Mechanisms of ARF6 function in Shigella entry

 描述(申请人提供)：志贺氏菌是全世界细菌性腹泻的常见原因，特别是在发展中国家。这些革兰氏阴性病原体通常通过受污染的供水传播，能够在人类结肠的粘膜细胞内入侵和复制。进入宿主细胞对志贺氏菌的毒力至关重要，但人们对此还不完全了解。显然，细菌通过III型分泌系统(T3SS)分泌的一系列蛋白质来操纵宿主细胞骨架和信号通路。作为阐明细菌进入过程的一部分，我的初步工作表明，在缺乏宿主小GTP酶ADP-核糖化因子6(ARF6)的情况下，福氏志贺菌的进入减少，表明ARF6在志贺氏菌的发病中起重要作用。已知ARF6作用于宿主细胞表面和内小体，调节膜转运、脂代谢和肌动蛋白重塑。我的初步研究还表明，T3SS效应器IpgD对于有效进入和激活ARF6至关重要。利用荧光显微镜和生化技术，我建议确定ARF6如何有助于福氏志贺氏菌的有效进入，并表征IpgD在福氏志贺氏菌进入过程中促进ARF6激活的机制。我的具体目标是：1.确定ARF6促进福氏志贺氏菌高效进入的机制(S)。2.确定福氏志贺氏菌激活ARF6的机制(S)。这一建议结合了细胞生物学、生化和遗传学的方法来检验我的假设，并进一步了解志贺氏菌进入的机制(S)。除了增加对基本细胞生物学过程和信号传递的现有知识的了解外，这项工作还将有助于我们对细胞内细菌感染的理解，并可能改进治疗方法。 这类感染不仅包括福氏志贺氏菌和沙门氏菌引起的腹泻疾病，而且还包括立克次体和结核病等其他传染病。

 DESCRIPTION (provided by applicant): Shigella species are a common cause of bacterial diarrhea worldwide, particularly in developing countries. Usually spread by contaminated water supply, these Gram-negative pathogens are capable of invading and replicating within mucosal cells of the human colon. Entry into the host cell is critical for Shigella virulence but incompletly understood. It is apparent that the bacteria manipulates host cytoskeleton and signaling pathways by means of a repertoire of proteins secreted by a type III secretion system (T3SS). As part of an effort to elucidate the bacterial entry process, my preliminary work shows that Shigella flexneri entry is decreased in the absence of host small GTPase ADP-ribosylation factor 6 (ARF6), indicating that ARF6 is important for Shigella pathogenesis. ARF6 is known to act at the host cell surface and at endosomes to regulate membrane trafficking, lipid metabolism, and actin remodeling. My preliminary studies also indicate that the T3SS effector IpgD is critical for efficient entry and ARF6 activation. Using fluorescence microscopy-based and biochemical techniques, I propose to determine how ARF6 contributes to efficient S. flexneri entry and to characterize the mechanism by which IpgD enhances activation of ARF6 during S. flexneri entry. My specific aims are to: 1. Determine the mechanism(s) by which ARF6 contributes to efficient S. flexneri entry. 2. Determine the mechanism(s) of ARF6 activation by S. flexneri. This proposal combines cell biological, biochemical, and genetic approaches to test my hypothesis and further our understanding of the mechanism(s) of Shigella entry. In addition to adding insight to current knowledge of basic cell biological processes and signaling, this work will contribute to our understanding of intracellular bacterial infection and may improve treatment of such infections, which include not only diarrheal disease caused by S. flexneri and Salmonella species but other infectious diseases such as rickettsia and tuberculosis.