High-Throughput Identification of Mycobacterium tuberculosis Persistence Mechanis

描述（由申请人提供）：结核分枝杆菌（Mtb）是肺部感染结核病的病原体，由于其在潜伏感染个体中持续存在并 产生耐缺氧的持续细胞。我们假设，不同的分子机制占这两种类型的结核分枝杆菌的持久性，并提出新的高通量遗传筛选，以确定和表征这些机制。我们将使用一种新的技术，Tn-seq，同时识别和量化转座子（Tn）插入突变体在大型随机Tn突变池大规模并行测序的Tn基因组连接。Tn-seq先前已用于分析细菌Tn突变体在体外各种培养条件下和在体内一些动物感染模型中的适应性。但是Tn-seq对于研究存在狭窄定殖瓶颈的生长条件或感染模型的实用性有限。我们建议使用条形码序列标签和多路复用来调整Tn-seq方法，以实现较小Tn突变池的成本效益分析。我们将使用Tn-seq与转基因小鼠的感染相结合，以在全基因组范围内定义Mtb在特定宿主适应性免疫防御中持续存在所需的因素。我们还将使用体外抗生素选择策略结合Tn-seq来定义Mtb最佳形成耐药性持续细胞所需的因素。最终，我们将把这些结果扩展到相关的动物感染模型中，以证明我们确定的Mtb持久性因素是可行的药物靶点。针对这些持久性因素的治疗将代表结核病控制的新方法，该方法将使Mtb对天然宿主免疫防御或现有抗生素敏感。

DESCRIPTION (provided by applicant): Mycobacterium tuberculosis (Mtb), the causative agent of the pulmonary infection tuberculosis, has evaded eradication from the human population due to its extraordinary ability to both persist in latently infected individuals and to generate antibiotic-tolerant persister cells. We hypothesize that different molecular mechanisms account for these two types of Mtb persistence and propose novel high-throughput genetic screens to identify and characterize these mechanisms. We will use a new technology, Tn-seq, that simultaneously identifies and quantifies transposon (Tn) insertion mutants within large random Tn mutant pools by massively parallel sequencing of Tn-genome junctions. Tn-seq has previously been used to analyze fitness of bacterial Tn mutants in various culture conditions in vitro and in some animal infection models in vivo. But Tn-seq has limited utility for studying growth conditions or infection models in which there are narrow colonization bottlenecks. We propose to adapt the Tn-seq method using bar-code sequence tags and multi-plexing to enable cost-effective analysis of smaller Tn mutant pools. We will use Tn-seq combined with infection of genetically modified mice to define on a genome- wide scale the factors that Mtb requires for persistence in the face of specific host adaptive immune defenses. We will additionally use an in vitro antibiotic selection strategy combined with Tn-seq to define factors that Mtb requires for optimal formation of antibiotic-tolerant persister cells. Ultimately, we will expand these results o relevant animal infection models, to demonstrate that the Mtb persistence factors we identify are viable drug targets. Therapeutics targeting these persistence factors would represent novel approaches to tuberculosis control that would sensitize Mtb either to natural host immune defenses or to existing antibiotics.