High throughput Genetic Analysis of Mycobacterium tuberculosis

描述（申请人提供）：本项目将基因组和高通量遗传策略应用于结核病的全球卫生紧急情况。它创造了新的资源，将连接诊所和实验室，并加快新的，更便宜，更有效的治疗方法和疫苗的研究。结核病是世界上最致命的细菌感染，每年造成200万人死亡。据估计，世界上约有二分之一到三分之一的人口潜伏感染M。TB.在美国，目前每年有10，000至20，000例活动性结核病例。即使是完全药物敏感的菌株的治疗目前也需要每天服用6个月的多种抗生素，并且通常涉及有害的副作用。耐一些、大多数或甚至所有当前化疗的多重耐药或MDR）和广泛耐药XDR菌株越来越常见。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和更有效的化学疗法和疫苗。M.结核病与问题的规模或"模型"微生物如E.大肠杆菌和酵母菌。该项目将建立一个新的遗传基础设施，为全球防治结核病服务。该项目的核心是将联合收割机基因工程与新改良的遗传方法相结合，在M. tb制作三套大的，每套4,332 M。结核菌株，每个菌株以确定的方式删除单个基因。此外，每个缺失由独特的条形码序列标记，允许定量混合群体中每个基因型的比例。将在两种不同的遗传背景中构建条形码缺失文库：1）H37Rv，然而，是标准实验室菌株; 2）mc26320，H37Rv的非致病性衍生物。H37Rv是一种病原体，必须在BSL 3条件下使用，而mc26320需要较不严格的BSL 2遏制，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是M.结核病遗传学该项目创建的遗传资源将通过NIH指定的保管和分发中心提供给全球所有合格的研究人员。已知某些M.结核杆菌基因是抗生素耐受性、生物膜形成、结核分枝杆菌相互作用等问题的关键。结核病与宿主的免疫系统有关，并有致病性生长。应用该库将最终允许穷举所有M。结核病基因与结核病及其治疗的这些关键方面有关。 公共卫生相关性：结核分枝杆菌是结核病（TB）的病原体，是全球健康负担的原因。需要遗传工具来更好地了解结核杆菌，以便开发更好的治疗方法，包括药物和疫苗。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方法。

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.