Shigella regulation of N-WASP mediated actin assembly

描述(申请人提供)：志贺氏菌属通过入侵引起腹泻和痢疾，并通过结肠粘膜传播。志贺氏菌属是CDC/NIAID的优先病原体。在细胞质中，志贺氏菌通过组装推进的肌动蛋白尾巴移动到细胞外围并进入邻近细胞。在细胞体内，肌动蛋白尾巴的组装涉及细胞内N-WASP的招募和激活。在静息细胞中，N-WASP以折叠的自抑制构象维持。志贺氏菌胞内激活N-WASP的分子机制在很大程度上还不清楚。 我们最近已经证明，细胞因子TOCA-1是福氏志贺氏菌激活N-WASP所必需的，特别是为了解除N-WASP的自身抑制。然而，一旦肌动蛋白尾部组装开始，肌动蛋白尾部的持续聚合似乎不再需要TOCA-1。我们的初步数据表明，在没有N-WASP调节蛋白WASP相互作用蛋白(WIP)的情况下，福氏志贺氏菌的肌动蛋白尾部组装更有效。与福氏志贺氏菌结合的N-WASP结合的WIP很少，在SDS-PAGE上迁移较慢。此外，TOCA-1在细胞内细菌表面的募集依赖于细菌III型分泌系统，这表明III型分泌蛋白介导了TOCA-1的募集。 我们的长期目标是了解福氏志贺氏菌操纵细胞肌动蛋白细胞骨架机制以增强致病作用的分子机制。在R21的应用中，我们提出了对福氏志贺氏菌调节N-WASP介导的肌动蛋白组装激活的机制的探索性研究，重点是WIP、TOCA-1和N-WASP修饰对N-WASP激活的作用。我们的目标是： 1.研究WIP在福氏志贺氏菌激活N-WASP中的作用。 2.表征N-WASP的Flexneri修饰及其修饰是否导致开放活性构象的稳定。 3.研究TOCA-1在福氏志贺氏菌表面对N-WASP的募集机制。 揭示福氏志贺氏菌胞内调控细胞内N-WASP激活的机制，很可能对福氏志贺氏菌和其他细菌病原体的致病机制以及参与细胞内N-WASP激活和调控的基本过程产生重要而新颖的见解。与公共卫生相关：人类病原体志贺氏菌是一种通过感染人体肠道细胞并通过肠道组织传播的细菌，其机制尚不清楚。细菌通过产生分子来促进传播，这些分子使细菌能够利用细胞的支架蛋白在组织中移动；我们建议对使细菌能够劫持细胞的支架蛋白以进行传播的分子机制进行详细研究。我们的结果可能有助于更好地理解病原体与人体组织的相互作用，并开发更好的治疗方法。

DESCRIPTION (provided by applicant): Shigella sp. cause diarrhea and dysentery by invasion and spread through the colonic mucosa. Shigella sp. are CDC/NIAID priority pathogens. In the cell cytoplasm, Shigella move to the cell periphery and into adjacent cells by assembling propulsive actin tails. Within the cell body, assembly of actin tails involves the recruitment and activation of cellular N-WASP. In resting cells, N-WASP is maintained in a folded autoinhibited conformation. The molecular mechanisms by which intracellular Shigella activate N-WASP have previously been largely unknown. We have recently demonstrated that the cellular factor Toca-1 is required for S. flexneri activation of N-WASP, specifically for the relief of N-WASP autoinhibition. However, once actin tail assembly is initiated, Toca- 1 appears to no longer be required for ongoing polymerization of the actin tail. Our preliminary data indicate that actin tail assembly by S. flexneri is more efficient in the absence of the N-WASP regulatory protein WASP-interacting protein (WIP). N-WASP bound to S. flexneri has little WIP bound to it and migrates slower on SDS- PAGE. In addition, Toca-1 recruitment to the surface of intracellular bacteria depends on the bacterial type III secretion system, suggesting that a type III secreted protein is mediates Toca-1 recruitment. Our long-term goals are to understand the molecular mechanisms by which S. flexneri manipulate the cellular actin cytoskeletal machinery to enhance pathogenesis. In this R21 application, we propose exploratory studies on the mechanisms by which S. flexneri regulates the activation of N-WASP mediated actin assembly, with a focus on the role of WIP, Toca-1, and N-WASP modification on N-WASP activation. Our aims are to: 1. Characterize the role of WIP in S. flexneri activation of N-WASP. 2. Characterize S. flexneri modification of N-WASP and whether its modification leads to stabilization of the open active conformation. 3. Characterize the mechanism of Toca-1 recruitment to N-WASP on the surface of intracellular S. flexneri. Uncovering the mechanisms by which intracellular S. flexneri regulate the activation of cellular N-WASP is highly likely to generate important and novel insights both into the mechanisms of pathogenesis of S. flexneri and other bacterial pathogens and into the fundamental processes involved in activation and regulation of N-WASP in the cell. PUBLIC HEALTH RELEVANCE: The human pathogen Shigella is a bacterium that causes diarrhea by infecting cells that line the human intestinal tract and disseminating through intestinal tissue by mechanisms that are poorly understood. The bacterium promotes spread by producing molecules that enable the bacterium to use the scaffolding proteins of the cell to move through the tissue; we propose detailed studies into the molecular mechanisms enabling the bacterium to hijack the cell's scaffolding proteins in order to spread. Our results could lead to an improved understanding of how pathogens interact with human tissue and the development of better therapeutics.