Lipid raft effects on Shigella type III secretion system-based interactions

描述(申请人提供)：福氏志贺氏菌是细菌性痢疾的病原体，它利用其III型分泌系统(TTSS)作为管道，将效应蛋白从细菌运送到宿主细胞细胞质，在那里它们颠覆正常的宿主功能。虽然关于TTSS的功能已经研究得很多，但关于初始宿主相互作用的细节还不是很清楚。已证明III型分泌器(TTSA)的组成部分与胆固醇(Chol)相互作用，最近的证据表明，含有Chol和鞘磷脂(Sm)的脂质体可诱导TTS。这些和其他数据表明，脂筏微域在志贺氏菌的接触、稳定的相互作用和效应器注射到宿主细胞中发挥作用，然而，还没有持续的尝试来直接探索病原体和脂筏样结构域之间的联系。我的假设是，人类细胞中的类脂筏结构域提供了一个位置，志贺氏菌TTSS可以锚定在细胞表面，同时提供诱导TTS的信号，并允许将效应蛋白注入宿主细胞的细胞质。为了更好地了解脂筏在志贺氏菌毒力中的作用，本研究的具体目的是：1)确定宿主细胞脂筏成分和膜相对福氏志贺菌最初附着和进入宿主细胞的作用；2)确定脂膜动力学对活志贺氏菌与模型膜相互作用的影响。这些研究将使用一系列不同的技术和方法，包括：使用模型膜系统对纯化蛋白质进行生化研究，对志贺氏菌与培养细胞相互作用的实时成像，以及开发改进大分子组件成像的方法。为了完成这些目标，我将提供第一次全面检查这种病原体第一次遇到宿主细胞时的微环境，以及脂质对这一过程的影响。这将使我们深入了解TTSS作为宿主细胞表面锚和宿主-病原体串扰所需的信号平台的ROE。从这些研究中获得的信息将为志贺氏菌TTS的致病机制提供基本信息，并可能对其他TTS产生影响。这最终可能导致开发实用的化合物或方法来预防表达TTSS的人类病原体的感染。

DESCRIPTION (provided by applicant): Shigella flexneri, the causative agent of bacillary dysentery, uses its type III secretion system (TTSS) as a conduit through which effector proteins are shuttled from bacterial to host cell cytoplasm where they subvert normal host functions. While much about TTSS function has been worked out, details on initial host interaction are not well understood. It has been shown that integral components of the type III secretion apparatus (TTSA) interact with cholesterol (Chol) and recent evidence indicates that liposomes containing Chol and sphingomyelin (Sm) induce TTS. These and other data suggest a role for lipid raft microdomains in Shigella contact, stable interaction, and effector injection into host cells, however, there has been no sustained attempts to directly explore the link between the pathogen and lipid raft-like domains. It is my hypothesis that lipid raft- like domains in human cells provide a site at which the Shigella TTSS can anchor to the surface of the cell while simultaneously supplying the signal that induces TTS and allows the injection of effector proteins into the host cell's cytoplasm. To better understand the role of lipid rafts in the virulece of Shigella, the specific aims of this investigation are to: 1) determine the role of host cell lipd raft components and membrane phases on S. flexneri initial attachment to and entry into host cells; and 2) determine the effects of lipid membrane dynamics on the interaction between live Shigella and model membranes. These studies will use a diverse array of techniques and approaches including: biochemical study of purified proteins using model membrane systems, real-time imaging of Shigella interaction with cultured cells, and developing methods for improving imaging of macromolecular assemblies. In completing these aims, I will provide the first comprehensive examination of this pathogen's microenvironment as it first encounters a host cell and the influence that lipids have on this process. This will provide insight into the roe of the TTSS as both an anchor on the host cell surface and as a signaling platform that is needed for host-pathogen crosstalk. The information obtained from these studies will provide basic information on the pathogenic mechanisms of Shigella TTS with likely implications with regard to other TTSS. This could eventually lead to the development of practical compounds or methods for preventing infections by TTSS-expressing human pathogen.