Decoding the Chlamydia inclusion membrane protein-host protein interactome

解码衣原体包涵膜蛋白-宿主蛋白相互作用组

• 批准号: 9185266
• 负责人: Joanne N. Engel
• 金额: $ 61.31万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-12-01 至 2020-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9185266
• 关键词: Affinity; Affinity Chromatography; Algorithms; Antibiotics; Bacteria; Binding; Biochemical; Bioinformatics; Biological; Catalogs; Cells; Cellular biology; Chlamydia; Chlamydia Infections; Chlamydia trachomatis; Chronic Disease; Complex; Data; Data Set; Developing Countries; Disease; Dynein ATPase; Endosomes; Environment; Epitopes; Eye Infections; Family; Genetic; Golgi Apparatus; Grant; Health Care Costs; Human; Infection; Knowledge; Length; Life Cycle Stages; Ligase; Link; Mass Spectrum Analysis; Mediating; Membrane; Membrane Proteins; Microbe; Microtubules; Modeling; Molecular; Morbidity - disease rate; Pathogenesis; Pharmaceutical Preparations; Positioning Attribute; Prevalence; Process; Protein Subunits; Proteins; Proteome; Proteomics; Recruitment Activity; Reproducibility; Research; Research Personnel; Respiratory Tract Infections; Role; SNAP receptor; Streptavidin; System; Techniques; Technology; Testing; Ubiquitin; Vaccines; Virulence; base; cost effective; dynactin; follow-up; genital infection; human disease; mutant; novel; obligate intracellular parasite; pathogen; programs; protein protein interaction; public health relevance; response; scaffold; sorting nexins; syntaxin; tool; trafficking; ubiquitin ligase

 DESCRIPTION (provided by applicant): Chlamydia species are important causes of human disease for which no vaccine exists. An important gap in our knowledge is how this obligate intracellular parasite establishes a privileged niche--a membrane bound compartment termed the inclusion--in order to survive and replicate in the hostile intracellular environment. Chlamydiae encode a distinctive family of secreted effectors, the Incs (Inclusion membrane proteins) that are translocated from the bacteria and inserted into the inclusion membrane. We hypothesize that these effectors are ideally positioned at the host-pathogen interface to mediate interactions between the inclusion and the host and contribute to successful intracellular survival. This grant builds on our extensive preliminary studies in which we used large-scale affinity purification/mass spectrometry (AP-MS) to comprehensively identify protein- protein interactions (PPIs) between all C. trachomatis Incs and the human proteome. From amongst 404 interactions for 38/62 Incs, we identified a plethora of new potential interactions. We propose to use biochemical, cell biological, and newly developed Chlamydial genetic strategies to validate our highest priority Inc-host PPIs and explore their role in C. trachomatis infections. In Aim 1, we follow-up at the detailed molecular level our novel observation that IncE subverts retromer components and possibly a subset of syntaxins to modulate host cell vesicular trafficking. In aim 2, we will investigate the mechanism and functional significance of the interaction of the Inc CT192 with the dynactin complex. We propose that either CT192 sequesters dynactin to interfere with dynein-dependent transport or that it allows the inclusion to hitch a ride onto dynein-dependent microbule transport. The proposed approaches are applicable to other high confidence PPIs that we have identified and determined to be high priority. In aim 3, we team up with our Co-investigator, Dr. Nevan Krogan, to apply powerful new proteomic technologies to globally profile changes in the host ubiquitome in response to pathogens. Our finding that up to 12 Incs appear to interact with various components of the host cell ubiquitin machinery suggests that Chlamydia reprograms the host ubiquitin program to facilitate infection. We prioritized study of Inc CT383, as it is expressed early, predicted to interact with 3 different Ub ligases, and has the potential to substantially remodel the host ubiquitinome. Together, these aims build upon our extensive preliminary data and allow us to comprehensively understand how Chlamydia employs Incs to create a unique intracellular niche and reprogram the host.

 描述（由申请方提供）：衣原体属是人类疾病的重要原因，目前尚无疫苗。我们知识中的一个重要空白是这种专性细胞内寄生虫如何建立一个特权小生境-一个被称为内含物的膜结合区室-以便在敌对的细胞内环境中生存和复制。衣原体编码一个独特的分泌效应子家族，Inc（包涵体膜蛋白），其从细菌移位并插入包涵体膜。我们假设这些效应子理想地位于宿主-病原体界面，以介导包涵体和宿主之间的相互作用，并有助于成功的细胞内存活。这项资助建立在我们广泛的初步研究基础上，在这些研究中，我们使用大规模亲和纯化/质谱（AP-MS）来全面鉴定所有C之间的蛋白质-蛋白质相互作用（PPI）。沙眼衣原体和人类蛋白质组。从38/62英寸的404个相互作用中，我们发现了大量新的潜在相互作用。我们建议使用生物化学，细胞生物学，和新开发的衣原体遗传策略，以验证我们的最高优先级的Inc-宿主PPI，并探讨其在C。沙眼感染 在目标1中，我们在详细的分子水平上跟踪我们的新观察，即因斯颠覆retromer组件和可能的突触融合蛋白的子集来调节宿主细胞囊泡运输。目的二是研究IncCT 192与dynactin复合物相互作用的机制和功能意义。我们认为，CT 192要么隔离动力蛋白以干扰动力蛋白依赖性转运，要么允许包含 搭上了动力蛋白依赖性微管转运的顺风车。所提出的方法适用于我们已经确定并确定为高优先级的其他高置信度PPI。在目标3中，我们与我们的合作研究者Nevan Krogan博士合作，将强大的新蛋白质组学技术应用于全球范围内对宿主泛素组响应病原体的变化。我们的发现，多达12 Incs似乎与宿主细胞泛素机制的各种成分相互作用，这表明衣原体重新编程宿主泛素程序，以促进感染。我们优先研究Inc CT 383，因为它表达较早，预测与3种不同的Ub连接酶相互作用，并有可能大幅重塑宿主泛素组。总之，这些目标建立在我们广泛的初步数据基础上，使我们能够全面了解衣原体如何利用Incs创造一个独特的细胞内生态位并重新编程宿主。