Genome Scale Discovery of Mycobacterial Gene Function by Synthetic Genetic Arrays

描述（由申请人提供）：分枝杆菌疾病，主要是结核病，每年造成近200万人死亡。无效疫苗以及多药和极耐药结核分枝杆菌菌株加剧了这一慢性全球危机。显然，需要新的有效药物来填补日益增长的治疗空白。合理的药物设计始于基因组序列信息的基础。建立在这个基因组基础上，需要对基因产物活性进行有意义的注释，更重要的是，编码的蛋白质和它们所包含的途径如何聚集在一起产生一个有活力的分枝杆菌。筛选已经在模式生物中发展起来，通过基因组尺度上的上位相互作用来连接基因。两种突变可能单独存在，但结合在一起是致命的。这种“合成致死”的相互作用将各自的基因产物在一个基本过程中联系在一起。随着分析基因数量的增加，合成基因分析的能力呈指数级增长，因此在合成基因阵列（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成千上万的合成相互作用，通常一个基因显示约30个相互作用的伙伴。大规模的方法需要一种遗传上易于处理的生物体，以容易地产生单个突变，一种有效地组合单个突变的过程，以及一种可靠的分析，所有这些都符合高通量格式。耻垢分枝杆菌满足所有这些要求，并将成为第一个全面分枝杆菌SGA （mSGA）的基础。即使所有的基本组成部分都已到位，评估方法的可行性和建立一个功能管道是启动一个完整的mSGA的先决条件。我们将使用配偶DNA转移来组合耻垢分枝杆菌的个体突变。如果这些双突变体不能生长，则表明这两个突变基因具有合成致死率。我们将进行有限的筛选，从具有已知合成致命性的候选基因对，到具有已知和可能未知相互作用的更复杂的系统，最后是具有代表性的“假设”基因，没有已知的活性或相互作用。这一进展将提供必要的积极控制，以表明该系统正按预期工作，并为评估扩展后的mSGA可能产生的潜在信息财富提供了机会。整合来自扩展的mSGA的上位信息与来自蛋白质组学和转录研究的其他元数据将提供分枝杆菌的系统生物学观点。该应用程序将建立一个mSGA管道，并将确定一组初步的合成致命相互作用，作为其潜力的原理证明。使用无偏倚的全基因组方法确定合成致死相互作用，确定代表新的高价值潜在药物靶点的基本功能。

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.