Exploration of the Mycobacterial Cell Envelope Proteome by Protein Labeling in Live Cells

项目摘要 结核分枝杆菌多重耐药和广泛耐药菌株的发病率上升 引起结核病(TB)的结核杆菌(MTB)威胁着我们目前的治疗库的效力。因此， 世卫组织消除结核病的战略计划强调开发新药。单元内的进程 包膜作为潜在的新药靶点很有吸引力，因为Mtb调节体内的代谢活动 在感染过程中对环境变化的反应，这些适应是必不可少的 结核分枝杆菌的生存能力和发病机制。然而，我们对细胞被膜途径的探索受到我们 对细胞被膜中蛋白质的定位和功能的了解有限，而且缺乏用于 审问他们。此应用程序的目标是通过开发一种标签来克服这一障碍 可以针对特定的亚细胞隔间，包括细胞包膜的方法学。这个 理论基础是，研究蛋白质定位和相互作用的新工具将使我们第一次能够(1) 准确地盘点细胞被膜蛋白质组和(2)询问细胞中的蛋白质-蛋白质相互作用 信封。我们的创新是应用了一种活细胞蛋白质标记方法，据我们所知，这种方法 适用于分枝杆菌。与现有方法相比，这种方法保留了蜂窝结构和 天然蛋白质相互作用。从我们的初步研究中，我们有证据表明这种方法会成功 来实现我们在分枝杆菌方面的目标。因此，我们建议将非规范分泌的 Mtb的蛋白质组，并阐明一种推定的非规范分泌蛋白的相互作用组，该蛋白对 细胞在分裂中生长。作为这些研究的结果，我们将拥有研究细胞包膜的新技术 并将深入了解有助于结核分枝杆菌生长的蛋白质定位和相互作用 适应。这些结果有望对细胞包膜蛋白的评估产生积极影响。 作为抗结核治疗的靶点，并增进我们对分枝杆菌细胞的基础知识 包络过程和路径。

Project Summary The rising incidence of multi-drug- and extensively drug-resistance strains of Mycobacterium tuberculosis (Mtb), which causes tuberculosis (TB), threatens the efficacy of our current therapeutic arsenal. Thus, the WHO strategic plan to eliminate TB emphasizes the development of new drugs. Processes within the cell envelope are attractive as potential novel drug targets because Mtb modulates metabolic activities within the cell envelope in response to environmental changes during infection, and these adaptations are essential to Mtb viability and pathogenesis. However, our exploration of cell envelope pathways is constrained by our limited knowledge of protein localization and function in the cell envelope and the lack of tools with which to interrogate them. The objective of this application is to overcome this obstacle by developing a labeling methodology that can be targeted to specific subcellular compartments, including the cell envelope. The rationale is that new tools to study protein localization and interactions will allow us to, for the first time, (1) accurately inventory the cell envelope proteome and (2) interrogate protein-protein interactions in the cell envelope. Our innovation is to apply a live-cell protein labeling method that has not, to our knowledge, been applied to mycobacteria. In contrast to existing methods, this approach preserves cellular architecture and native protein interactions. From our preliminary studies we have evidence that the method will be successful in our achieving our goals in mycobacteria. We therefore propose to characterize the non-canonical secreted proteome of Mtb and elucidate the interactome of a putative non-canonical secreted protein that is essential for cell growth in division. As a result of these studies we will have novel techniques to study cell envelope proteins and will gain insight into protein localization and interactions that contribute to Mtb growth and adaptation. These results are anticipated to have a positive impact on the evaluation of cell envelope proteins as targets for anti-tuberculosis therapy and to advance our fundamental knowledge of mycobacterial cell envelope processes and pathways.