Decreased Methylenetetrahydrofolate Reductase Activity Leads to Increased Sensitivity to para-Aminosalicylic Acid in Mycobacterium tuberculosis

Tuberculosis (TB), caused by Mycobacterium tuberculosis, is one of the most fatal diseases in the world. Methylenetetrahydrofolate reductase (MTHFR) catalyzes the production of 5-methyltetrahydrofolate (5-CH(3)-THF), which is required for the de novo biosynthesis of methionine in bacteria. Here, we identified Rv2172c as an MTHFR in M. tuberculosis through in vitro and in vivo analyses and determined that the protein is essential for the in vitro growth of the bacterium. Subsequently, we constructed rv2172c R159N and L214A mutants in M. tuberculosis and found that these mutants were more sensitive to the antifolates para-aminosalicylic acid (PAS) and sulfamethoxazole (SMX). Combining biochemical and genetic methods, we found that rv2172c R159N or L214A mutation impaired methionine production, leading to increased susceptibility of M. tuberculosis to PAS, which was largely restored by adding exogenous methionine. Moreover, overexpression of rv2172c in M. tuberculosis could increase methionine production and lead to PAS resistance. This research is the first to identify an MTHFR in M. tuberculosis and reveals that the activity of this enzyme is associated with susceptibility to antifolates. These findings have particular value for antitubercular drug design for the treatment of drug-resistant TB.