Mutations outside the rifampicin resistance-determining region associated with rifampicin resistance in Mycobacterium tuberculosis
Mutations outside the rifampicin resistance-determining region associated with rifampicin resistance in Mycobacterium tuberculosis
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DOI：
10.1093/jac/dkq519
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发表时间：
2011-04-01
影响因子：
5.2
通讯作者：
Yam, Wing-Cheong
中科院分区：
文献类型：
--
作者：
Siu, Gilman Kit Hang;Zhang, Ying;Yam, Wing-Cheong
目的：结核分枝杆菌中96%的利福平耐药与位于rpoB基因中心的81 bp利福平耐药决定区（RRDR）内的突变有关。通过靶向RRDR检测利福平耐药性，在4%的病例中未能与耐药表型匹配。我们的研究旨在确定与M利福平耐药相关的RRDR以外的突变。方法和结果：对50株利福平耐药和20株利福平敏感的结核分枝杆菌临床分离株进行检测。在结核病中，2个利福平耐药分离株在RRDR中没有任何已知的突变。对整个rpoB基因的测序分析确定了两个罕见的突变，V146 F和I572 F。基于嗜热栖热菌RpoB的分子结构模型表明，这两个取代的氨基酸都位于非常接近的β-亚基的利福平结合口袋。用简单的氨基酸取代大的氨基酸可能会影响蛋白质与药物的相互作用。突变型rpoB基因的克隆及在野生型耻垢分枝杆菌和M.结论：本研究表明，结核分枝杆菌中146和572位氨基酸与利福平耐药相关。结核病除了RRDR。利福平耐药M.通过包括这两个位置，可以在准确性方面提高结核病。
Objectives: Ninety-six percent of rifampicin resistance in Mycobacterium tuberculosis was shown to be associated with mutations inside the 81 bp rifampicin resistance-determining region (RRDR) located in the centre of the rpoB gene. The detection of rifampicin resistance by targeting the RRDR failed to match with a resistant phenotype in 4% of all cases. Our study aims to identify the mutations outside the RRDR that are associated with rifampicin resistance in M. tuberculosis.Methods and results: Among 50 rifampicin-resistant and 20 rifampicin-susceptible clinical isolates of M. tuberculosis, 2 of the rifampicin-resistant isolates did not harbour any known mutations in the RRDR. Sequencing analysis of the whole rpoB gene identified two rare mutations, V146F and I572F. A molecular structure model based on Thermus thermophilus RpoB revealed that both these substituted amino acids are located in close proximity to the rifampicin-binding pocket of the beta-subunit. Substitutions of simple amino acids for bulky ones are likely to affect the protein-drug interaction. Cloning and transformation of the mutated rpoB gene into wild-type Mycobacterium smegmatis and M. tuberculosis successfully elevated the MIC of rifampicin and conferred the rifampicin resistance phenotype.Conclusions: Our study showed that amino acid positions 146 and 572 are associated with rifampicin resistance in M. tuberculosis in addition to the RRDR. Molecular assays for identifying rifampicin-resistant M. tuberculosis might be improved in terms of accuracy by including these two positions.