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 (Mtb)导致结核病（TB），威胁着我们目前治疗武器库的功效。因此 世卫组织消除结核病战略计划强调开发新药。细胞内的过程 包膜作为潜在的新型药物靶点是有吸引力的，因为Mtb调节包膜内的代谢活性。 在感染过程中，细胞被膜对环境变化的反应，这些适应是必不可少的， 结核分枝杆菌生存力和发病机制。然而，我们对细胞包膜途径的探索受到我们的限制。 对蛋白质在细胞包膜中的定位和功能的了解有限，并且缺乏工具， 审问他们。本申请的目的是通过开发标签来克服这一障碍， 该方法可以靶向特定的亚细胞区室，包括细胞包膜。的 基本原理是，研究蛋白质定位和相互作用的新工具将使我们第一次，（1） 准确地清点细胞包膜蛋白质组和（2）询问细胞中的蛋白质-蛋白质相互作用 信封.我们的创新是应用一种活细胞蛋白质标记方法，据我们所知， 应用于分枝杆菌与现有方法相比，这种方法保留了细胞结构， 天然蛋白质相互作用根据我们的初步研究，我们有证据表明这种方法是成功的 我们在分枝杆菌方面的目标。因此，我们建议描述非典型分泌 Mtb的蛋白质组，并阐明一个假定的非典型分泌蛋白质的相互作用组，这是必不可少的 细胞分裂生长。作为这些研究的结果，我们将有新的技术来研究细胞包膜 蛋白质，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价产生积极的影响 作为抗结核治疗的靶点，并促进我们对分枝杆菌细胞的基础知识 包封过程和路径。

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.