Genome Scale Discovery of Mycobacterial Gene Function by Synthetic Genetic Arrays

通过合成基因阵列在基因组规模上发现分枝杆菌基因功能

• 批准号: 8567025
• 负责人: KEITH M DERBYSHIRE
• 金额: $ 15.74万
• 依托单位: WADSWORTH CENTER
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-05-25 至 2015-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8567025
• 关键词: Address; Animal Model; Antibiotics; Bacterial Genome; Biochemical Pathway; Biological Assay; Biology; Caenorhabditis elegans; Candidate Disease Gene; Chronic; Collection; Complex; DNA; Data; Development; Disease; Drug Design; Drug Targeting; Drug resistance; Epistatic Gene; Escherichia coli; Evolution; Foundations; Genes; Genetic; Genetic Screening; Genome; Genomics; Genus Mycobacterium; Goals; Growth; HIV; Individual; Knowledge; Libraries; Link; Methodology; Morphologic artifacts; Mutagenesis; Mutation; Mycobacterium smegmatis; Mycobacterium tuberculosis; Organism; Partner in relationship; Pathogenesis; Pathway interactions; Pharmaceutical Preparations; Phenotype; Process; Proteins; Proteomics; Relative (related person); Research; Resources; Role; Series; Surveys; System; Systems Biology; Testing; Therapeutic; Tuberculosis; Vaccines; Virulence; Work; Yeasts; base; density; expectation; gene function; genetic analysis; genetic manipulation; genome-wide; global health; innovation; killings; meetings; mutant; mycobacterial; next generation; novel; pathogen; public health relevance; rapid growth; research study; resistant strain; sex; synergism; tool; tuberculosis treatment

DESCRIPTION (provided by applicant): Mycobacterial disease, primarily tuberculosis, kills nearly two million people annually. Ineffective vaccines, as well as multi-drug and extremely-drug resistant strains of M. tuberculosis, exacerbate this chronic global crisis. Clearly, new efficacious drugs are needed to fill the growing therapeutic void. Rational drug design begins with a foundation of genomic sequence information. Building upon this genomic foundation requires meaningful annotation of gene product activity and, more importantly, how the encoded proteins and the pathways they comprise come together to produce a viable mycobacterium. Screens have been developed in model organisms to connect genes by epistatic interactions on a genomic scale. Two mutations may be viable separately, but lethal when brought together. Such "synthetic lethal" interactions link the respective gene products together in an essential process. The power of synthetic genetic analysis grows exponentially with the number of genes analyzed, so testing many non-essential genes in a synthetic genetic array (SGA) is ideal. Hundreds of thousands of synthetic interactions have been described in yeasts and E. coli, and typically a single gene displays ~30 interacting partners. Large-scale approaches require a genetically tractable organism to easily create individual mutants, a process to efficiently combine individual mutations, and a reliable assay, all amenable to a high-throughput format. Mycobacterium smegmatis meets all of these requirements and will form the basis for the first comprehensive mycobacterial SGA (mSGA). Even though all of the essential components are in place, evaluating the feasibility of the approach and the establishment of a functional pipeline are prerequisites to initiating a full mSGA. We will use conjugal DNA transfer to combine individual mutations in M. smegmatis. An inability of these double mutants to grow will indicate synthetic lethality, linking the two mutant genes. We will perform limited screens progressing from candidate gene pairs with known synthetic lethality, to a more complex system with both known and likely unknown interactions, and finally with representative "hypothetical" genes with no known activity or interaction. This progression will provide the necessary positive controls to show that the system is working as intended, and it provides an opportunity to assess the potential wealth of information that an expanded mSGA could yield. Integration of epistatic information from an expanded mSGA with other meta-data from proteomic and transcriptional studies will provide a systems biology view of a mycobacterium. This application will establish an mSGA pipeline, and will identify a preliminary set of synthetic lethal interactions as proof-of-principle for its potential. Synthetic lethal interactions, identified using an unbiased genome-wid approach, identify essential functions that represent new high-value potential drug targets.

描述（由申请人提供）：分枝杆菌疾病，主要是结核病，每年杀死近200万人。无效的疫苗以及多药结核分枝杆菌的多药抗菌菌株，加剧了这种慢性全球危机。显然，需要新的有效药物来填补日益增长的治疗空隙。理性药物设计始于基因组序列信息的基础。在这个基因组基础的基础上，需要对基因产物活性的有意义的注释，更重要的是，编码的蛋白质及其构成的途径如何结合在一起以产生可行的分枝杆菌。在模型生物中已经开发了筛选，以通过基因组量表上的上皮相互作用来连接基因。两个突变可能是分别可行的，但是当汇集时致命。这种“合成致死”相互作用将各个基因产物连接在一起，在基本过程中将其联系在一起。合成遗传分析的能力随着分析的基因数量的指数增长，因此在合成遗传阵列（SGA）中测试许多非必需基因是理想的。在酵母和大肠杆菌中已经描述了数十万个合成相互作用，通常显示一个〜30个相互作用的伴侣。大规模的方法需要一种可遗传的有机体，才能轻松创建单个突变体，有效地结合单个突变的过程以及可靠的测定，所有这些都可以符合高通量格式。分枝杆菌Smegmatis符合所有这些要求，并将为第一个全面的分枝杆菌SGA（MSGA）构成基础。即使所有基本要素都到位，也可以评估方法的可行性和功能管道的建立是启动完整MSGA的先决条件。我们将使用链式DNA转移来组合smegmatis中的个体突变。这些双重突变体无法生长的能力将表明合成的杀伤力，将两个突变基因联系起来。我们将执行有限的屏幕，从具有已知合成杀伤力的候选基因对发展，到具有已知相互作用和可能未知相互作用的更复杂的系统，最后具有代表性的“假设”基因，没有已知的活性或相互作用。这种进展将提供必要的积极控制，以表明该系统正在按预期工作，并提供了一个机会来评估扩展的MSGA可能产生的潜在信息。从扩展的MSGA与蛋白质组学和转录研究的其他元数据的集成将提供分枝杆菌的系统生物学视图。该应用程序将建立MSGA管道，并将确定一组合成致命相互作用集作为其潜力的原理证明。使用无偏基因组方法鉴定的合成致死相互作用确定代表新的高价值药物靶标的基本功能。