The role of the anaplerotic node in redox homeostasis and pathogenesis of Mycobacterium tuberculosis and its exploitation as a therapeutic target

摘要回补结在结核分枝杆菌氧化还原稳态和发病机制中的作用及其作为治疗靶点的开发我们迫切需要新的治疗方法来控制结核病的流行，结核分枝杆菌（Mtb，结核病的病原体）的耐药菌株的出现以及与HIV的灾难性协同作用加剧了结核病的流行。结核分枝杆菌是一种不常见的细菌病原体，它具有引起急性危及生命的疾病和潜伏性结核感染的显著能力，这种感染可以持续人类宿主的一生。据估计，全球有23%的人患有潜伏性结核病，仅2017年就有1000万例结核病病例，150万例结核病死亡，使结核病再次成为全球主要传染病。结核病作为一种人类疾病成功的关键在于结核分枝杆菌非凡的代谢灵活性。这促进了在人类宿主内的可变和恶劣环境中的生存，其中包括暴露于还原和氧化（氧化还原）应激。结核杆菌能够监测人类宿主的这些压力，并协调适当的反应以生存。氧化还原稳态的维持对于结核分枝杆菌引起疾病的能力至关重要，然而在该研究领域中的许多问题仍然没有答案。一个基本的问题是代谢在维持氧化还原平衡中的作用是什么？我们有初步证据表明，由四种酶组成的中央代谢中心（ANA节点）参与维持这种平衡，并确定了一种靶向该节点并杀死Mtb的药物。我们以前的工作表明，这个枢纽是Mtb在其人类宿主细胞中生存所必需的，我们现在想测试我们的假设，即这个节点对于维持氧化还原平衡至关重要，导致疾病的能力，因此代表了药物靶标。我们建议使用我们的缺乏完整ANA节点的Mtb突变株来验证节点在氧化还原控制中的功能。首先，我们将研究具有不完整的ANA结对Mtb在暴露于还原或氧化应激期间的存活、氧化还原平衡和即时代谢反应的影响。对于这项工作，我们将使用一个定制的系统，报告结核分枝杆菌的氧化还原状态。下一个问题是ANA结在氧化还原应激期间如何重新配置代谢？为了解决这个问题，我们将使用我们在萨里大学率先采用的基于尖端系统的代谢组学技术，直接分析参与介导氧化还原应激的代谢途径。通过利用结核病感染的人类细胞和小鼠模型，我们将探讨ANA结在宿主环境中维持氧化还原稳态和毒力的重要性。最后，我们将对一种药物进行化学修饰，我们在初步工作中已经表明这种药物靶向ANA结并杀死Mtb，以提高抗微生物活性。通过探索这些工具化合物在氧化还原稳态的背景下的作用模式，我们将验证ANA结作为药物靶标。通过这种方式，我们将阐明ANA结在这种重要病原体的氧化还原控制和毒力中的作用，并合成可能被开发成未来结核病治疗方法的化合物。

Summary. The role of the anaplerotic node in redox homeostasis and pathogenesis of Mycobacterium tuberculosis and its exploitation as a therapeutic targetWe desperately need new treatments to control the tuberculosis pandemic, which is fuelled by the emergence of drug-resistant strains of Mycobacterium tuberculosis (Mtb, the causative agent of tuberculosis) as well as a catastrophic synergy with HIV. Mtb is an unusual bacterial pathogen, which has the remarkable ability to cause both acute life threatening disease and also symptomless latent tuberculosis infections that can persist for the lifetime of the human host. It is estimated that 23% of the world has latent tuberculosis, and in 2017 alone, there were 10.0 million cases of tuberculosis and 1.5 million tuberculosis deaths, making TB once again the leading infectious disease globally. A key to the success of tuberculosis as a human disease is ascribed to the extraordinary metabolic flexibility of Mtb. This promotes survival in the variable and harsh environments within the human host, which include exposure to REDuctive and OXidative (redox) stresses. Mtb is able to monitor these stresses in the human host and co-ordinate an appropriate response in order to survive. Maintenance of redox homeostasis is critical to the ability of Mtb to cause disease however many questions in this area of research remain unanswered. A fundamental question is what is the role of metabolism in maintaining redox balance? We have preliminary evidence that a central metabolic hub (the ANA node) consisting of four enzymes is involved in maintaining this balance and have identified a drug which targets this node and kills Mtb. Our previous work showed that this hub is required for the survival of Mtb within its human host cell and we now want to test our hypothesis that this node is essential for the maintenance of redox balance, the ability to cause disease and therefore represents a drug target. We propose to characterise the function of the node in redox control using our mutant strains of Mtb which lack a complete ANA node. Firstly we will investigate the effect of having an incomplete ANA node on the survival, redox balance and immediate metabolic responses of Mtb during exposure to reductive or oxidative stresses. For this work we will use a bespoke system which reports on the redox status of Mtb. The next question is how does the ANA node reconfigure metabolism during redox stress? To tackle this question we will directly analyse the metabolic pathways involved in mediating redox stress using cutting-edge systems-based metabolomics techniques that we have spearheaded at the University of Surrey. By utilising a human cell and mouse model of tuberculosis infection we will probe the essentiality of the ANA node to maintaining redox homeostasis and virulence in the host environment. Finally we will chemically modify a drug which we have shown in preliminary work targets the ANA node and kills Mtb in order to improve the antimicrobial activity. By exploring the mode of action of these tool compounds in the context of redox homeostasis we will validate the ANA node as a drug target. In this way we will elucidate the role of the ANA node in redox control and virulence of this important pathogen and synthesise compounds which can potentially be developed into future treatments for tuberculosis.