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intracellular parasite; pathogen; protein complex; protein function; protein protein interaction; recruit; tool; unpublished works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

项目总结/摘要 衣原体感染是人类疾病的重要原因，目前还没有疫苗。他们非凡 患病率、相关发病率、卫生保健费用以及与各种慢性病状态的联系使其公开化 至关重要的问题。虽然感染可以用抗生素治疗，但没有一种药物是划算的 足以广泛消除疾病。我们知识中的一个重要空白是， 空泡细菌建立了一个特殊的生态位--一个被称为内含物的膜结合区室-- 避免了宿主的先天免疫反应衣原体编码一个独特的家族的分泌效应，Inc （包涵体膜蛋白），通过III型分泌系统从细菌转运 并插入到包合膜中。我们假设这些效应器中的一些，由于它们的位置 在宿主-病原体界面，调节先天免疫反应。然而，由于易于处理的遗传工具 直到最近才针对衣原体开发出来，但大多数Inc的具体功能仍然未知。 我们率先使用高通量亲和纯化-质谱（AP-MS）策略， 用转染鉴定2/3的C.沙眼在最近未发表的 工作，我们已经发现了一些意想不到的作用，这些效应器，这表明，不仅可以Incs 作为蛋白质的支架，但它们可能会建立更高级别的新的蛋白质复合物的包容性。 尽管如此，我们的“转染”相互作用组可能受到Inc的非天然呈递或Inc的非天然呈递的限制。 要求一些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 高通量策略，以克服这些限制，通过调整我们的AP-MS筛选， 在感染的背景下验证宿主相互作用伙伴，并识别可能已经被感染的新的Inc-宿主PPI。 在我们的初始屏幕中丢失。在初步数据中，我们验证了这种“感染”相互作用组方法， 使我们能够优先考虑进一步研究两个迷人的效应器，CT 226和CT 224，这可能会影响 宿主对C.沙眼感染，一个未充分研究的领域，一般为细胞内 空泡状人类病原体。我们已经发现，预测的卷曲螺旋结构域的C-末端的 CT 226结合由Flightless-1和LRRFIP 1和/或LRRFIP 2组成的复合物（L/F复合物）。功能 该复合物的结构尚不完全清楚，但据报道可以调节炎性小体和IRF 3 和NFkB信号传导。我们已经发现，预测的卷曲螺旋结构域在C- CT 224的末端与TRAF 7结合，TRAF 7是TNF受体相关因子的一个独特成员， NFkB信号传导、细胞因子产生和细胞凋亡。我们假设CT 226：L/F和CT 224：TRAF 7 相互作用调节宿主细胞对C.沙眼感染在目标2和3中，我们将 使用生物化学、细胞生物学和遗传学策略分别解码CT 226和CT 224的功能 探讨它们在C.沙眼感染