Surface protein dynamics in live bacterial pathogens

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，这种模式可能会随着 细胞生长。在细菌感染和发病机制的背景下，蛋白质在细菌中的动态行为 细胞膜可能特别重要，因为它是细菌细胞的外表面， 直接联系主机。技术壁垒使得直接检查分布和 革兰氏阴性菌表面蛋白，尤其是完整外膜蛋白的迁移率 细菌病原体。这份提案中描述的项目的目的是利用最近的几个 开发的技术允许观察活的细菌细胞中的表面蛋白质动态，以研究 膜蛋白迁移率在几种肠道致病机制中的作用 细菌，包括B类致病菌福氏志贺氏菌、肠沙门氏菌、耶尔森氏菌和 致病性大肠埃希菌(EPEC)。这些新技术，基于对 视频显微镜图像，能够在蛋白质变化时追踪它们的大规模分布 时间和单个蛋白质分子在细菌表面的小范围运动。他们将被用来 在细菌感染的背景下回答关于表面蛋白流动性的三个具体问题：1)如何 IcsAA/IRG在外膜上的迁移性是否有助于志贺菌表面的蛋白质极化 福氏杆菌？2)沙门氏菌外膜毒力因子的流动性和活性如何， 耶尔森氏菌和EPEC受感染相关的脂多糖重塑的影响？以及，3)什么是 革兰氏阴性菌多药耐药外排泵的组织和动态行为 细菌，在药物暴露之前和期间？这个项目的第四个目标是开发一套高度 吞吐量、自动化计算图像/分析技术，可促进蛋白质分析 活菌实验中的动力学和细胞间变异，我们将免费提供给 研究社区。

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 enterics, 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 IcsAA/irG 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.