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intracellular parasite; pathogen; protein complex; protein function; protein protein interaction; recruit; tool; unpublished works

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 衣原体感染是人类疾病的重要原因，目前尚不存在疫苗。他们的非凡之处 流行率、相关发病率、医疗保健费用以及与各种慢性病州的联系使其公之于众 至关重要的关切。虽然感染可以用抗生素治疗，但没有一种药物是划算的。 足以广泛根除疾病。我们知识中的一个重要缺口是这种细胞内的义务是如何 液泡细菌建立了一个特权的生态位--一个被膜包裹的隔间，称为包涵体--以及 避免宿主的先天免疫反应。衣原体编码一个独特的分泌型效应器家族，即Incs (包涵膜蛋白)，通过III型分泌系统从细菌转移 并插入到包涵膜中。我们假设这些效应器中的一些根据它们的位置 在宿主-病原体界面，调节先天免疫反应。然而，由于易驯服的遗传工具 尽管最近才开发出针对衣原体的INC，但大多数INC的具体功能仍不清楚。 我们率先将高通量亲和纯化-质谱学(AP-MS)策略与 通过转染法鉴定2/3的沙眼衣原体可能的宿主结合伙伴。在最近未发表的文章中 工作中，我们发现其中几个效应器具有意想不到的作用，这表明Incs不仅可以 作为蛋白质支架的功能，但它们可能会在包涵体上建立更高阶的新蛋白质复合体。 然而，我们的“转基因”交互作用组可能受到incs或incs的非自然呈现的限制。 要求某些INC-宿主蛋白质-蛋白质相互作用(PPI)需要多个INC。在目标1中，我们提出了一个 高吞吐量战略，通过调整我们的AP-MS屏幕来快速识别和 在感染环境中验证主机交互合作伙伴，并确定新的Inc.主机PPI 在我们最初的屏幕上错过了。在初步数据中，我们验证了这种“感染”交互作用组方法，它具有 使我们能够优先进一步研究两个令人着迷的效应器，CT226和CT224，这可能会影响 宿主对沙眼衣原体感染的先天免疫反应，这是一个普遍研究较少的细胞内领域 空泡状的人类病原体。我们发现，预测的螺旋线圈结构域在C-末端 CT226结合到由LRRFIP1和/或LRRFIP2组成的复合体(L/F复合体)。该功能 这个复合体的作用还不完全清楚，但已有报道调节炎症体和IRF3。 和哺乳动物细胞中的NFkB信号。我们已经发现，预测的盘绕线圈结构域在C- CT224的末端与TRAF7结合，TRAF7是肿瘤坏死因子受体相关因子中的一个独特成员 NFkB信号、细胞因子的产生和细胞凋亡。我们假设CT226：L/F和CT224：TRAF7 相互作用调节宿主细胞对沙眼衣原体感染的先天免疫反应。在目标2和目标3中，我们将 分别使用生化、细胞生物学和遗传策略破译CT226和CT224的功能 探讨它们在沙眼衣原体感染中的作用。