M. tuberculosis genes required for pulmonary survival revealed by TraCS

描述(申请人提供)：结核病(TB)是由结核分枝杆菌(Mtb)感染引起的，是世界主要的公共卫生问题之一，每年导致200多万人死亡。同时出现的艾滋病毒流行以及广泛耐药的结核分枝杆菌菌株的出现，极大地放大了结核分枝杆菌感染的影响，造成了一场全球医疗危机。那些负责治疗结核病的人面临的问题之一是，即使是由药物敏感的结核分枝杆菌菌株引起的感染也需要长期治疗，部分原因是目前使用的抗分枝杆菌药物在杀死宿主适应细菌方面效率低下。这项工作的主要目标是确定在肺中适应生存所必需的结核分枝杆菌基因，特别是那些在早期筛查方法中没有发现的基因。前提是，对感染期间有机体的生理学了解得越多，就越有机会设计出针对体内基本细菌酶和途径的成功治疗干预措施。这项工作使用了通过气雾剂给豚鼠注射的Mtb转座子突变文库的全基因组筛选。豚鼠宿主复制了人类结核病的许多方面，包括在所有模型系统中找不到的坏死性、缺氧性肉芽肿，例如小鼠。该提案有两个目的。AIM1将转座子捕获和测序或TraCS分析应用于现有的豚鼠感染来源文库。TRACS是一种基于深度测序的可靠和高度敏感的方法，它允许检测使细菌无法在肺环境中生存的MTB基因突变。第二个目的是对筛查的验证性分析，其中在原始筛查中确定的两个基因中将构建单独的缺失等位基因。在低剂量气雾化豚鼠和小鼠结核病感染模型中，将检测缺失突变株的体内表型，并将结果与野生型和互补突变株的感染结果进行比较。纳入小鼠感染队列可以进行物种间比较和验证特定病毒的潜在排他性 在由豚鼠模拟的类人肺环境中维持疾病的基因。最终，这项工作将有助于针对细菌在宿主环境中生存所必需的途径的治疗学的发展。

DESCRIPTION (provided by applicant): Tuberculosis (TB), caused by infection with Mycobacterium tuberculosis (Mtb) is one of the world's leading public health concerns, responsible for the deaths of more than 2 million people annually. The concurrent HIV epidemic coupled with the emergence of extensively drug resistant strains of Mtb have greatly amplified the impact of M. tuberculosis infection and created a global healthcare crisis. One of the problems facing those tasked with treating tuberculosis is that even infections caused by drug susceptible Mtb strains require long-term treatment in part because the anti-mycobacterial agents used currently are inefficient at killing host adapted bacteria. The primary objective of this work is to identify Mtb genes essential for adaptation to survival in the lung particularly those not identified in earlier screening approaches. The premise is that the more known about the physiology of the organism during infection, the greater is the opportunity to design successful therapeutic interventions which target in vivo essential bacterial enzymes and pathways. This work employs a whole genome screen of an Mtb transposon mutant library administered via aerosol to guinea pigs. The guinea pig host reproduces many aspects of human tuberculosis including the necrotic, hypoxic granuloma not found in all model systems, for example the mouse. The proposal has two AIMS. AIM1 is to apply Transposon Capture and Sequencing or TraCS analysis to existing guinea pig infection derived libraries. TraCs is a robust and highly sensitive deep sequencing based methodology, which allows the detection of mutations in Mtb genes which render the bacilli incapable of survival in the pulmonary environment. The second AIM is a confirmatory analysis of the screen wherein individual deletion alleles will be constructed in two genes identified in the original screen. The in vivo phenotype of the deletion mutants will be examined in the low dose aerosol guinea pig and murine tuberculosis infection models, and results will be compared to those observed with infection of wild type and complemented mutant strains. The inclusion of a murine infection cohort allows interspecies comparison and validation of the potential exclusivity of the particular gene for maintenance of disease in the human-like pulmonary environment modeled by the guinea pig. Ultimately this work will contribute to the development of therapeutics which target pathways essential for bacilli survival in the host environment.