Surface protein dynamics in live bacterial pathogens

描述（由申请人提供）：现在人们普遍认识到，细菌细胞具有极其复杂的结构组织，许多单独的蛋白质以高度不均匀的模式分布在细胞中，这些模式可能随着细胞生长而迅速变化。在细菌感染和发病机制的背景下，细胞包膜中蛋白质的动态行为可能特别重要，因为它是直接接触宿主的细菌细胞的外表面。技术障碍使得直接检查细菌表面蛋白的分布和流动性变得困难，特别是革兰氏阴性细菌病原体中的完整外膜蛋白。该提案中描述的项目的目的是利用几种最近开发的技术，这些技术可以观察活细菌细胞中的表面蛋白动态，以研究膜蛋白流动性在几种肠道细菌引起的疾病的持续性和发病机制中的作用，包括B类病原体福氏志贺氏菌、肠道沙门氏菌、耶尔森氏菌和肠病性大肠杆菌（EPEC）。这些新技术基于视频显微镜图像的定量分析，能够追踪随时间变化的大规模蛋白质分布以及细菌表面上单个蛋白质分子的小规模运动。它们将用于回答有关细菌感染情况下表面蛋白流动性的三个具体问题：1) IcsA/VirG 在外膜中的流动性如何促进福氏志贺氏菌表面的蛋白质极化？ 2) 沙门氏菌、耶尔森氏菌和 EPEC 外膜毒力因子的移动性和活性如何受到与感染相关的脂多糖重塑的影响？以及，3）在药物暴露之前和期间，革兰氏阴性细菌中的多药耐药性（MDR）外排泵的组织和动态行为是什么？该项目的第四个目标是开发一套高通量、自动化计算图像/分析技术，可以促进活细菌实验中蛋白质动力学和细胞间变异的分析，我们将免费向研究界提供这些技术。

DESCRIPTION (provided by applicant): It is now widely appreciated that bacterial cells have a dramatically complex structural organization, with many individual proteins distributed in the cells in a highly nonuniform pattern that may change rapidly as the cell grows. In the context of bacterial infection and pathogenesis, the dynamic behavior of proteins in the cell envelope is likely to be particularly important, since it is the outside surface of the bacterial cell that directly contacts the host. Technical barriers have made it difficult to directly examine the distribution and mobility of bacterial surface proteins, particularly integral outer membrane proteins in Gram-negative bacterial pathogens. The aim of the project described in this proposal is to exploit several recently developed techniques that allow observation of surface protein dynamics in living bacterial cells to study the role of membrane protein mobility in the persistence and pathogenesis of disease caused by several enteric bacteria, including the category B pathogens Shigella flexneri, Salmonella enterica, Yersinia spp., and enteropathogenic Escherichia coli (EPEC). These new techniques, based on quantitative analysis of videomicroscopy images, are capable of tracing both large-scale protein distributions as they change over time and small-scale movements of individual protein molecules on the bacterial surface. They will be used to answer three specific questions about surface protein mobility in the context of bacterial infection: 1) How does mobility of IcsA/VirG in the outer membrane contribute to protein polarization on the surface of Shigella flexneri? 2) How are the mobility and activity of virulence factors in the outer membrane of Salmonella, Yersinia, and EPEC affected by the lipopolysaccharide remodeling associated with infection? and, 3) What is the organization and dynamic behavior of multidrug resistance (MDR) efflux pumps in Gram-negative bacteria, prior to and during drug exposure? A fourth goal of this project is to develop a suite of high-throughput, automated computational image/analysis techniques that can facilitate analysis of protein dynamics and cell-to-cell variation in experiments on live bacteria, which we will make freely available to the research community.