The T120P or M172V mutation on rv2172c confers high level para-aminosalicylic acid resistance in Mycobacterium tuberculosis.

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.

Genome-wide DNA methylation and transcriptome and proteome changes in Mycobacterium tuberculosis with para-aminosalicylic acid resistance.

Previous studies have reported that genome-wide DNA methylation and differentially expressed genes and proteins are closely associated with drug resistance in Mycobacterium tuberculosis (M. tuberculosis). However, no reports have explored such associations in para-aminosalicylic acid (PAS)-resistant M. tuberculosis H37Rv. Here, we investigated genome-wide methylation and transcriptome and proteome changes to explore the associations between specific genes and PAS resistance in M. tuberculosis H37Rv. The results revealed that 1,388 differentially methylated (1,161 hypermethylated and 227 hypomethylated) genes, 214 significantly differentially expressed (103 up- and 111 down-regulated) genes and 137 differentially expressed (48 up- and 89 down-regulated) proteins were regulated by PAS in M. tuberculosis H37Rv. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis revealed that metabolic pathways and ABC transporters were closely associated with differentially methylated and expressed genes, respectively. In addition, correlation analysis revealed that differentially methylated genes were negatively correlated with their transcriptional levels in PAS-resistant M. tuberculosis H37Rv. Furthermore, the existence of five hypermethylated candidate genes (esxC, fabG3, fbpB, papA1 and pks2) in PAS-resistant M. tuberculosis H37Rv was verified using protein-protein interaction analysis in the STRING database. The integrated DNA methylation and transcriptome and proteome analysis could provide valuable resources for epigenetics studies in PAS-resistant M. tuberculosis H37Rv.