Novel Genetic Analyses of Shigella virulence proteins

描述(由申请人提供)：确定细菌蛋白在发病机制中的作用的一个主要限制是对哺乳动物细胞进行遗传操作的困难。这项资助提出了新的和有效的全基因组筛选，使用遗传易驯化的酵母酿酒酵母来识别致病志贺氏菌蛋白靶向的真核细胞途径。在以前的工作中，首席研究员已经证明了宿主细胞与耶尔森氏菌和沙门氏菌毒力蛋白之间的相互作用在酵母和哺乳动物中是保守的。考虑到所有真核生物的基本细胞过程高度保守，这并不令人惊讶。值得注意的是，当在酵母中表达时，最近被认为由志贺氏菌III型分泌系统直接输送到哺乳动物细胞的18种蛋白质中，有7种赋予了有毒的酵母表型和/或定位于特定的亚细胞室。具体目标1将比较在酵母和哺乳动物细胞中观察到的志贺氏菌蛋白的亚细胞定位模式。特定目的2描述了全基因组半自动表型筛选，以确定其过度表达或表达缺失抑制或加剧个别致病志贺氏菌蛋白所产生的毒性表型的真核蛋白。对这些互补筛选结果的计算分析将产生对单个志贺氏菌蛋白靶向的真核细胞通路的系统水平的洞察。虽然这些方法本质上是探索性的，但初步研究表明，它们可能会产生关于特定致病蛋白的目标的假设，以在更复杂的疾病生理模型中进行测试。虽然这些研究集中在可能的志贺氏菌毒力蛋白上，但这些分析适用于任何以真核细胞内过程为目标的微生物病原体。因此，在这笔赠款中开发的建立酵母作为全基因组分析的模型系统以确定致病蛋白目标的方法，应该被证明对难以进行遗传操作或生长危险的生物特别有用。

DESCRIPTION (provided by applicant): A major limitation in determining the roles in pathogenesis of bacterial proteins is the difficulty of genetically manipulating mammalian cells. This grant proposes novel and efficient genome-wide screens with the genetically tractable yeast Saccharomyces cerevisiae to identify eukaryotic cellular pathways targeted by pathogenic Shigella proteins. In previous work the Principal Investigator has demonstrated that interactions between host cells and virulence proteins of Yersinia and Salmonella are conserved among yeast and mammals. This is not surprising given the high degree of conservation of the basic cellular processes among all eukaryotes. Remarkably, when expressed in yeast, 7 of the 18 proteins recently proposed to be delivered by the Shigella type III secretion system directly into mammalian cells confer a toxic yeast phenotype and/or localize to a specific subcellular compartment. Specific Aim 1 will compare the subcellular localization patterns of the Shigella proteins observed in yeast and mammalian cells. Specific Aim 2 describes genome-wide semi-automated phenotypic screens to identify eukaryotic proteins whose overexpression or loss of expression suppresses or exacerbates the toxic phenotypes conferred by individual pathogenic Shigella proteins. Computational analyses of the results of these complementary screens will generate systems-level insights into the eukaryotic cellular pathways targeted by individual Shigella proteins. Although these approaches are exploratory in nature, preliminary studies suggest that they are likely to generate hypotheses as to the targets of specific pathogenic proteins to test in more complex physiologic models of disease. While these studies focus on probable Shigella virulence proteins, these analyses are applicable to any microbial pathogen that targets eukaryotic intracellular processes. Thus, approaches developed in this grant to establish yeast as a model system for genome-wide analyses to determine the targets of pathogenic proteins, should prove particularly useful for organisms that are difficult to genetically manipulate or dangerous to grow.