Molecular and Experimental Mycobacteriology, Research Center Borstel Leibniz Lung Center, Borstel, Germany.
Medical Scientist Training Program, Stanford University, Stanford, CA, USA; Nuffield Department of Medicine, University of Oxford, Oxford, UK.
Lancet Microbe. 2023 May;4(5):e358-e368. doi: 10.1016/S2666-5247(23)00002-2. Epub 2023 Mar 29.
Bedaquiline is a core drug for the treatment of multidrug-resistant tuberculosis; however, the understanding of resistance mechanisms is poor, which is hampering rapid molecular diagnostics. Some bedaquiline-resistant mutants are also cross-resistant to clofazimine. To decipher bedaquiline and clofazimine resistance determinants, we combined experimental evolution, protein modelling, genome sequencing, and phenotypic data.
For this in-vitro and in-silico data analysis, we used a novel in-vitro evolutionary model using subinhibitory drug concentrations to select bedaquiline-resistant and clofazimine-resistant mutants. We determined bedaquiline and clofazimine minimum inhibitory concentrations and did Illumina and PacBio sequencing to characterise selected mutants and establish a mutation catalogue. This catalogue also includes phenotypic and genotypic data of a global collection of more than 14 000 clinical Mycobacterium tuberculosis complex isolates, and publicly available data. We investigated variants implicated in bedaquiline resistance by protein modelling and dynamic simulations.
We discerned 265 genomic variants implicated in bedaquiline resistance, with 250 (94%) variants affecting the transcriptional repressor (Rv0678) of the MmpS5-MmpL5 efflux system. We identified 40 new variants in vitro, and a new bedaquiline resistance mechanism caused by a large-scale genomic rearrangement. Additionally, we identified in vitro 15 (7%) of 208 mutations found in clinical bedaquiline-resistant isolates. From our in-vitro work, we detected 14 (16%) of 88 mutations so far identified as being associated with clofazimine resistance and also seen in clinically resistant strains, and catalogued 35 new mutations. Structural modelling of Rv0678 showed four major mechanisms of bedaquiline resistance: impaired DNA binding, reduction in protein stability, disruption of protein dimerisation, and alteration in affinity for its fatty acid ligand.
Our findings advance the understanding of drug resistance mechanisms in M tuberculosis complex strains. We have established an extended mutation catalogue, comprising variants implicated in resistance and susceptibility to bedaquiline and clofazimine. Our data emphasise that genotypic testing can delineate clinical isolates with borderline phenotypes, which is essential for the design of effective treatments.
Leibniz ScienceCampus Evolutionary Medicine of the Lung, Deutsche Forschungsgemeinschaft, Research Training Group 2501 TransEvo, Rhodes Trust, Stanford University Medical Scientist Training Program, National Institute for Health and Care Research Oxford Biomedical Research Centre, Oxford University Hospitals NHS Foundation Trust, Bill & Melinda Gates Foundation, Wellcome Trust, and Marie Skłodowska-Curie Actions.
贝达喹啉是治疗耐多药结核病的核心药物；然而，对耐药机制的了解甚少，这阻碍了快速分子诊断。一些贝达喹啉耐药突变体对氯法齐明也交叉耐药。为了解读贝达喹啉和氯法齐明的耐药决定因素，我们结合了实验进化、蛋白质建模、基因组测序和表型数据。
在这项体外和计算机数据分析中，我们使用了一种新的体外进化模型，使用亚抑菌药物浓度选择贝达喹啉耐药和氯法齐明耐药突变体。我们确定了贝达喹啉和氯法齐明的最小抑菌浓度，并进行了 Illumina 和 PacBio 测序，以对选定的突变体进行特征描述并建立突变目录。该目录还包括来自全球 14000 多株临床结核分枝杆菌复合体分离株和公开可用数据的表型和基因型数据。我们通过蛋白质建模和动态模拟研究了与贝达喹啉耐药相关的变体。
我们发现了 265 个与贝达喹啉耐药相关的基因组变异，其中 250 个（94%）变异影响 MmpS5-MmpL5 外排系统的转录抑制剂（Rv0678）。我们在体外鉴定了 40 个新的变体，以及由大规模基因组重排引起的新的贝达喹啉耐药机制。此外，我们在体外检测到 14 个（16%）迄今为止在临床贝达喹啉耐药分离株中发现的与氯法齐明耐药相关的突变。从我们的体外工作中，我们检测到 14 个（16%）迄今为止在临床氯法齐明耐药菌株中发现的与氯法齐明耐药相关的突变，并且在临床耐药菌株中也发现了这些突变，并对 35 个新的突变进行了编目。Rv0678 的结构建模显示了四种主要的贝达喹啉耐药机制：DNA 结合能力受损、蛋白稳定性降低、蛋白二聚化破坏和与脂肪酸配体的亲和力改变。
我们的发现提高了对结核分枝杆菌复合体菌株药物耐药机制的理解。我们建立了一个扩展的突变目录，其中包括与贝达喹啉和氯法齐明耐药和敏感性相关的变体。我们的数据强调了基因分型测试可以描绘出具有临界表型的临床分离株，这对于设计有效的治疗方法至关重要。
莱布尼茨肺部进化医学科学学院、德国研究基金会、2501 跨进化研究组、罗兹信托基金、斯坦福大学医学科学家培训计划、国家卫生与保健卓越研究中心牛津生物医学研究中心、牛津大学医院 NHS 基金会信托基金、比尔和梅林达盖茨基金会、惠康信托基金和居里夫人行动。
