MmpS5-MmpL5 Transporters Provide <i>Mycobacterium smegmatis</i> Resistance to imidazo[1,2-<i>b</i>][1,2,4,5]tetrazines

The emergence and spread of drug-resistant Mycobacterium tuberculosis strains (including MDR, XDR, and TDR) force scientists worldwide to search for new anti-tuberculosis drugs. We have previously reported a number of imidazo[1,2- b ][1,2,4,5]tetrazines - putative inhibitors of mycobacterial eukaryotic-type serine-threonine protein-kinases, active against M. tuberculosis . Whole genomic sequences of spontaneous drug-resistant M. smegmatis mutants revealed four genes possibly involved in imidazo[1,2- b ][1,2,4,5]tetrazines resistance; however, the exact mechanism of resistance remain unknown. We used different approaches (construction of targeted mutants, overexpression of the wild-type ( w.t. ) and mutant genes, and gene-expression studies) to assess the role of the previously identified mutations. We show that mutations in MSMEG_1380 gene lead to overexpression of the mmpS5-mmpL5 operon in M. smegmatis , thus providing resistance to imidazo[1,2- b ][1,2,4,5]tetrazines by increased efflux through the MmpS5-MmpL5 system, similarly to the mechanisms of resistance described for M. tuberculosis and M. abscessus . Mycobacterial MmpS5-MmpL5 transporters should be considered as an MDR-efflux system and they should be taken into account at early stages of anti-tuberculosis drug development.