Association between mutations in a <i>thyX-hsdS.1</i> region and <i>para</i>-aminosalicylic acid resistance in <i>Mycobacterium tuberculosis</i> clinical isolates

Although <i>para</i>-aminosalicylic acid (PAS) has been used to treat tuberculosis agent for decades, its mechanisms of resistance are still not thoroughly understood. Previously, sporadic studies showed that certain mutations in the <i>thyX-hsdS.1</i> region caused PAS resistance in <i>M. tuberculosis</i>, but a comprehensive analysis is lacking. Recently, we found a G–10A mutation in <i>thyX-hsdS.1</i> in a PAS-resistant clinical isolate, but it did not cause PAS resistance. SNPs in <i>thyX-hsdS.1</i> in 6550 clinical isolates were analyzed, and 153 SNPs were identified. C–16 T was the most common SNP identified (54.25%, 83/153), followed by C–4T (7.19%, 11/153) and G–9A (6.54%, 10/153). Subsequently, the effects of those SNPs on the promoter activity of <i>thyX</i> were tested, and the results showed that mutations C–1T, G–3A, C–4T, C–4G, G–7A, G–9A, C–16T, G–18C, and C–19G led to increased promoter activity compared with the wild-type sequence, but other mutations did not. Then, <i>thyX</i> and wild-type <i>thyX-hsdS.1</i>, or <i>thyX-hsdS.1</i> containing specific SNPs, were overexpressed in <i>M. tuberculosis</i> H37Ra. The results showed that mutations resulting in increased promoter activity also caused PAS resistance. Moreover, the results of an electrophoretic mobility shift assay showed that <i>thyX-hsdS.1</i> containing the C–16T mutation had a higher binding capacity to RNA polymerase than did the wild-type sequence. Taken together, our data demonstrated that among the SNPs identified in <i>thyX-hsdS.1</i> of <i>M. tuberculosis</i> clinical isolates, only those able to increase the promoter activity of <i>thyX</i> caused PAS resistance and therefore can be considered as molecular markers for PAS resistance.