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.

尽管对氨基水杨酸（para-aminosalicylic acid, PAS）已被用于结核病治疗数十年，但其耐药机制仍未被完全阐明。既往零星研究表明，结核分枝杆菌（M. tuberculosis）thyX-hsdS.1区域的特定突变可引发PAS耐药，但目前仍缺乏系统性综合分析。近期我们在1株PAS耐药临床分离株的thyX-hsdS.1区域中发现了G–10A突变，但该突变并未引发PAS耐药。本研究对6550株临床分离株的thyX-hsdS.1区域进行单核苷酸多态性（Single Nucleotide Polymorphism, SNP）分析，共鉴定出153个SNP。其中C–16T为最常见的突变位点（占比54.25%，83/153），其次为C–4T（7.19%，11/153）与G–9A（6.54%，10/153）。随后，我们检测了上述SNP对thyX启动子活性的影响，结果显示：与野生型序列相比，C–1T、G–3A、C–4T、C–4G、G–7A、G–9A、C–16T、G–18C及C–19G突变可提升thyX的启动子活性，其余突变则无此效果。随后，我们在结核分枝杆菌H37Ra中分别过表达thyX、野生型thyX-hsdS.1以及携带特定SNP的thyX-hsdS.1，结果显示：可提升启动子活性的突变同样可引发PAS耐药。此外，电泳迁移率变动分析（Electrophoretic Mobility Shift Assay, EMSA）结果显示，携带C–16T突变的thyX-hsdS.1与RNA聚合酶的结合能力高于野生型序列。综上，本研究数据表明：在结核分枝杆菌临床分离株thyX-hsdS.1区域鉴定出的SNP中，仅可提升thyX启动子活性的突变可引发PAS耐药，因此可作为PAS耐药的分子标志物。