Redefining Mycobacterium tuberculosis genes essential for infection

重新定义感染所必需的结核分枝杆菌基因

• 批准号: 9977614
• 负责人: Anna DeGraff Tischler
• 金额: $ 23.1万
• 依托单位: UNIVERSITY OF MINNESOTA
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-02-21 至 2022-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9977614
• 关键词: Acute; Aerosols; Anabolism; Antibiotics; Biology; Cause of Death; Cell Wall; Chronic; Chronic Phase; Collection; Complement; Complex; Custom; Data; Development; Diagnosis; Dose; Drug Combinations; Drug Targeting; Drug resistance in tuberculosis; Enzymes; Essential Genes; Exhibits; Future; Gene Expression; Genes; Genetic Transcription; Goals; Growth; In Vitro; Individual; Infection; Knowledge; Libraries; Lung; Lung infections; Mammals; Metabolic; Methods; Mission; Modeling; Multi-Drug Resistance; Multidrug-Resistant Tuberculosis; Mus; Mycobacterium Infections; Mycobacterium tuberculosis; Nutrient; Opportunistic Infections; Pathway interactions; Patients; Pharmaceutical Preparations; Phase; Prevalence; Research; Resistance; Riboflavin; System; Systemic infection; Technology; Testing; Tetracyclines; Time; Tissues; Translations; Treatment Protocols; Tuberculosis; United States; United States National Institutes of Health; attenuation; drug development; fitness; in vivo; innovation; interest; knock-down; mutant; new therapeutic target; non-tuberculosis mycobacteria; novel; novel therapeutics; prevent; transposon sequencing; tuberculosis drugs; tuberculosis treatment

PROJECT SUMMARY/ABSTRACT Treatment of tuberculosis (TB) caused by Mycobacterium tuberculosis (Mtb) and pulmonary infections caused by non-tuberculous mycobacteria (NTM) is lengthy and complex, requiring patients to take multiple drugs for a minimum of 6 months. There is a critical need to define new targets for development of antibiotics to shorten and simplify treatment of TB and NTM infections. Most current antibiotics target essential functions. While genes essential for Mtb growth in standard in vitro conditions have been defined, these genes may not be required during infection. The overall goal of this proposal is to determine if specific pathways annotated as essential for in vitro growth are required for Mtb growth or persistence in the host. The proposed research will test the central hypothesis that only a subset of functions essential for in vitro growth will be essential during infection due to differences in the availability of metabolic precursors. Analyzing the importance of in vitro- essential genes to Mtb infection typically requires conditional expression methods. While these methods are valuable for individual analysis of gene essentiality they remain slow and inefficient, making them inappropriate for discovery. Specific Aim 1 will make use of a novel collection of transposon (Tn) insertion mutants in genes previously annotated as essential for in vitro growth generated using a custom rich medium. An innovative transposon sequencing (Tn-seq) approach will be taken using defined mini-libraries of these Tn mutants and a mouse high-dose aerosol infection model to rapidly and efficiently identify those in vitro-essential genes that Mtb requires for growth in the host. Preliminary data suggest that ribA2, encoding the first committed step in riboflavin biosynthesis, is one in vitro-essential gene required for Mtb infection. Mammals do not encode the riboflavin synthesis enzymes, making them promising drug targets. Specific Aim 2 will test the hypothesis that Mtb requires riboflavin biosynthesis for growth and persistence in the host using state-of-the-art conditional expression technologies and a mouse infection model. The proposed research is expected to identify several in vitro-essential genes that Mtb requires for infection and to demonstrate that de novo riboflavin biosynthesis is a function essential for both replication and persistence of Mtb in the host. This knowledge will be significant because it will define new targets for the development of antibiotics to treat TB and pulmonary NTM infections.

项目概要/摘要 治疗由结核分枝杆菌（Mtb）引起的结核病（TB）和引起的肺部感染 非结核分枝杆菌 (NTM) 治疗过程漫长且复杂，需要患者在一段时间内服用多种药物 至少 6 个月。迫切需要确定抗生素开发的新目标，以缩短 并简化结核病和非结核分枝杆菌感染的治疗。目前大多数抗生素都针对基本功能。尽管 标准体外条件下 Mtb 生长所必需的基因已被定义，但这些基因可能不是 感染期间需要。该提案的总体目标是确定特定路径是否注释为 体外生长所必需的，是结核分枝杆菌在宿主体内生长或持久存在所必需的。拟议的研究将 检验中心假设，即只有体外生长所必需的功能子集在 由于代谢前体的可用性差异而导致感染。分析体外实验的重要性 Mtb 感染的必需基因通常需要条件表达方法。虽然这些方法是 对于基因必要性的个体分析很有价值，但它们仍然缓慢且低效，因此不合适 为了发现。具体目标 1 将利用基因中一系列新型转座子 (Tn) 插入突变体 之前注释为使用定制丰富培养基产生的体外生长所必需的。一个创新的 将使用这些 Tn 突变体的已定义迷你文库和一个转座子测序 (Tn-seq) 方法 小鼠高剂量气溶胶感染模型，可快速有效地鉴定那些体外必需基因 Mtb 需要宿主的生长。初步数据表明，ribA2 编码了第一个承诺步骤 核黄素生物合成是结核分枝杆菌感染所需的体外必需基因之一。哺乳动物不编码 核黄素合成酶，使它们成为有前途的药物靶点。具体目标 2 将检验以下假设： Mtb 需要核黄素生物合成才能使用最先进的条件在宿主体内生长和持久存在 表达技术和小鼠感染模型。拟议的研究预计将确定几个 结核分枝杆菌感染所需的体外必需基因，并证明核黄素从头生物合成 是 Mtb 在宿主中复制和持久性所必需的功能。这些知识将很重要 因为它将确定开发治疗结核病和肺部 NTM 感染的抗生素的新目标。