Mutations in dfrA and its upstream region are associated with para -aminosalicylic acid resistance in Mycobacterium tuberculosis

Objectives para-Aminosalicylic acid is an antifolate prodrug activated in the folate biosynthesis pathway of Mycobacterium tuberculosis (Mtb), ultimately inhibiting dihydrofolate reductase encoded by dfrA. Mutations in dfrA coding and upstream regions have not been thoroughly investigated. This study aimed to assess mutations in dfrA and its upstream regulatory region in clinical Mtb isolates. Methods We retrieved and analysed 6547 whole-genome sequences of Mtb clinical isolates. We identified 29 SNPs in the dfrA coding region and eight mutations in the thyA–dfrA upstream region. The most prevalent coding mutations and upstream mutations were selected for functional evaluation using thermal stability analysis, GFP reporter assays and para-aminosalicylic acid drug susceptibility test (DST). Results The coding mutations Trp142→Gly, Gln28→Leu and Arg32→Pro were analysed for protein thermal stability and para-aminosalicylic acid susceptibility. Gln28→Leu showed increased thermal stability relative to the WT, whereas Trp142→Gly and Arg32→Pro exhibited reduced stability. Consistently, para-aminosalicylic acid DST demonstrated that Gln28→Leu conferred an 8-fold increase in para-aminosalicylic acid resistance, while Trp142→Gly and Arg32→Pro remained phenotypically susceptible. Moreover, GFP reporter assays revealed that G70T, G59T and C48A increased promoter activity by 13-fold, 4-fold and 3-fold, respectively, whereas the remaining mutations showed no significant effect. Findings were verified by para-aminosalicylic acid DST, where G70T, G59T and C48A showed para-aminosalicylic acid resistance compared with WT. Conclusions By integrating genomic analysis with functional assays of thermal stability, promoter activity and drug susceptibility, this study demonstrated that mutations in dfrA coding and upstream regulatory regions contribute to para-aminosalicylic acid resistance in Mtb clinical isolates.