Inclusion membrane protein (Inc) modulation of the innate immune response to Chlamydia trachomatis

包涵膜蛋白 (Inc) 调节沙眼衣原体先天免疫反应

• 批准号: 10246668
• 负责人: Joanne N. Engel
• 金额: $ 80.26万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-09-04 至 2022-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10246668
• 关键词: Affinity Chromatography; Antibiotics; Apoptosis; Area; Bacteria; Binding; Biochemical; Bioinformatics; Biological; Cells; Cellular biology; Chlamydia; Chlamydia Infections; Chlamydia trachomatis; Chronic Disease; Coiled-Coil Domain; Complex; Data; Data Set; Developing Countries; Disease; Environment; Epitopes; Event; Eye Infections; Family; Genetic; Grant; Health Care Costs; Human; Human Cell Line; IRF3 gene; Individual; Infection; Inflammasome; Innate Immune Response; Interferons; Knowledge; Link; Mammalian Cell; Mass Spectrum Analysis; Mediating; Membrane; Membrane Proteins; Methodology; Microbe; Morbidity - disease rate; Pathogenesis; Pharmaceutical Preparations; Positioning Attribute; Prevalence; Prevention; Process; Production; Proteins; Proteome; Publishing; Regulation; Reporting; Reproducibility; Respiratory Tract Infections; Role; Scaffolding Protein; Signal Transduction; TNF Receptor-Associated Factors; Testing; Time; Transfection; Type III Secretion System Pathway; Vaccines; Work; base; cost effective; cytokine; fascinate; genetic approach; genital infection; human disease; human pathogen; member; non-Native; novel; obligate intracellular parasite; pathogen; protein complex; protein function; protein protein interaction; recruit; tool; unpublished works; Affinity Chromatography; Antibiotics; Apoptosis; Area; Bacteria; Binding; Biochemical; Bioinformatics; Biological; Cells; Cellular biology; Chlamydia; Chlamydia Infections; Chlamydia trachomatis; Chronic Disease; Coiled-Coil Domain; Complex; Data; Data Set; Developing Countries; Disease; Environment; Epitopes; Event; Eye Infections; Family; Genetic; Grant; Health Care Costs; Human; Human Cell Line; IRF3 gene; Individual; Infection; Inflammasome; Innate Immune Response; Interferons; Knowledge; Link; Mammalian Cell; Mass Spectrum Analysis; Mediating; Membrane; Membrane Proteins; Methodology; Microbe; Morbidity - disease rate; Pathogenesis; Pharmaceutical Preparations; Positioning Attribute; Prevalence; Prevention; Process; Production; Proteins; Proteome; Publishing; Regulation; Reporting; Reproducibility; Respiratory Tract Infections; Role; Scaffolding Protein; Signal Transduction; TNF Receptor-Associated Factors; Testing; Time; Transfection; Type III Secretion System Pathway; Vaccines; Work; base; cost effective; cytokine; fascinate; genetic approach; genital infection; human disease; human pathogen; member; non-Native; novel; obligate intracellular parasite; pathogen; protein complex; protein function; protein protein interaction; recruit; tool; unpublished works

