Essentiality of Mycobacterium tuberculosis D-alanine racemase

结核分枝杆菌 D-丙氨酸消旋酶的本质

• 批准号: 8012690
• 负责人: OFELIA CHACON
• 金额: $ 21.48万
• 依托单位: UNIVERSITY OF NEBRASKA LINCOLN
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-05-18 至 2012-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8012690
• 关键词: Accounting; Acquired Immunodeficiency Syndrome; Address; Alanine; Alanine Racemase; Amino Acids; Anabolism; Antimycobacterial Agents; Antitubercular Agents; Binding; Biochemical; Biochemical Pathway; Cause of Death; Cell Wall; Centers for Disease Control and Prevention (U.S.); Cessation of life; Culture Media; Cycloserine; D-Alanine Metabolism Pathway; Diagnosis; Drug Delivery Systems; Drug Design; Enzymes; Essential Genes; Extreme drug resistant tuberculosis; Genes; Genus Mycobacterium; Goals; Growth; HIV; HIV Seropositivity; High Pressure Liquid Chromatography; Human; Incidence; Individual; Injectable; Knock-out; Knowledge; Laboratories; Ligase; Liquid Chromatography; Metabolic; Molecular Genetics; Morbidity - disease rate; Multidrug-Resistant Tuberculosis; Mutagenesis; Mycobacterium smegmatis; Mycobacterium tuberculosis; Nuclear Magnetic Resonance; Nutritional; Outcome; Pathogenesis; Pathway interactions; Patients; Peptidoglycan; Pharmaceutical Preparations; Physiological; Play; Population; Public Health; Racemases; Recovery; Reporting; Research; Resistance; Rifampin; Risk; Role; Route; Source; Supplementation; Tetracyclines; Time; Tuberculosis; Vertebral column; World Health Organization; analog; chemotherapy; crosslink; fluoroquinolone resistance; inhibitor/antagonist; innovation; isoniazid; macrophage; metabolomics; monocyte; mortality; mutant; mycobacterial; novel; pathogen; pressure; promoter; prototype; public health relevance; resistant strain; vaccine development

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.

描述（由申请人提供）：结核分枝杆菌（Mtb）是人类结核病（TB）的主要病原体，仍然是全世界发病率和死亡率的主要原因。新发结核病病例中艾滋病毒阳性诊断的比例正在上升，与艾滋病毒阴性者相比，艾滋病毒阳性者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主要担忧。在此背景下，我们的长期目标是对结核分枝杆菌肽聚糖生物合成的D-丙氨酸途径进行分子遗传学和生化分析，确定药物靶点，并开发新型、有效、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支柱。 D-丙氨酸在新生 PG 链的交联中发挥作用，我们的中心假设是 D-丙氨酸是 PG 的重要结构成分。因此，为了使分枝杆菌存活，必须在生长培养基中提供D-丙氨酸并有效内化，或通过内源生物合成产生。丙氨酸消旋酶 (Alr) 催化分枝杆菌中 D-丙氨酸生物合成的主要途径，并且即使在不存在 D-丙氨酸的情况下，alr 基因对于耻垢分枝杆菌的生长也是可有可无的。 不幸的是，目前针对 Mtb 的研究还不足以确定 Alr 是否是 Mtb 的重要功能以及原型抑制剂 D-环丝氨酸 (DCS)（一种 D-丙氨酸类似物）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，即利用 Alr 或针对 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 Mtb 代谢网络中的利基环境中研究 Alr。具体来说，我们将1）确定Alr在Mtb中的重要性； 2) 表征 DCS 治疗对 Mtb 的代谢影响。将在肉汤培养物和人单核细胞衍生的巨噬细胞中分析必要性。本研究的亮点将是使用四环素调节启动子来控制结核分枝杆菌中alr基因的表达，构建条件突变株。我们还将利用我们实验室已经开发的创新核​​磁共振代谢组学研究来确定稳态代谢池和通量。拟议的研究将提供 Mtb 中 D-丙氨酸代谢的基础知识。将生成有关该物种中 Alr 酶的生理必要性和药物靶点潜力的信息。该项目还将解决 Mtb 中 D-丙氨酸生物合成是否存在替代途径的问题，从而探索是否存在用于进一步药物和/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，每年导致约 200 万人死亡，也是 HIV 阳性个体最重要的死亡原因之一。全球范围内对一线和二线药物均耐药的广泛耐药菌株的发生率不断增加，凸显了开发新抗结核药物的迫切需要。 D-丙氨酸是 Mtb 的重要成分。作为拟议研究的结果，我们期望确定 D-丙氨酸消旋酶和 D-丙氨酸生物合成相关途径的相关性，以作为药物设计和/或疫苗开发的目标。