Cytoskeletal-Pathogen Interactions in Shigella Infection

描述（由申请人提供）：志贺氏菌是引起严重腹泻疾病的革兰氏阴性肠道病原体，已被列为B类生物制剂。志贺氏菌的发病机制需要细菌侵入结肠上皮并通过结肠粘膜扩散。志贺氏菌进入上皮细胞是由效应分子介导的，效应分子通过III型分泌装置分泌，激活Rho家族GTPase信号通路，诱导形成细胞表面突起和膜褶，通过巨噬细胞作用吞噬细菌。Cdc42和Rac都被认为在志贺氏菌进入过程中起作用。已知Cdc42可以激活Rac；目前尚不清楚Cdc42参与志贺氏菌进入是否完全通过这一联系介导。此外，Cdc42和/或Rac在志贺氏菌进入时激活的下游效应物是未知的。我们最近证实了主要的志贺氏菌途径是cdc42依赖性的。然而，我们也证明了一种新的不依赖cdc42的侵袭途径的存在。此外，我们已经表明，Cdc42唯一已知的下游效应激活肌动蛋白细胞骨架，N-WASP，不参与志贺氏菌的进入。一旦进入细胞质，志贺氏菌通过肌动蛋白尾部的主动组装移动。肌动蛋白尾部的形成是由志贺氏菌外膜蛋白IcsA介导的，IcsA结合并激活N-WASP。活化的N-WASP刺激Arp2/3复合物介导的肌动蛋白组装。IcsA结合和激活N-WASP的分子机制尚不清楚。

DESCRIPTION (provided by applicant): Shigella are gram negative enteric pathogens that cause severe diarrheal disease and have been classified as a Category B Biological Agent. Shigella pathogenesis requires bacterial invasion of the colonic epithelium and bacterial spread through the colonic mucosa. Shigella entry into epithelial cells is mediated by effector molecules, secreted through a type III secretion apparatus, that activate Rho family GTPase signaling pathways to induce the formation of cell surface projections and membrane ruffles that engulf the bacteria by macropinocytosis. Both Cdc42 and Rac have been implicated as having a role in the Shigella entry process. Cdc42 is known to activate Rac; it is not clear whether Cdc42 involvement in Shigella entry is mediated exclusively via this link. Moreover, the downstream effectors of Cdc42 and/or Rac activation during Shigella entry are unknown. We have recently confirmed that the major Shigella pathway is Cdc42-dependent. However, we have also demonstrated the existence of a novel Cdc42-independent invasion pathway. Furthermore we have shown that the only known downstream effector of Cdc42 that activates the actin cytoskeleton, N-WASP, is not involved in Shigella entry. Once in the cytoplasm, Shigella moves by active assembly of an actin tail. Actin tail formation is mediated by the Shigella outer membrane protein IcsA, which binds and activates N-WASP. Activated N-WASP stimulates Arp2/3 complex-mediated actin assembly. The molecular mechanism by which IcsA binds and activates N-WASP is poorly understood. Our goals in this proposal are to: 1. Define the specific roles of Cdc42 and Rac in Shigella entry; 2. Identify and characterize the downstream effectors of Rho family activation during Shigella entry; and, 3. Elucidate the mechanism(s) by which Shigella IcsA activates N-WASP and determine whether this mechanism mimics Cdc42 activation of N-WASP These studies will define the specific cellular signaling pathways required for Shigella entry and actin tail formation and will identify downstream pathways of Rho family activation.