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

总结。结核分枝杆菌在氧化还原动态平衡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大流行，这是由结核分枝杆菌耐药菌株(结核分枝杆菌，结核病的病原体)的出现以及与艾滋病毒的灾难性协同作用所加剧的。结核分枝杆菌是一种不常见的细菌病原体，它具有非凡的能力，既能引起危及生命的急性疾病，又能引起无症状的潜伏性结核病感染，这些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1000万结核病病例和150万结核病死亡病例，使结核病再次成为全球头号传染病。结核病作为一种人类疾病的成功关键归因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可变和恶劣环境中的生存，包括暴露在还原和氧化(氧化)压力下。结核分枝杆菌能够监测人类宿主的这些压力，并协调适当的反应以求生存。氧化还原动态平衡的维持对结核分枝杆菌致病能力至关重要，然而这一研究领域的许多问题仍未得到解答。一个基本的问题是，新陈代谢在维持氧化还原平衡中扮演什么角色？我们有初步证据表明，由四种酶组成的中央代谢中枢(ANA节点)参与维持这种平衡，并已确定了一种针对该节点并杀死结核分枝杆菌的药物。我们之前的工作表明，这个枢纽对于结核分枝杆菌在其人类宿主细胞内的生存是必需的，现在我们想验证我们的假设，即这个节点对于维持氧化还原平衡是必不可少的，从而具有致病能力，因此代表着药物靶点。我们建议使用我们的Mtb突变株来表征该节点在氧化还原控制中的功能，该突变株缺乏一个完整的ANA节点。首先，我们将研究ANA结节不完整对MTB在还原或氧化应激下的存活率、氧化还原平衡和即刻代谢反应的影响。对于这项工作，我们将使用一个定制的系统，报告MTB的氧化还原状态。下一个问题是ANA节点如何在氧化还原应激期间重新配置新陈代谢？为了解决这个问题，我们将使用萨里大学率先采用的尖端基于系统的代谢组学技术，直接分析参与调节氧化还原压力的代谢途径。通过利用人类细胞和小鼠的结核病感染模型，我们将探索ANA节点在维持宿主环境中的氧化还原动态平衡和毒力方面的重要性。最后，我们将对我们在前期工作中展示的一种药物进行化学修饰，以靶向ANA节点并杀死Mtb，以提高其抗菌活性。通过探索这些工具化合物在氧化还原动态平衡背景下的作用模式，我们将验证ANA节点作为药物靶点的有效性。通过这种方式，我们将阐明ANA节点在氧化还原控制和这种重要病原体的毒力中的作用，并合成可能被开发为未来结核病治疗的化合物。

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.