M. tuberculosis genes required for pulmonary survival revealed by TraCS

描述（由申请人提供）：结核（TB）是由结核分枝杆菌（Mtb）感染引起的，是世界上主要的公共卫生问题之一，每年造成200多万人死亡。同时流行的艾滋病毒加上广泛耐药的结核分枝杆菌菌株的出现，大大扩大了结核分枝杆菌感染的影响，并造成了全球卫生保健危机。负责治疗结核病的人面临的一个问题是，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适应宿主的细菌方面效率低下。这项工作的主要目的是确定结核分枝杆菌基因对适应肺部生存至关重要，特别是那些在早期筛查方法中未发现的基因。前提是，对感染期间生物体的生理学了解得越多，就越有机会设计成功的治疗干预措施，以体内必需的细菌酶和途径为目标。这项工作采用全基因组筛选结核分枝杆菌转座子突变文库通过气雾剂给豚鼠。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（例如小鼠）中没有发现的坏死性、缺氧肉芽肿。该提案有两个目的。AIM1是将转座子捕获和测序或TraCS分析应用于现有的豚鼠感染衍生文库。TraCs是一种强大且高度敏感的基于深度测序的方法，它允许检测结核分枝杆菌基因的突变，这些突变使杆菌无法在肺部环境中存活。第二个AIM是对筛选的验证性分析，其中将在原始筛选中鉴定的两个基因中构建单个缺失等位基因。缺失突变体的体内表型将在低剂量气溶胶豚鼠和小鼠结核感染模型中进行检测，并将结果与野生型和补充突变株感染观察到的结果进行比较。纳入小鼠感染队列允许种间比较和验证特定的潜在排他性

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.