Redefining Mycobacterium tuberculosis genes essential for infection

项目总结/摘要 治疗由结核分枝杆菌（Mtb）引起的结核病（TB）和由结核分枝杆菌（Mtb）引起的肺部感染 由非结核分枝杆菌（NTM）是漫长和复杂的，需要患者采取多种药物， 最少6个月。迫切需要确定开发抗生素的新靶点，以缩短 简化结核病和非结核病感染的治疗。目前大多数抗生素针对基本功能。而 已经定义了在标准体外条件下Mtb生长所必需的基因，这些基因可能不是 在感染期间需要。本提案的总体目标是确定注释为 Mtb在宿主中的生长或持久性需要体外生长所必需的。拟议的研究将 测试中心假设，即只有体外生长所必需的功能的子集在体外生长期间是必需的。 由于代谢前体可用性的差异而导致感染。分析了体外培养的重要性- Mtb感染的必需基因通常需要条件表达方法。虽然这些方法 它们对于基因重要性的个体分析是有价值的，但它们仍然缓慢而低效，使它们不合适。 为了发现。具体目标1将利用一个新的收集转座子（Tn）插入突变体的基因 先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 转座子测序（Tn-seq）方法将使用这些Tn突变体的确定的微型文库和一个 小鼠高剂量气溶胶感染模型，以快速有效地鉴定那些体外必需基因， 结核分枝杆菌在宿主体内生长所需的条件。初步数据表明，ribA 2，编码第一个承诺的步骤， 核黄素生物合成是结核分枝杆菌感染所必需的一个体外必需基因。哺乳动物不编码 核黄素合成酶，使其成为有前途的药物靶点。具体目标2将检验以下假设： 结核分枝杆菌需要核黄素的生物合成，以在宿主中生长和持久存在， 表达技术和小鼠感染模型。预计拟议的研究将确定几个 在体外Mtb感染所需的必需基因，并证明从头核黄素生物合成 是Mtb在宿主中的复制和持久性所必需的功能。这些知识将是重要的 因为它将为开发治疗结核病和肺部NTM感染的抗生素确定新的靶点。

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.