Abstract:
Isoniazid (INH), one of the first-line drugs used for the treatment of tuberculosis, is a prodrug which is activated by the intracellular KatG enzyme of Mycobacterium tuberculosis The activated drug hinders cell wall biosynthesis by inhibiting the InhA protein. INH-resistant strains of M. tuberculosis usually have mutations in katG, inhA, ahpC, kasA, and ndh genes. However, INH-resistant strains which do not have mutations in any of these genes are reported, suggesting that these strains may adopt some other mechanism to become resistant to INH. In the present study, we characterized Rv2170, a putative acetyltransferase in M. tuberculosis, to elucidate its role in inactivating isoniazid. The purified recombinant protein was able to catalyze the transfer of the acetyl group to INH from acetyl coenzyme A (acetyl-CoA). High-performance liquid chromatography (HPLC) and liquid chromatography-mass spectrometry (LC-MS) analyses showed that following acetylation by Rv2170, INH is broken down into isonicotinic acid and acetylhydrazine. A drug susceptibility assay and confocal analysis showed that Mycobacterium smegmatis, which is susceptible to INH, is not inhibited by INH acetylated with Rv2170. Mutant proteins of Rv2170 failed to acetylate INH. Recombinant M. smegmatis and M. tuberculosis H37Ra overexpressing Rv2170 were found to be resistant to INH at MICs that inhibited wild-type strains. Besides, intracellular M. tuberculosis H37Ra overexpressing Rv2170 survived better in macrophages when treated with INH. Our results strongly indicate that Rv2170 acetylates INH, and this could be one of the strategies adopted by at least some M. tuberculosis strains to overcome INH toxicity, although this needs to be tested in INH-resistant clinical strains.
