High throughput Genetic Analysis of Mycobacterium tuberculosis

描述（由申请人提供）：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事件。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，每年夺去200万人的生命。据估计，世界人口的一半到三分之一潜伏感染结核分枝杆菌。在美国，目前每年有1万到2万例活动性结核病病例。目前，即使对完全药物敏感的菌株进行治疗，也需要6个月每天服用多种抗生素，而且往往会产生有害的副作用。多重耐药（MDR）和广泛耐药（XDR）菌株对目前的一些、大多数甚至所有化疗药物都有耐药性，这种情况越来越普遍。结核病与艾滋病之间存在一种致命的相互作用，这使得这两种流行病的影响更加有害。因此，迫切需要新的和更有效的结核病化疗和疫苗。可用于结核分枝杆菌的研究工具与问题的规模或大肠杆菌和酵母菌等“模式”微生物的可用工具不相称。这个项目将创造一个新的基因基础设施，为全球防治结核病的斗争服务。该项目的核心是将结核分枝杆菌的基因工程和新改进的遗传方法结合起来，每组4332个结核分枝杆菌菌株，每个菌株以确定的方式删除单个基因。此外，每个缺失都被一个独特的条形码序列标记，允许在混合群体中量化每种基因型的比例。在两种不同的遗传背景下建立条形码缺失文库：1)H37Rv是标准的实验室菌株；2) mc26320， H37Rv的非致病性衍生物。H37Rv是一种病原体，必须在BSL3条件下处理，而mc26320对BSL2的要求不那么严格，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研究的重要工具。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全世界所有合格的研究人员。众所周知，某些结核分枝杆菌基因是抗生素耐受性、生物膜形成、结核分枝杆菌与宿主免疫系统相互作用以及致病性生长等问题的关键。该文库的应用将最终允许对与结核病及其治疗的这些关键方面相关的所有结核分枝杆菌基因进行详尽的枚举。

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.