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Abstract：
Although para-aminosalicylic acid (PAS) has been used to treat tuberculosis for decades, mechanisms of resistance to this drug in Mycobacterium tuberculosis (M. tuberculosis) clinical isolates have not been thoroughly investigated. Previously, we found that decreased methylenetetrahydrofolate reductase (MTHFR) activity of Rv2172c led to increased sensitivity to antifolates in M. tuberculosis. In this study, we collected the genome-sequencing data of 173 PAS-resistant and 803 PAS-sensitive clinical isolates and analyzed rv2172c mutations in those 976 isolates. The results showed that two mutations (T120P and M172V) on rv2172c could be identified in a certain proportion (6.36%) of PAS-resistant isolates. The results of AlphaFold2 prediction indicated that the T120P or M172V mutation might affect the enzymatic activity of Rv2172c by influencing nicotinamide adenine dinucleotide (NADH) binding, and this was verified by subsequent biochemical analysis, demonstrating the role of residues Thr120 and Met172 on NADH binding and enzymatic activity of Rv2172c. In addition, the effect of rv2172c T120P or M172V mutation on methionine production and PAS resistance was determined in M. tuberculosis. The results showed that both T120P and M172V mutations caused increased intracellular methionine concentrations and high level PAS resistance. In summary, we discovered new molecular markers and also a novel mechanism of PAS resistance in M. tuberculosis clinical isolates and broadened the understanding of the NADH-dependent MTHFR catalytic mechanism of Rv2172c in M. tuberculosis, which will facilitate the molecular diagnosis of PAS resistance and also the development of new drugs targeting Rv2172c.
摘要：
尽管对氨基水杨酸（PAS）已被用于治疗结核病数十年，结核分枝杆菌对这种药物的耐药机制(M.结核病）尚未对临床分离株进行彻底调查。以前,我们发现,Rv2172c亚甲基四氢叶酸还原酶(MTHFR)活性降低导致结核分枝杆菌抗叶酸药物敏感性增加.在这项研究中,我们收集了173株PAS耐药和803株PAS敏感临床分离株的基因组测序数据,并分析了这976株分离株中的rv2172c突变.结果表明，在一定比例（6.36％）的PAS抗性分离株中，可以在rv2172c上鉴定出两个突变（T120P和M172V）。AlphaFold2预测结果表明，T120P或M172V突变可能通过影响烟酰胺腺嘌呤二核苷酸（NADH）结合来影响Rv2172c的酶活性，随后的生化分析证实了这一点，证明残基Thr120和Met172对NADH结合和Rv2172c的酶活性的作用。此外,在结核分枝杆菌中确定了rv2172cT120P或M172V突变对甲硫氨酸产生和PAS抗性的影响.结果表明，T120P和M172V突变均导致细胞内蛋氨酸浓度增加和高水平的PAS抗性。总之,我们在结核分枝杆菌临床分离株中发现了新的分子标记和PAS耐药的新机制，并拓宽了对结核分枝杆菌中Rv2172c的NADH依赖性MTHFR催化机制的理解，这将有助于PAS耐药性的分子诊断以及靶向Rv2172c的新药的开发。
