Binding pocket alterations in dihydrofolate synthase confer resistance to para-aminosalicylic acid in clinical isolates of Mycobacterium tuberculosis

Abstract

The mechanistic basis for the resistance of Mycobacterium tuberculosis to para-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 precursor para-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 in M. tuberculosis strain H37Rv. Here, we demonstrate that various missense mutations within the coding sequence of the dihydropteroate (H2Pte) binding pocket of dihydrofolate synthase (FolC) confer PAS resistance in laboratory isolates of M. tuberculosis and Mycobacterium bovis. From a panel of 85 multidrug-resistant M. tuberculosis clinical isolates, 5 were found to harbor mutations in the folC gene within the H2Pte binding pocket, resulting in PAS resistance. While these alterations in the H2Pte 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 of folC fully restored PAS susceptibility in folC mutant strains. Confirmation of this novel PAS resistance mechanism will be beneficial for the development of molecular method-based diagnostics for M. tuberculosis clinical isolates and for further defining the mode of action of this important tuberculosis drug.

FIG 1

(A) Ribbon diagram of the N-domain FolC (PDB code 2VOS). The locations of six mutations are shown as thick colored bands (Ile43, Arg49, Ser150, Phe152, Glu153, and Ala183), while the ADP is shown as fine bands. (B) As shown, all the mutated residues either reside in the H₂Pte binding loop (α1-α2, residues 36 to 50) or are situated in the α4-α5 loop. The residue Glu40 was not included in the model.

FIG 2

DHFS activity of FolC mutants. The DHFS activities of the purified wild-type FolC and FolC mutants were determined using H₂Pte as the substrate. The enzyme activity was assayed as described in Materials and Methods. The data represent the means ± the standard deviations (SD) from three independent experiments.

FIG 3

HPLC-MS analysis of H₂PtePAS and H₂PtePAS-Glu. (A) H₂PtePAS and H₂PtePAS-Glu coexisted in the reaction system when not all of the H₂PtePAS was consumed by wild-type FolC. (B and C) Wild-type FolC could catalyze ligation of glutamate to H₂PtePAS to produce H₂PtePAS-Glu in the presence of ATP and Mg²⁺ (B); however, the FolC mutants could not catalyze this reaction (C). (D) For H₂PtePAS, the calculated weight was 330.30 and found weight was ∼329.21 ([M-H]⁻). (E) For H₂PtePAS-Glu, the calculated weight was 459.42 and found weight was ∼458.27 ([M-H]⁻). All MS data were acquired in the negative mode.

FIG 4

H₂Pte antagonized the effect of PAS on M. tuberculosis H37Ra. The survival of the bacterial cells after 36 h of treatment was determined by the XTT reduction assay as described in Materials and Methods. H37Ra (H₂Pte) indicates that bacterial cells were treated with H₂Pte at different concentrations (0, 0.1, 0.3, 1, 3, and 10 μg/ml), while H37Ra (H₂Pte + PAS) indicates that bacterial cells were treated with both H₂Pte (0, 0.1, 0.3, 1, 3, and 10 μg/ml) and PAS (0.1 μg/ml). The data represent the means (bars) ± the SD (error bars) from three independent experiments.

FIG 5

Schematic of FolC-mediated PAS resistance in M. bovis and M. tuberculosis. PAS is incorporated into the folate pathway by FolP1 via competition with PABA, yielding H₂PtePAS. (A) Both wild-type FolC and the FolC mutants can catalyze the formation of H₂Pte-Glu. (B) In the case of H₂PtePAS, the wild-type FolC further incorporates it to produce H₂PtePAS-Glu, whereas the FolC mutants do not. FolC_wt, wild-type FolC; FolC_mut, FolC mutants. The dotted arrow indicates the enzymatic activities of the FolC mutants are greatly reduced compared with that of wild-type FolC, and the fork indicates that this pathway is blocked.