Cytoskeletal-Pathogen Interactions in Shigella Infection

描述（由申请方提供）：志贺氏菌属是革兰氏阴性肠道病原体，可引起严重腹泻病，被归类为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和细菌通过结肠粘膜传播。志贺氏菌进入上皮细胞是由效应分子介导的，通过III型分泌装置分泌，其激活Rho家族GT3信号传导途径以诱导细胞表面突起和膜皱褶的形成，所述突起和膜皱褶通过巨胞饮吞噬细菌。Cdc 42和Rac都被认为在志贺氏菌进入过程中发挥作用。已知Cdc 42激活Rac;尚不清楚Cdc 42参与志贺氏菌进入是否仅通过此链接介导。此外，Cdc 42和/或Rac激活的下游效应在志贺氏菌进入是未知的。我们最近证实志贺氏菌的主要途径是Cdc 42依赖性的。然而，我们也证明了一个新的Cdc 42独立的入侵途径的存在。此外，我们已经表明，唯一已知的下游效应Cdc 42，激活肌动蛋白细胞骨架，N-WASP，不参与志贺氏菌进入。一旦进入细胞质，志贺氏菌通过肌动蛋白尾的主动组装而移动。肌动蛋白尾的形成是由志贺氏菌外膜蛋白IcsA介导的，其结合并激活N-WASP。活化的N-WASP刺激Arp 2/3复合物介导的肌动蛋白组装。IcsA结合和激活N-WASP的分子机制知之甚少。 我们在本提案中的目标是： 1.明确Cdc 42和Rac在志贺菌进入中的具体作用; 2.鉴定和表征志贺氏菌进入期间Rho家族激活的下游效应物;以及， 3.阐明志贺氏菌IcsA激活N-WASP的机制，并确定该机制是否模拟Cdc 42激活N-WASP 这些研究将确定志贺氏菌进入和肌动蛋白尾形成所需的特定细胞信号传导途径，并将确定Rho家族激活的下游途径。

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.