para-Aminosalicylic acid is a prodrug targeting dihydrofolate reductase in Mycobacterium tuberculosis

Abstract

para-Aminosalicylic acid (PAS) is one of the antimycobacterial drugs currently used for multidrug-resistant tuberculosis. Although it has been in clinical use for over 60 years, its mechanism(s) of action remains elusive. Here we report that PAS is a prodrug targeting dihydrofolate reductase (DHFR) through an unusual and novel mechanism of action. We provide evidences that PAS is incorporated into the folate pathway by dihydropteroate synthase (DHPS) and dihydrofolate synthase (DHFS) to generate a hydroxyl dihydrofolate antimetabolite, which in turn inhibits DHFR enzymatic activity. Interestingly, PAS is recognized by DHPS as efficiently as its natural substrate para-amino benzoic acid. Chemical inhibition of DHPS or mutation in DHFS prevents the formation of the antimetabolite, thereby conferring resistance to PAS. In addition, we identified a bifunctional enzyme (riboflavin biosynthesis protein (RibD)), a putative functional analog of DHFR in a knock-out strain. This finding is further supported by the identification of PAS-resistant clinical isolates encoding a RibD overexpression mutation displaying cross-resistance to genuine DHFR inhibitors. Our findings reveal that a metabolite of PAS inhibits DHFR in the folate pathway. RibD was shown to act as a functional analog of DHFR, and as for DHFS, both were shown to be associated in PAS resistance in laboratory strains and clinical isolates.

Keywords: Antibiotic Action; Antibiotic Resistance; Drug Development; Folate Metabolism; Microbiology; Mycobacterium tuberculosis.

FIGURE 1.

dfrA contributes to PAS resistance in M. tuberculosis. A, H37Rv overexpressing dfrA but not folC is resistant to PAS. OD, optical density. B, PAS does not inhibit rDHFR enzymatic activity as compared with the specific DHFR inhibitor WR99210 with the DHFR assay. The values represent the means ± S.D. from one representative experiment performed with triplicate samples.

FIGURE 2.

PAS is activated intracellularly to target DHFR. A and B, CLFTs from PAS-treated M. bovis BCG (A) and M. tuberculosis (B) inhibit rDHFR activity. WR99210 and streptomycin (Strep) were used as positive and negative controls, respectively. OD, optical density. C, PAS intracellular activation is time-dependent. D, PAS inhibition was decreased upon the addition of pABA. E, providing pABA in PAS-treated mycobacterial cells neutralized the inhibition of CLFT on rDHFR. The values represent the means ± S.D. from one representative experiment performed with triplicate samples. The experiments were carried out in three independent biological replicates resulting in the same conclusion.

FIGURE 3.

A model of PAS mechanism of action in Mycobacterium. The normal folate pathway is depicted on the left side. As a pABA analog, PAS is incorporated into the folate pathway by competing with pABA in the reaction catalyzed by DHPS, the product of which is further processed by DHFS to generate hydroxyl dihydrofolate (right side). This antimetabolite in turn inhibits DHFR activity (denoted by the T bar) and thus blocks the folate pathway (denoted by the cross).

FIGURE 4.

PAS is incorporated into the folate pathway and bioactivated into an antimetabolite at the level of DHPS. A and B, DHPS catalyzes the reaction of H₂PtPP and its natural substrate pABA (A) and PAS (B) with a similar affinity. The Michaelis-Menten values (K_m values) of pABA and PAS to DHPS are labeled. O.D., optical density. C, LC/MS/MS chromatography revealed a peak at 329 (m/z) corresponding to hydroxyl dihydropteroate identified from the reaction mixture by the Q1 full scan. D, the ion was further fragmented into two peaks at 176 (m/z) and 151 (m/z) corresponding to H₂PtPP and PAS, respectively, in the Q3 scan. E, the chromatograms monitoring this ion demonstrated a single peak, suggesting that hydroxyl dihydropteroate was produced by DHPS with PAS and H₂PtPP as substrate.

FIGURE 5.

Enzymes in the folate pathway are required for PAS activation. A, inhibition of DHPS antagonizes PAS-mediated inhibition, as shown by the growth inhibition curves of PAS in the presence of increasing sulfathiazole (Sul) concentrations. OD, optical density. B, the addition of sulfathiazole to PAS-treated mycobacterial cultures suppresses the inhibition of rDHFR activity by CLFT. C, mutation in folC (folCE40A) reduces M. tuberculosis susceptibility to PAS, and complementation with wild-type copy of folC (in pMV262) restores its susceptibility to wild-type level. The values in B and C represent the means ± S.D. from one representative experiment performed with triplicate samples.

FIGURE 6.

RibD is responsible for M. tuberculosis PAS resistance. A, chemical structure of NITD344. B, NITD344 inhibits M. tuberculosis rDHFR enzymatic activity demonstrated in the DHFR enzyme inhibition assay. OD, optical density. C, Western blot showing increased RibD expression in spontaneous NITD344-resistant mutant M. tuberculosis R7. D and E, both M. tuberculosis R7 (red squares) and H37Rv overexpressing ribD in pM262 (blue triangles) are resistant to NITD344 (D) and PAS (E). The values in B, D, and E represent the means ± S.D. from one representative experiment performed with triplicate samples.

FIGURE 7.

Overexpression of RibD compensates for the knock-out of DHFR in Mycobacterium. A, schematic representation of the genome region of the H37Rv wild type (WT) and knock-out mutant (KO) showing the relative gene organization of dfrA and its flanking genes, the locations of the probes for Southern blot analyses and PvuII restriction sites, and the corresponding fragment lengths. B, dfrA could be successfully knocked out only while overexpressing RibD in M. tuberculosis. The knock-out mutant of dfrA was confirmed by Southern blot using probes 1 and 2.