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.

项目摘要/摘要 结核分枝杆菌所致结核病及肺部感染的治疗 由非结核分枝杆菌(NTM)引起的细菌感染既漫长又复杂，需要患者服用多种药物来治疗 至少6个月。迫切需要为抗生素的开发确定新的目标，以缩短 并简化结核病和非传染性支气管炎感染的治疗。大多数目前的抗生素针对的是基本功能。而当 在标准的体外条件下，结核分枝杆菌生长所必需的基因已经被定义，这些基因可能不是 在感染期间需要。该提案的总体目标是确定特定路径是否被注释为 对体外生长至关重要的是结核分枝杆菌在宿主中的生长或持久性所必需的。拟议的研究将 测试核心假设，即只有对体外生长至关重要的功能子集在 由于代谢前体的可获得性不同而引起的感染。分析体外培养的重要性-- 结核分枝杆菌感染的基本基因通常需要条件表达方法。虽然这些方法是 对于基因重要性的个体分析很有价值，它们仍然缓慢和低效，使它们不合适 为了发现。特殊目标1将利用基因中一组新的转座子(TN)插入突变体 先前被注明为使用定制富含介质产生的体外生长所必需的。创新的 将采用转座子测序(TN-SEQ)方法，使用这些TN突变体的已定义微型文库和 小鼠大剂量气溶胶感染模型快速有效地识别那些体外必需的基因 MTB需要主机中的增长。初步数据表明，编码第一个承诺步骤的riA2 核黄素生物合成是结核分枝杆菌感染所必需的体外必需基因。哺乳动物并不编码 核黄素合成酶，使它们成为有希望的药物靶点。《特定目标2》将检验这一假设 结核分枝杆菌需要核黄素的生物合成来在宿主中生长和持久，使用最先进的条件 表达技术和小鼠感染模型。这项拟议的研究预计将确定几个 体外-结核分枝杆菌感染所需的必需基因并证明从头开始核黄素的生物合成 是在主机中复制和持久保存结核分枝杆菌所必需的功能。这一知识将是非常重要的 因为它将为开发治疗结核病和肺部NTM感染的抗生素定义新的目标。