Target and Non-target Based Resistance to Bedaquiline in South Africa

结核病是世界范围内由一种可治愈的传染病造成的主要死亡原因，并正在成为一种主要的 由于耐药结核分枝杆菌(Mtb)菌株的传播而引起的关切。2015年，南非 国家结核病规划引入贝达奎兰(BDQ)以加强现有的治疗方案 对利福平耐药的结核病。这一倡议对数以千计的南方人民具有直接和深远的影响 患有结核病的非洲人。令人担忧的是，与BDQ耐药性相关的突变(rv0678和atpe)具有 在从从未接受过BDQ或氯法齐明(CFZ)治疗的患者分离的结核分枝杆菌克隆中被发现。 有趣的是，BDQ和CFZ都瞄准了Mtb电子传输链。这些发现，再加上事实 结核分枝杆菌可以持续处于休眠的、耐药的状态，有时会在几十年后重新激活而导致结核病 原发感染，提示临床上迫切需要更好地了解BDQ/CFZ的耐药机制 结核分枝杆菌的菌株。 我们的长期目标是了解结核分枝杆菌耐药性的机制，以及这种知识如何 在南非用于预防和治疗目的。在这个提议中，我们的中心假设是 Rv0678、atpe和结核分枝杆菌基因组其他地方的突变会扰乱中枢新陈代谢。 对BDQ和CFZ的抗性。为了验证这一假设，我们建立了一个全球合作努力， 南非Sefako Makgatho健康科学大学(SMU)的基础和临床研究人员 阿拉巴马大学伯明翰分校。作为这次合作的一部分，我们建立了一系列 特异性目的是确定rv0678和ATPE突变在分离的特定结核分枝杆菌谱系中的患病率 南非的病人。我们还将使用一种名为细胞外通量(XF96)分析的新技术 我们已经适应了对MTB生物能量学的实时研究。这项技术将得到13C的补充 液-质联用法进行稳定同位素分析。最后，我们将进一步推进我们的 令人兴奋的初步发现和确定BDQ耐药相关变异是否对杆菌有贡献 生物能量灵活性。 这一贡献是重大的，因为它有可能确定一种新的范式，这将导致 对BDQ/CFZ抗性出现的机理的理解，以及这一过程如何中断 被利用来杀灭结核分枝杆菌。我们认为这项提议是创新的，因为新采用的技术 这是由全基因组测序，实时生物能量学和代谢组学支持的，使自己与众不同 与研究病原微生物耐药性的传统方法不同。

Tuberculosis is the leading cause of death worldwide from a curable infectious agent and is becoming a major concern due to the spread of drug resistant Mycobacterium tuberculosis (Mtb) strains. In 2015, the South African National Tuberculosis Programme introduced Bedaquiline (BDQ) to strengthen existing regimens for the therapy of rifampicin-resistant TB. This initiative has immediate and far-reaching implications for thousands of South Africans suffering from TB. Concerningly, mutations associated with BDQ resistance (rv0678 and atpE) have been identified in Mtb clones isolated from patients who have never been treated with BDQ or clofazimine (CFZ). Intriguingly, both BDQ and CFZ target the Mtb electron transport chain. These findings, together with the fact that Mtb can persist in a dormant, drug-tolerant state, sometimes reactivating to cause TB decades after the primary infection, indicate an urgent need to better understand the mechanisms of BDQ/CFZ resistance in clinical strains of Mtb. Our long-term goal is to understand the mechanisms of Mtb drug resistance and how this knowledge can be used for prophylactic and therapeutic purposes in South Africa. In this proposal, our central hypothesis is that mutations in rv0678, atpE and elsewhere in the Mtb genome dysregulate central metabolism that contributes to BDQ and CFZ resistance. To test this hypothesis, we have established a global collaborative effort between basic and clinical investigators at Sefako Makgatho Health Sciences University (SMU) in South Africa and the University of Alabama at Birmingham (UAB). As part of this collaboration, we have established a series of specific aims to determine the prevalence of rv0678 and atpE mutations in specific Mtb lineages isolated from patients in South Africa. We will also make use of a novel technology termed extracellular flux (XF96) analysis that we have adapted for studying Mtb bioenergetics in real time. This technology will be complemented by 13C stable isotope analyses using liquid chromatography mass spectrometry. Lastly, we will further pursue our exciting preliminary findings and determine whether BDQ resistance associated variants contribute to the bacilli's bioenergetic flexibility. This contribution is significant, because it has the potential to identify a new paradigm that will lead to a mechanistic understanding of the emergence of BDQ/CFZ resistance, and how disruption of this process could be exploited to sterilize Mtb. This proposal is innovative in our opinion, because the newly adapted technology that is supported by whole genome sequencing, real-time bioenergetics and metabolomics, distinguishes itself from conventional approaches for studying drug resistance in pathogenic microbes.