Remodeling of the Host Ubiquitin Landscape by Chlamydia trachomatis

项目总结 沙眼衣原体是性传播疾病、非先天性不孕症和 一种叫做沙眼的致盲眼病。这种专有的胞内细菌在膜结合的环境中复制 包含体，在那里它利用一种特殊的蛋白质输出系统，称为III型分泌 系统将效应蛋白转移到宿主细胞的细胞质中。一个独特的III型效应器亚家族 蛋白质包涵膜蛋白(Incs)，插入包涵膜，其N-和C-。 Termini理想地处于宿主-病原体界面，与宿主蛋白质和细胞器相互作用。然而，我们的 对Incs功能的理解一直是有限的，因为它们彼此之间或彼此之间几乎没有同源性 已知的蛋白质，以及对衣原体的遗传操作直到最近才成为可能。为了定义C. 沙眼衣原体-人类蛋白相互作用组，恩格尔实验室进行了高通量亲和纯化 预测的58个INC的质谱图。值得注意的是，INC的一个子集被识别为目标 宿主泛素(Ub)机制的组成部分，这促使人们假设衣原体的一个子集 INCS可能在感染过程中调节宿主的泛Q化，这些变化可能对 沙眼衣原体感染的发病机制。泛素化，它涉及Ub与一个 通过E1Ub激活酶、E2Ub结合酶和E3Ub连接酶的靶蛋白改变 Ub修饰蛋白的功能、稳定性或在细胞内的位置。调控蛋白质泛素化的研究进展 感染是细胞内病原体的一个新兴主题。事实上，以前的研究已经证实，C. 全球沙眼衣原体感染改变宿主蛋白稳定性；然而，Inc-Ub机制如何相互作用 重塑宿主蛋白质组是未知的。该项目的长期目标是了解交互如何 在特定INC和宿主Ub机制之间改变宿主Ub环境并促进衣原体 发病机制。在目标1中，一种新开发的基于MS的泛素残基分析(URP)方法和 生物信息学将被用来在全球范围内定义沙眼衣原体引起的宿主泛喹组的变化。这些 变化将与通过类似管道获得的其他细胞内病原体的数据集进行比较，以确定 衣原体感染特异的蛋白质泛素化以及蛋白质泛素化的变化 这些事件是细胞内病原体的常见目标。在目标2中，URP、宿主和细菌的组合 遗传学，以及细胞和分子生物学将被用来确定宿主泛喹组的变化 依赖于INC(CT383)及其与主机Ub机器组件的交互。加在一起，这些 研究将为沙眼衣原体对宿主细胞重新编程的机制提供关键的新见解 通过泛素化促进发病。 好了！

PROJECT SUMMARY Chlamydia trachomatis is the leading cause of sexually transmitted diseases, non-congenital infertility, and the blinding eye disease called trachoma. This obligate intracellular bacterium replicates within a membrane bound compartment, the inclusion, where it utilizes a specialized protein export system called the type III secretion system to translocate effector proteins into the cytoplasm of the host cell. A unique subfamily of type III effector proteins the Inclusion membrane proteins (Incs), is inserted into the inclusion membrane, with its N- and C- termini ideally poised at the host-pathogen interface to interact with host proteins and organelles. However, our understanding of the function of Incs has been limited, as they share little homology to each other or to other known proteins, and genetic manipulation of Chlamydia only recently become possible. To define the C. trachomatis Inc-human protein interactome, the Engel lab performed a high-throughput affinity-purification mass spectroscopy screen of 58 predicted Incs. Remarkably, a subset of Incs was identified to target components of the host ubiquitin (Ub) machinery, which prompts the hypothesis that a subset of Chlamydia Incs may modulate host ubiqutylation during infection and that these changes may be critical to the pathogenesis of C. trachomatis infection. Ubiquitylation, which involves the covalent attachment of Ub to a target protein by an E1 Ub-activating enzyme, an E2 Ub-conjugating enzyme, and an E3 Ub ligase, alters the function, stability, or location of the Ub-modified protein within the cell. Modulating protein ubiquitylation during infection is an emerging theme for intracellular pathogens. Indeed, previous studies have established that C. trachomatis infection globally alters the host protein stability; however, how Inc-Ub machinery interactions reshape the host proteome is unknown. The long-term goal of this project is to understand how interactions between specific Incs and the host Ub machinery alters the host Ub landscape and promotes Chlamydia pathogenesis. In aim 1, a newly developed MS-based ubiquitin remnant profiling (URP) approach and bioinformatics will be used to globally define C. trachomatis-induced changes in the host ubiquinome. These changes will be compared to data sets for other intracellular pathogens obtained by a similar pipeline to identify changes in protein ubiquitylation that are specific to Chlamydia infections as well as protein ubiquitylation events that are common targets of intracellular pathogens. In aim 2, a combination of URP, host and bacterial genetics, and cell and molecular biology will be used to identify changes in the host ubiquinome that are dependent on the Inc (CT383) and its interactions with components of host Ub machinery. Together, these studies will provide critical new insights on the mechanisms by which C. trachomatis reprograms the host cell through ubiquitylation to promote pathogenesis. !