Competition between H4PteGlu and H2PtePAS Confers para-Aminosalicylic Acid Resistance in Mycobacterium tuberculosis

Abstract

Tuberculosis remains a serious challenge to human health worldwide. para-Aminosalicylic acid (PAS) is an important anti-tuberculosis drug, which requires sequential activation by Mycobacterium tuberculosis (M. tuberculosis) dihydropteroate synthase and dihydrofolate synthase (DHFS, FolC). Previous studies showed that loss of function mutations of a thymidylate synthase coding gene thyA caused PAS resistance in M. tuberculosis, but the mechanism is unclear. Here we showed that deleting thyA in M. tuberculosis resulted in increased content of tetrahydrofolate (H₄PteGlu) in bacterial cells as they rely on the other thymidylate synthase ThyX to synthesize thymidylate, which produces H₄PteGlu during the process. Subsequently, data of in vitro enzymatic activity experiments showed that H₄PteGlu hinders PAS activation by competing with hydroxy dihydropteroate (H₂PtePAS) for FolC catalysis. Meanwhile, over-expressing folC in ΔthyA strain and a PAS resistant clinical isolate with known thyA mutation partially restored PAS sensitivity, which relieved the competition between H₄PteGlu and H₂PtePAS. Thus, loss of function mutations in thyA led to increased H₄PteGlu content in bacterial cells, which competed with H₂PtePAS for catalysis by FolC and hence hindered the activation of PAS, leading to decreased production of hydroxyl dihydrofolate (H₂PtePAS-Glu) and finally caused PAS resistance. On the other hand, functional deficiency of thyA in M. tuberculosis pushes the bacterium switch to an unidentified dihydrofolate reductase for H₄PteGlu biosynthesis, which might also contribute to the PAS resistance phenotype. Our study revealed how thyA mutations confer PAS resistance in M. tuberculosis and provided new insights into studies on the folate metabolism of the bacterium.

Keywords: Mycobacterium tuberculosis; para-aminosalicylic acid; tetrahydrofolate; thyA.

Figure 1

Schematic diagram of the mechanism of PAS action. PAS, para-aminosalicylic acid; pABA, para-aminobenzoic acid; H₂PtePAS, hydroxy dihydropteroate; H₂Pte, dihydropteroate; H₂PtePAS-Glu, hydroxy dihydrofolate; H₂PteGlu, dihydrofolate; H₄PteGlu, tetrahydrofolate; 5, 10-m-H₄PteGlu, 5, 10-methylenetetrahydrofolate; DHPS/FolP1, dihydropteroate synthetase; DHFS/FolC, dihydrofolate synthase; DHFR/DfrA, dihydrofolate reductase; ThyA, thymidylate synthase; ThyX, thymidylate synthase; RibD, bifunctional diaminohydroxyphosphoribosylaminopyrimidine deaminase/5-amino-6-(5-phosphoribosylamino) uracil reductase.

Figure 2

The expression of folC remains unchanged in ThyA functional deficient strain. (A) Comparison of the expressional level of FolC during the exponential phase in H37Ra (WT) and H37Ra ΔthyA (thyA⁻) by Western blot assay. Upper part: Total protein was normalized to 25 μg of each strain, then electrophoresed by SDS-PAGE and stained by Coomassie brilliant blue. Lower part: Western blot analysis of total protein immunoblotted with rabbit FolC polyclonal antibody. Experiments were repeated at least three times, and were performed three biological replicates each time. (B) Relative quantitative of FolC product by Western blot assay. ns, no significance. (C) Comparison of the transcriptional level of the gene folC during the exponential phase in H37Ra (WT) and H37Ra ΔthyA (thyA⁻) by RNA-seq. ns, no significance.

Figure 3

The quantitative detection of H₄PteGlu by UPLC-MS/MS in thyA deletion strain. Cell-associated H₄PteGlu was extracted from H37Ra (WT) and H37Ra ∆thyA (thyA^-). The experiments were performed using six biological replicates. p-values (p) were calculated using t-tests. ** p < 0.01.

Figure 4

Catalytic utilization of H₂Pte, H₄PteGlu, and H₂PtePAS by DHFS. The experiments were performed using three biological replicates. p-values (p) were calculated using t-tests. ** p < 0.01, *** p < 0.001.

Figure 5

H₄PteGlu hinders the activation of PAS. (A) H₂PtePAS was identified based on HPLC-MS/MS. Retention time 3.193 min, ion channel 331.0 -> 178.0. (B) H₂PtePAS-Glu was identified based on HPLC-MS/MS. Retention time 3.181 min, ion channel 460.0 -> 178.0. (C) Extracted ion chromatograms of H₂PtePAS-Glu showing H₄PteGlu reduced the catalytic efficiency of FolC on H₂PtePAS.