Tuberculosis Omics Research Consortium, Family Medicine and Population Health, Institute of Global Health, Faculty of Medicine and Health Sciences, University of Antwerp, Antwerp, Belgium.
Department of Science and Innovation (DSI)-National Research Foundation (NRF) Centre of Excellence for Biomedical Tuberculosis Research, South African Medical Research Council Centre for Tuberculosis Research, Division of Molecular Biology and Human Genetics, Faculty of Medicine and Health Sciences, Stellenbosch University, Cape Town, South Africa.
Sci Rep. 2023 Jun 6;13(1):9212. doi: 10.1038/s41598-023-35563-0.
Molecular detection of bedaquiline resistant tuberculosis is challenging as only a small proportion of mutations in candidate bedaquiline resistance genes have been statistically associated with phenotypic resistance. We introduced two mutations, atpE Ile66Val and Rv0678 Thr33Ala, in the Mycobacterium tuberculosis H37Rv reference strain using homologous recombineering or recombination to investigate the phenotypic effect of these mutations. The genotype of the resulting strains was confirmed by Sanger- and whole genome sequencing, and bedaquiline susceptibility was assessed by minimal inhibitory concentration (MIC) assays. The impact of the mutations on protein stability and interactions was predicted using mutation Cutoff Scanning Matrix (mCSM) tools. The atpE Ile66Val mutation did not elevate the MIC above the critical concentration (MIC 0.25-0.5 µg/ml), while the MIC of the Rv0678 Thr33Ala mutant strains (> 1.0 µg/ml) classifies the strain as resistant, confirming clinical findings. In silico analyses confirmed that the atpE Ile66Val mutation minimally disrupts the bedaquiline-ATP synthase interaction, while the Rv0678 Thr33Ala mutation substantially affects the DNA binding affinity of the MmpR transcriptional repressor. Based on a combination of wet-lab and computational methods, our results suggest that the Rv0678 Thr33Ala mutation confers resistance to BDQ, while the atpE Ile66Val mutation does not, but definite proof can only be provided by complementation studies given the presence of secondary mutations.
结核分枝杆菌中贝达喹啉耐药相关基因突变与表型耐药之间仅有很小的比例具有统计学关联，因此，贝达喹啉耐药的分子检测极具挑战性。本研究通过同源重组或重组的方式，在结核分枝杆菌 H37Rv 参考株中引入两个突变(atpE Ile66Val 和 Rv0678 Thr33Ala)，并检测这些突变的表型效应。通过 Sanger 测序和全基因组测序对获得的菌株的基因型进行了确认，并用最低抑菌浓度 (MIC) 测定法评估了贝达喹啉的敏感性。采用突变截止扫描矩阵 (mCSM) 工具预测了突变对蛋白稳定性和相互作用的影响。atpE Ile66Val 突变并未使 MIC 升高至临界浓度 (MIC 0.25-0.5 µg/ml) 以上，而 Rv0678 Thr33Ala 突变株的 MIC(>1.0 µg/ml) 则将该菌株分类为耐药株，与临床发现相符。计算机分析证实，atpE Ile66Val 突变对贝达喹啉-ATP 合酶的相互作用影响最小，而 Rv0678 Thr33Ala 突变则极大地影响了 MmpR 转录阻遏物的 DNA 结合亲和力。基于湿实验和计算方法的综合结果，我们的研究结果表明，Rv0678 Thr33Ala 突变赋予了 BDQ 耐药性，而 atpE Ile66Val 突变则没有，但鉴于存在次要突变，只有通过互补研究才能提供明确的证据。
