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

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.