A New Dehydratase Conferring Innate Resistance to Thiacetazone and Intra‐amoebal Survival of Mycobacterium Smegmatis

Nontuberculous mycobacteria are innately resistant to most antibiotics, although the mechanisms responsible for their drug resistance remain poorly understood. They are particularly refractory to thiacetazone (TAC), a second‐line antitubercular drug. Herein, we identified MSMEG_6754 as essential for the innate resistance of Mycobacterium smegmatis to TAC. Transposon‐mediated and targeted disruption of MSMEG_6754 resulted in hypersusceptibility to TAC. Conversely, introduction of MSMEG_6754 into Mycobacterium tuberculosis increased resistance 100‐fold. Resolution of the crystal structure of MSMEG_6754 revealed a homodimer in which each monomer comprises two hot‐dog domains characteristic of dehydratase‐like proteins and very similar to the HadAB complex involved in mycolic acid biosynthesis. Gene inactivation of the essential hadB dehydratase could be achieved in M. smegmatis and M. tuberculosis only when the strains carried an integrated copy of MSMEG_6754, supporting the idea that MSMEG_6754 and HadB share redundant dehydratase activity. Using M. smegmatis‐Acanthamoeba co‐cultures, we found that intra‐amoebal growth of the MSMEG_6754 deleted strain was significantly reduced compared with the parental strain. This in vivo growth defect was fully restored upon complementation with catalytically active MSMEG_6754 or HadABC, indicating that MSMEG_6754 plays a critical role in the survival of M. smegmatis within the environmental host.