Shigella regulation of N-WASP mediated actin assembly

描述（由申请方提供）：志贺氏菌属通过侵入和通过结肠粘膜传播引起腹泻和痢疾。志贺氏菌属是CDC/NIAID优先病原体。在细胞质中，志贺氏菌通过组装推进性肌动蛋白尾向细胞周边移动并进入相邻细胞。在细胞体内，肌动蛋白尾部的组装涉及细胞N-WASP的募集和激活。在静息细胞中，N-WASP保持折叠的自抑制构象。细胞内志贺氏菌激活N-WASP的分子机制以前在很大程度上是未知的。 我们最近证明了细胞因子Toca-1是S. N-WASP的弗氏激活，特别是用于减轻N-WASP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，肌动蛋白尾组装由S。弗氏杆菌在不存在N-WASP调节蛋白WASP相互作用蛋白（WASP-interacting protein，WASP）的情况下更有效。N-WASP与S结合。弗氏杆菌几乎不与其结合，并且在SDS-PAGE上迁移较慢。此外，Toca-1募集至细胞内细菌表面取决于细菌III型分泌系统，表明III型分泌蛋白介导Toca-1募集。 我们的长期目标是了解S。Flexneri操纵细胞肌动蛋白细胞骨架机制以增强发病机制。在这个R21的应用中，我们提出了探索性的研究机制，S。flexneri调节N-WASP介导的肌动蛋白组装的活化，重点是N-WASP、Toca-1和N-WASP修饰对N-WASP活化的作用。我们的目标是： 1.描述了S. flexneri激活N-WASP。 2.表征S. N-WASP的弗氏修饰以及其修饰是否导致开放活性构象的稳定。 3.表征Toca-1募集至细胞内S表面N-WASP的机制。弗莱克斯内里。 揭示了细胞内S。flexneri调节细胞N-WASP的激活，这对于研究S. flexneri和其他细菌病原体，并进入参与细胞中N-WASP激活和调节的基本过程。公共卫生相关性：人类病原体志贺氏菌是一种细菌，通过感染人体肠道细胞并通过尚不清楚的机制通过肠道组织传播而引起腹泻。细菌通过产生使细菌能够使用细胞的支架蛋白在组织中移动的分子来促进传播;我们建议详细研究使细菌能够劫持细胞的支架蛋白以传播的分子机制。我们的研究结果可能会让我们更好地了解病原体如何与人体组织相互作用，并开发出更好的治疗方法。

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.