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（由申请方提供）：结核分枝杆菌（Mtb）是人类结核病（TB）的主要病原体，仍然是全球发病率和死亡率的主要原因。在新的结核病病例中，艾滋病毒阳性诊断的百分比正在上升，艾滋病毒阳性者患结核病的风险至少比艾滋病毒阴性者高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关注点。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物化学分析，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的重要结构组分。因此，对于分枝杆菌的生存，D-丙氨酸必须在生长培养基中提供并有效内化，或通过内源性生物合成。酶Dalanine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alr基因即使在不存在D-丙氨酸的情况下也能在耻垢分枝杆菌中生长。 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）的致死靶标。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或靶向PG生物合成中的替代酶。因此，我们的方法的概念创新是研究Alr不是孤立的，而是在其生态位内的结核分枝杆菌代谢网络的背景下。具体而言，我们将1）确定Alr在Mtb中的重要性;和2）表征DCS治疗对Mtb的代谢作用。将在肉汤培养物和人单核细胞源性巨噬细胞中分析细胞毒性。本研究的重点将是利用四环素调控的启动子来控制alr基因在结核分枝杆菌中的表达的条件突变株的构建。我们还将使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。这些研究将为结核分枝杆菌的D-丙氨酸代谢提供基础知识。将生成关于该种属中Alr酶的生理学必要性和药物靶点潜力的信息。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，从而探索进一步药物和/或疫苗开发的新靶点来源的存在。 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，并且是HIV阳性个体死亡的最重要原因之一。对一线和二线药物都具有耐药性的广泛耐药菌株在世界范围内的发病率增加，突出表明迫切需要开发新的抗结核药物。D-丙氨酸是Mtb的重要成分。作为所提出的研究的结果，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/或疫苗开发的靶点。