High throughput Genetic Analysis of Mycobacterium tuberculosis

描述(由申请人提供)：该项目将基因组和高通量遗传策略应用于全球结核病紧急卫生事件。它创造了连接临床和实验室的新资源，并加快了新的、更便宜、更有效的治疗和疫苗的研究。结核病是世界上最致命的细菌感染，每年导致200万人死亡。据估计，世界上有一半到三分之一的人口潜伏感染结核分枝杆菌。在美国，目前每年有10,000至20,000个活跃的结核病病例。即使是完全对药物敏感的菌株，目前的治疗也需要每天服用6个月的多种抗生素，而且往往会产生有害的副作用。耐多药(或多药耐药)和广泛耐药的XDR菌株对目前的一些、大多数甚至所有化疗药物耐药的情况越来越普遍。结核病与艾滋病之间存在致命的相互作用，这使得这两种流行病的影响更加有害。因此，迫切需要针对结核病的新的更有效的化疗方法和疫苗。可用于结核分枝杆菌的研究工具与问题的规模或可用于“模拟”微生物(如大肠杆菌和酵母)的工具不相称。该项目将创建一个新的基因基础设施，为全球抗击结核病提供服务。该项目的核心是将基因工程和新近改进的结核分枝杆菌遗传方法结合起来，制造出三个大集合，每个集合包含4332个结核分枝杆菌菌株，每个菌株以确定的方式删除一个单一基因。此外，每个缺失都由唯一的条形码序列标记，允许量化混合人群中每个基因型的比例。条码缺失文库将在两个不同的遗传背景下建立：1)H37Rv是标准的实验室毒株；2)Mc26320，H37Rv的非致病衍生品。H37Rv是一种病原体，必须在BSL3条件下工作，而Mc26320对BSL2的遏制要求较低，是疫苗细胞研究的重要基础菌株。这些菌株集和相关资源将成为结核分枝杆菌遗传学的重要工具。该项目创造的遗传资源将通过美国国立卫生研究院指定的储存和分配中心提供给世界各地所有合格的研究人员。已知某些结核分枝杆菌基因对抗生素耐受性、生物被膜的形成、结核分枝杆菌与宿主免疫系统的相互作用以及病原生长等问题起着关键作用。该文库的应用最终将使我们能够详尽地列举与结核病及其治疗的这些关键方面有关的所有结核分枝杆菌基因。 公共卫生相关性：结核分枝杆菌是结核病(TB)的病原体，对全世界的健康负担负有责任。需要基因工具来更好地了解结核杆菌，以便开发包括药物和疫苗在内的更好的治疗方法。为了更好地了解结核分枝杆菌的耐药性和发病机制，我们建议建立一套针对结核分枝杆菌每个基因的单基因敲除。

DESCRIPTION (provided by applicant): This project applies genomic and high-throughput genetic strategies to the global health emergency of tuberculosis. It creates new resources that will bridge clinic and laboratory, and quicken research for new, cheaper, and more effective treatments and vaccines. Tuberculosis (TB) is the world's most deadly bacterial infection and kills two million persons each year. It is estimated that between one-half and one-third of the world's population is latently infected with M. tb. In the US there are currently between 10,000 and 20,000 active TB cases per year. Treatment of even completely drug-susceptible strains currently requires 6 months of multiple antibiotics taken daily and often involves deleterious side effects. Multiply-drug-resistant or MDR) and extensively drug resistant XDR strains that resist some, most, or even all current chemotherapies are increasingly common. There is a deadly interaction of TB with AIDS, which makes the effect of both epidemics more harmful. Thus, the need for new and more effective chemotherapies and vaccines against TB is urgent. The research tools available for M. tb are not commensurate with the scale of the problem or what is available in "model" microorganisms such as E. coli and yeast. This project will create a new genetic infrastructure in service of the global fight against tuberculosis. The core of the project is to combine genetic engineering and newly refined genetic methods in M. tb to make three large sets each of 4,332 M. tb strains, with each strain deleted in a defined way for a single gene. Furthermore, each deletion is marked by a unique barcode sequence allowing quantification of the proportion of each genotype in a mixed population. Bar coded deletion libraries will be built in two different genetic backgrounds: 1) H37Rv, however, is the standard laboratory strain; 2) mc26320, a nonpathogenic derivative of H37Rv. H37Rv is a pathogen and it must be worked with under BSL3 conditions, whereas mc26320 requires less stringent BSL2 containment and is an important base strain for vaccine cell studies. These strain sets and related resources will be important tools for M. tb genetics. The genetic resources created by this project will be made available to all qualified researchers worldwide via an NIH-designated depository and distribution center. It is already known that certain M. tb genes are key to questions of antibiotic tolerance, biofilm formation, interaction of M. tb with the host immune system, and pathogenic growth. Application of the library will finally allow an exhaustive enumeration of all M. tb genes that are relevant for these key aspects of tuberculosis and its treatment. PUBLIC HEALTH RELEVANCE: Mycobacterium tuberculosis, the causative agent of Tuberculosis (TB) is responsible for a worldwide health burden. Genetic tools are needed to better understand the tubercle bacilli so better therapeutics including drugs and vaccines can be developed. We propose to create a set of single gene knockout for every gene of Mycobacterium tuberculosis in order to better understand drug resistance and pathogenesis.