Project Summary/Abstract Chlamydia infections are important causes of human disease for which no vaccine exists. Their extraordinary prevalence, associated morbidity, health care costs, and link to various chronic disease states make them public concerns of critical importance. Although infections can be treated with antibiotics, no drug is cost-effective enough for widespread elimination of disease. An important gap in our knowledge is how this obligate intracellular vacuolar bacterium establishes a privileged niche--a membrane bound compartment termed the inclusion--and avoids the host innate immune response. Chlamydia encode a distinctive family of secreted effectors, the Incs (Inclusion membrane proteins), which are translocated from the bacteria through the type III secretion system and inserted into the inclusion membrane. We hypothesize that some of these effectors, by virtue of their position at the host-pathogen interface, modulate the innate immune response. However, since tractable genetic tools have only recently been developed for Chlamydia, the specific function of the majority of Incs remains unknown. We pioneered using a high throughput affinity purification-mass spectroscopy (AP-MS) strategy in conjunction with transfection to identify putative host binding partners for 2/3 of the C. trachomatis Incs. In recent unpublished work, we have discovered unexpected roles for several of these effectors that suggests that not only can Incs function as proteins scaffolds, but that they may establish higher order novel protein complexes at the inclusion. Nonetheless, our “transfection” interactome is potentially limited by the non-native presentation of Incs or the requirement that some Inc-host protein-protein interactions (PPIs) require multiple Incs. In aim 1, we propose a high throughput strategy to overcome these limitations by adapting our AP-MS screen to rapidly identify and validate host interacting partners in the context of infection and to identify new Inc-host PPIs that may have been missed in our initial screen. In preliminary data, we validate this “infection” interactome approach, which has enabled us to prioritize the further study of two fascinating effectors, CT226 and CT224, that may influence the host innate immune response to C. trachomatis infections, an understudied area in general for intracellular vacuolar human pathogens. We have discovered that the predicted coiled-coil domain in the C-terminus of CT226 binds to a complex comprised of Flightless-1 and LRRFIP1 and/or LRRFIP2 (L/F complex). The function of this complex is incompletely understood but has been reported to modulate inflammasome as well as IRF3 and NFkB signaling in mammalian cells. We have discovered that the predicted coiled-coil domain in the C- terminus of CT224 binds to TRAF7, a unique member of the TNF receptor associated factors that modulates NFkB signaling, cytokine production, and apoptosis. We hypothesize that the CT226:L/F and the CT224:TRAF7 interactions modulate the host cell innate immune response to C. trachomatis infection. In aims 2 and 3, we will decode the function of CT226 and CT224, respectively, using biochemical, cell biological, and genetic strategies to explore their role in C. trachomatis infections.

项目摘要/摘要 衣原体感染是不存在疫苗的人类疾病的重要原因。他们的非凡 患病率，相关的发病率，医疗保健费用以及与各种慢性疾病状态的联系使它们公开 关注至关重要的关注。尽管感染可以用抗生素治疗，但没有药物具有成本效益 足以释放疾病的宽度。我们知识上的一个重要差距是这种细胞内的义务 真空细菌建立了一个特权的小境 - 一种称为包容的膜结合室，并 避免宿主先天免疫反应。衣原体编码一个独特的分泌效果系列，Incs （包含膜蛋白），该蛋白通过细菌通过III型分泌系统易位 并插入包含膜。我们假设其中一些效果是由于它们的位置 在宿主 - 病原体界面，调节先天免疫反应。但是，由于可处理的遗传工具 直到最近是针对衣原体开发的，大多数INC的具体功能仍然未知。 我们使用高吞吐量亲和力纯化质量光谱（AP-MS）策略进行了启动 通过翻译以识别2/3的三尖型肠骨c。在最近的未出版中 工作，我们发现了其中几种效果的意外角色，这表明不仅可以 充当蛋白质支架，但它们可以在包含物中建立高阶新型蛋白质复合物。 但是，我们的“转染”相互作用可能受到INC或INC的非本地介绍的限制 要求某些Inc-宿主蛋白质蛋白质相互作用（PPI）需要多个INC。在AIM 1中，我们建议 高吞吐量策略通过调整我们的AP-MS屏幕来快速识别和 在感染的背景下验证主机相互作用的伴侣，并确定可能是 在我们的初始屏幕中错过了。在初步数据中，我们验证了这种“感染”互动方法，该方法具有 使我们能够优先考虑两种引人入胜的效果CT226和CT224，这可能会影响该效果 宿主先天免疫反应对沙眼梭状芽胞庭感染，这是细胞内的一个理解区域 液泡人病原体。我们发现，在C末端预测的盘绕螺旋域 CT226与由Flightless-1和LRRFIP1和/或LRRFIP2（L/F复合物）组成的复合物结合。功能 该复合物的中有不完全理解，但据报道调节炎症体和IRF3 哺乳动物细胞中的NFKB信号传导。我们已经发现C-中预测的盘绕螺旋域 CT224的末端与TRAF7结合，TRAF7是TNF接收器相关因素的独特成员 NFKB信号传导，细胞因子产生和凋亡。我们假设CT226：L/F和CT224：TRAF7 相互作用调节宿主细胞对沙眼梭菌感染的先天免疫反应。在目标2和3中，我们将 分别使用生化，细胞生物学和遗传策略分别解码CT226和CT224的功能 探索它们在沙眼梭状芽胞庭感染中的作用。