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.

The existence of 5-hydroxymethylcytosine and 5-formylcytosine in both DNA and RNA in mammals.

We developed a novel strategy by oxidation-derivatization combined mass spectrometry analysis for the determination of 5-hydroxymethylcytosine and 5-formylcytosine in both DNA and RNA. We reported the presence of 5-formylcytosine in RNA of mammals and found that ascorbic acid and hydroquinone can increase the oxidation of 5-methylcytosine to 5-hydroxymethylcytosine in DNA and RNA.