown to have two mutations in the MSMEG_1380 encoding a transcriptional regulator. The remaining 2 mutant strains had mutations in MSMEG_0641 and MSMEG_2087 genes encoding transporter-proteins. No mutations were found in STPK genes, meaning that they might be not the primary targets of the studied compounds. Further investigation of MSMEG_1601 function may be of interest as this protein might be the biological target or a part of a new mechanism underlying resistance to antituberculosis drug candidates.