Binding Pocket Alterations in Dihydrofolate Synthase Confer Resistance to para -Aminosalicylic Acid in Clinical Isolates of Mycobacterium tuberculosis

The mechanistic basis for the resistance ofMycobacterium tuberculosis topara -aminosalicylic acid (PAS), an important agent in the treatment of multidrug-resistant tuberculosis, has yet to be fully defined. As a substrate analog of the folate precursorpara -aminobenzoic acid, PAS is ultimately bioactivated to hydroxy dihydrofolate, which inhibits dihydrofolate reductase and disrupts the operation of folate-dependent metabolic pathways. As a result, the mutation of dihydrofolate synthase, an enzyme needed for the bioactivation of PAS, causes PAS resistance inM. tuberculosis strain H37Rv. Here, we demonstrate that various missense mutations within the coding sequence of the dihydropteroate (H2 Pte) binding pocket of dihydrofolate synthase (FolC) confer PAS resistance in laboratory isolates ofM. tuberculosis andMycobacterium bovis . From a panel of 85 multidrug-resistantM. tuberculosis clinical isolates, 5 were found to harbor mutations in thefolC gene within the H2 Pte binding pocket, resulting in PAS resistance. While these alterations in the H2 Pte binding pocket resulted in reduced dihydrofolate synthase activity, they also abolished the bioactivation of hydroxy dihydropteroate to hydroxy dihydrofolate. Consistent with this model for abolished bioactivation, the introduction of a wild-type copy offolC fully restored PAS susceptibility infolC mutant strains. Confirmation of this novel PAS resistance mechanism will be beneficial for the development of molecular method-based diagnostics forM. tuberculosis clinical isolates and for further defining the mode of action of this important tuberculosis drug.