Protein-protein association in mycobacteria

描述（由申请人提供）：结核分枝杆菌（Mtb）是全球主要死亡原因之一，每年夺去数百万人的生命。全世界约有17亿人无症状感染结核杆菌，这对全球公共卫生控制措施构成了重大障碍。Mtb H37Rv完整的基因组序列强调了我们在了解分枝杆菌疾病及其发病机制方面的不足，并揭示了需要一种将原始基因组序列数据转化为功能信息的方法。我们认为，在Mtb功能的表征中存在一个重大的瓶颈，主要是因为积累了更复杂和详细的输出（例如，分枝杆菌基因组序列，DNA微阵列实验，大规模蛋白质表达测量和基因缺失），而不是首先关注产生这些输出的原因。此外，越来越清楚的是，毒力和耐药途径是由相互作用的蛋白质网络介导的。为了解决这个问题，我们开发了一种简单快速的方法，称为分枝杆菌蛋白片段互补（M-PFC），使我们能够分析分枝杆菌细胞中的蛋白-蛋白关联。在Specific Aim 1中，我们将全面评估和优化M-PFC，并开发定量分析方法来测量相互作用的强度，确定M-PFC在分枝杆菌膜中检测蛋白-蛋白关联的能力，使用M-PFC研究氧化还原剂对蛋白-蛋白关联的影响，构建Gateway克隆载体，使研究人员能够快速克隆和分析他们感兴趣的基因。最后，我们将在Mtb中测试我们的系统。在特异性目标2中，我们将通过筛选与已知毒力因子相互作用的蛋白质的结核分枝杆菌文库来测试和验证我们的系统。该申请具有创新性，因为它描述了一种在分枝杆菌领域尚未描述的新型实验系统的发展，并且具有很强的潜力，可以导致鉴定新的药物靶点，以及与结核分枝杆菌的持久性和发病机制有关的毒力因素。这项工作在功能基因组学、结构生物学、药物设计和系统生物学方面的影响也具有创新性。我们将把我们的试剂提供给更广泛的科学界。

DESCRIPTION (provided by applicant): M. tuberculosis (Mtb) is one of the leading causes of death worldwide and claims millions of lives annually. Approximately ~1.7 billion people worldwide are asymptomatically infected with the tubercle bacillus and constitute a major impediment to worldwide public health control measures. The completed genome sequence of Mtb H37Rv emphasizes our inadequacies in understanding mycobacterial disease and pathogenesis and has revealed the need for an approach to convert raw genome sequence data into functional information. We believe that a significant bottleneck exits in the characterization of Mtb function, primarily because of the accumulation of more complex and detailed outputs (e.g., mycobacterial genome sequences, DNA microarrays experiments, large-scale protein expression measurements, and gene deletions), rather than focusing on what create these outputs in the first place. Furthermore, it has become increasingly clear that virulence and drug resistance pathways are mediated by networks of interacting proteins. To address this problem, we developed a simple and rapid method termed, Mycobacterial Protein Fragment Complementation (M-PFC) that allows us to analyze protein-protein association in mycobacterial cells. In Specific Aim 1, we will thoroughly evaluate and optimize M-PFC and develop quantitative assays to measure the strength of interaction, determine how well M-PFC can detect protein-protein association in the mycobacterial membrane, use M-PFC to study the effect of redox agents on protein-protein association, construct Gateway cloning vectors that will enable investigators to rapidly clone and analyze their genes of interest. Finally, we will test our system in Mtb. In Specific Aim 2, we will test and validate our system by screening a Mtb library for proteins that interact with a known virulence factor. This application is innovative in that it describes the development of a novel experimental system not yet described in the mycobacterial field and has strong potential to lead to the identification of new drug targets, and virulence factors involved in the persistence and pathogenesis of Mtb. This effort is also innovative in the impact that it will have on functional genomics, structural biology, drug design and systems biology. We will make our reagents available to the wider scientific community. M. tuberculosis (Mtb) is one of the leading causes of death worldwide and claims millions of lives annually. The completed genome sequence of Mtb H37Rv emphasizes our inadequacies in understanding mycobacterial disease and pathogenesis and has revealed the need for an approach to convert raw genome sequence data into functional information. We have developed a novel method termed M-PFC to study protein-protien association in mycobacteria. This system should allow the identification of virulence pathways involved in persistence and pathogenesis, which will improve control of TB.