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Mycobacterium tuberculosis acetylates and inactivates isoniazid - a novel mechanism of isoniazid resistance

K. B. Arun, Aravind Madhavan and R. Ajay Kumar#

Mycobacterium Research Laboratory, Rajiv Gandhi Centre for Biotechnology, Thiruvananthapuram - 695014. Kerala, India.

#Corresponding Author

Dr. R. Ajay Kumar, Mycobacterium Research Laboratory, Rajiv Gandhi Centre for Biotechnology, Thiruvananthapuram - 695014. Kerala, India; rakumar@rgcb.res.in; Tel. +91-471-2529-513  

Isoniazid (INH), a first-line drug used for the treatment of tuberculosis, is a pro-drug and is activated by the KatG enzyme of Mycobacterium tuberculosis, and inhibits the bacterium by interfering with its cell wall biosynthetic pathway. Mutations in katG, inhA, ahpC, kasA, and ndh genes are identified in INH resistant strains of M. tuberculosis. However, INH resistant strains without mutations in any of these genes are also identified, indicating that these strains may adopt some other mechanism to resist INH toxicity. We characterised Rv2170, a putative acetyltransferase in M. tuberculosis, and propose a novel mechanism to explain its role in INH inactivation. Purified recombinant Rv2170 was able to catalyze the transfer of the acetyl group from acetyl CoA to INH. HPLC and LC-MS analyses of the acetylation reaction mixture revealed that acetylated INH is broken down into isonicotinic acid and acetylhydrazine. INH acetylated in vitro with recombinant Rv2170 was unable to inhibit susceptible M. smegmatis. Mutations at the INH-binding pocket of Rv2170 prevented the enzyme from acetylating INH. Overexpression of Rv2170 in M. smegmatis and M. tuberculosis H37Ra renders them resistant to INH at minimum inhibitory concentrations that inhibited wild-type strains. Recombinant intracellular M. tuberculosis H37Ra overexpressing Rv2170 survived significantly better in macrophages treated with INH. Our data strongly indicate that acetylation of INH by Rv2170 could be a strategy adopted by at least some M. tuberculosis strains to escape INH toxicity, although this needs to be validated in INH resistant clinical strains.

Keywords: Mycobacterium tuberculosis, isoniazid, drug resistance, acetyltransferase, acetylation