Essentiality of Mycobacterium tuberculosis D-alanine racemase

描述(申请人提供)：结核分枝杆菌(结核分枝杆菌)，人类结核病(TB)的主要病原体，仍然是全球发病率和死亡率的主要原因。新的结核病病例中艾滋病毒阳性诊断的百分比正在上升，与艾滋病毒阴性的人相比，艾滋病毒阳性的人患结核病的风险至少高出20倍。对化疗高度耐药的结核分枝杆菌菌株的出现也是艾滋病患者的一大担忧。在此背景下，我们的长期目标是对结核分枝杆菌多肽多糖生物合成的D-丙氨酸途径进行分子遗传学和生化分析，确定药物靶点，并开发新的、有效的、更安全的抑制剂来治疗艾滋病患者的结核病。肽聚糖(PG)是分枝杆菌细胞壁的骨架。D-丙氨酸在新生PG链的交联中起作用，我们的中心假设是D-丙氨酸是PG的重要结构成分。因此，为了让分枝杆菌存活，D-丙氨酸必须在生长介质中提供，并有效地内化，或由内源生物合成产生。D-丙氨酸消旋酶(ALR)催化分枝杆菌合成D-丙氨酸的主要途径，即使在没有D-丙氨酸的情况下，ALR基因对于污垢分枝杆菌的生长也是必不可少的。不幸的是，目前对Mtb的研究不足以确定ALR是否是Mtb的基本功能和D-丙氨酸类似物D-环丝氨酸(DC)原型抑制剂的致死靶点。拟议的研究将使我们能够确定药物设计的最佳策略，既可以利用ALR，也可以针对PG生物合成中的替代酶。因此，我们方法的概念创新是不是孤立地研究ALR，而是在它在MTB代谢网络中的利基的背景下进行研究。具体地说，我们将1)确定ALR在结核分枝杆菌中的重要性；以及2)表征DCS治疗对结核分枝杆菌的代谢影响。将在肉汤培养和人类单核细胞来源的巨噬细胞中分析其重要性。这项研究的重点将是利用四环素调控启动子来控制ALR基因在结核分枝杆菌中的表达，构建条件突变株。我们还将使用我们实验室已经开发的创新的核磁共振代谢组学研究来确定稳态代谢池和代谢通量。建议的研究将提供有关结核分枝杆菌D-丙氨酸代谢的基础知识。将产生关于ALR酶在该物种中的生理重要性和药物靶向潜力的信息。该项目还将解决结核分枝杆菌中是否存在D-丙氨酸生物合成的替代途径的问题，从而探索是否存在进一步药物和/或疫苗开发的新靶点来源。 公共卫生相关性：结核分枝杆菌(Mtb)是世界上最可怕的细菌病原体之一，每年造成约200万人死亡，也是导致艾滋病毒阳性者死亡的最重要原因之一。世界范围内广泛耐药菌株的发病率增加，对一线和二线药物都有耐药性，这突显了开发新的抗结核药物的迫切需要。D-丙氨酸是一种重要的Mtb成分。作为拟议研究的结果，我们期望确定D-丙氨酸消旋酶和D-丙氨酸生物合成的相关途径的相关性，以作为药物设计和/或疫苗开发的目标。

DESCRIPTION (provided by applicant): Mycobacterium tuberculosis (Mtb), the main etiologic agent of human tuberculosis (TB), remains a leading cause of worldwide morbidity and mortality. The percentage of HIV positive diagnosis among new TB cases is on the rise and the risk of developing TB is at least 20 times higher in HIV positive compared to HIV negative individuals. The emergence of Mtb strains highly resistant to chemotherapy is also a major concern to AIDS patients. In this context, our long term goal is to perform molecular genetics and biochemical analyses of the D-alanine pathway of peptidoglycan biosynthesis in Mtb, identify drug targets, and develop novel, effective, safer inhibitors to treat TB in AIDS patients. Peptidoglycan (PG) is the backbone of the mycobacterial cell wall. D-alanine plays a role in the cross-linking of nascent PG strands and our central hypothesis is that D-alanine is an essential structural component of PG. Thus, for mycobacteria to survive, D-alanine must be provided in the growth medium and efficiently internalized, or made by endogenous biosynthesis. The enzyme Dalanine racemase (Alr) catalyzes the primary route of D-alanine biosynthesis in mycobacteria and the alr gene is dispensable for growth in Mycobacterium smegmatis, even in the absence of D-alanine. Unfortunately, current studies in Mtb are insufficient to determine whether Alr is an essential function of Mtb and the lethal target of the prototype inhibitor D- cycloserine (DCS), a D-alanine analog. The proposed research will allow us to determine the best strategy for drug design that either exploits Alr or targets alternative enzymes in PG biosynthesis. Thus, the conceptual innovation of our approach is to study Alr not in isolation but in the context of its niche within the Mtb metabolic network. Specifically, we will 1) Determine the essentiality of Alr in Mtb; and 2) Characterize metabolic effects of DCS treatment on Mtb. Essentiality will be analyzed both in broth cultures and human monocyte-derived macrophages. The highlight of this study will be the construction of a conditional mutant strain using a tetracycline regulated promoter to control the expression of the alr gene in Mtb. We will also use innovative Nuclear Magnetic Resonance metabolomic studies already developed in our laboratories to determine steady state metabolic pools and fluxes. The proposed studies will provide fundamental knowledge on D-alanine metabolism in Mtb. Information will be generated on the physiological essentiality and drug target potential of the Alr enzyme in this species. This project will also address the issue of whether or not there is an alternative pathway of D-alanine biosynthesis in Mtb, thus exploring the existence of a novel source of targets for further drug and/or vaccine development. PUBLIC HEALTH RELEVANCE: Mycobacterium tuberculosis (Mtb) is one of the most dreaded bacterial pathogens in the world, accounting for approximately 2 million deaths per year, and one of the most important causes of death in HIV-positive individuals. The world wide increase in the incidence of extensively drug-resistant strains, resistant to both first and second line drugs, underscores the urgent need to develop new anti-tuberculosis agents. D-alanine is an essential Mtb component. As an outcome of the proposed studies, we expect to determine the relevance of D-alanine racemase and related pathways of D-alanine biosynthesis to serve as targets of drug design and/or vaccine development.