鹦鹉热衣原体包涵体膜蛋白生物学特性及功能研究

Chlamydia psittaci (C. psittaci) is an obligate intracellular zoonotic bacterium with a broad host range of animal and humans, thus reveal the targets of C. psittaci pathogenesis is crucial for preventive drug designed. Absence of Chlamydia Inclusion membrane proteins (Incs) can directly affects their developmental cycle, and triggers premature Inclusion membrane lysis to disrupt their pathogenicity, however, the specific mechanism of Inc proteins is still unclear. In this work, we focus on five C. psittaci Inc proteins. First, we expect to adopt indirect immunofluorescence and laser scanning confocal to study their time course and intracellular localization. Then, detecting C. psittaci Inc proteins induced inflammatory cytokines expression, cell apoptosis and autophagy as well as pathological injury degree of lungs to analyze their toxicity. After then, use both yeast two hybrid and targeted proteomic to screen host cell interaction partner, and further analysis the influence of silencing target proteins on C. psittaci development cycle, infection capacity and host cell apoptosis. By Targe Tron, we further generate individual Inc gene deletion strain to clarify its roles on the survival and pathogenesis of chlamydia. Our study will reveal the crucial targe of C. psittaci pathogenesis, and thus for further development of C. psittaci vaccine.

鹦鹉热衣原体（Cps）是一种严格细胞内寄生、多宿主人兽共患病病原体，寻求Cps致病的关键靶点对于设计防治性药物至关重要。衣原体包涵体膜蛋白（Incs）的缺失会直接影响衣原体的生长周期，使包涵体膜提前裂解而终止发育进而影响其致病过程，但作用机制仍不清楚。本课题以5个Cps Inc蛋白为切入点，拟采用间接免疫荧光、激光共聚焦等方法分析其表达时相及细胞内定位，检测Cps Inc蛋白诱导炎性因子表达、宿主细胞凋亡、自噬及肺组织的病理损伤情况；酵母双杂交及靶向蛋白质组学技术筛选鉴定与Cps Inc蛋白相互作用的宿主靶蛋白，研究干扰靶蛋白表达对于Cps发育周期、感染能力及宿主细胞凋亡的影响；通过Targe Tron技术构建Cps Inc单基因缺失的突变体，体内外实验明确Inc基因对衣原体生存与致病的作用。本项目的开展将明确Cps生存致病相关的重要靶点，并为开发Cps疫苗奠定基础。

鹦鹉热衣原体(Chlamydia psittaci, Cps)是一类专性胞内寄生，多宿主人兽共患病病原体，能感染包括人类在内的多种哺乳动物以及鸟类，引起严重的疾病。包涵体膜蛋白是维持衣原体生长发育的关键因子，能够参与衣原体与宿主细胞相互作用、疾病发生与宿主免疫反应过程，在诊断、疫苗和机制方面研究广泛。然而对鹦鹉热衣原体包涵体膜蛋白鲜有报道，其生物学特性和功能仍然不清楚。本项目根据特征性双叶疏水结构域，采用生物信息学预测Cps包涵体膜蛋白，通过体外基因克隆表达了Cps包涵体蛋白，采用间接免疫荧光、RT-PCR、Western Blot、RNA干扰、透射电镜和流式细胞术等技术确定Cps关键包涵体膜蛋白，探究Cps包涵体膜蛋白在参与宿主炎症反应、诱导宿主细胞自噬、凋亡以及参与衣原体免疫逃逸中的作用及机制。研究结果表明：CPSIT_0556和CPSIT_0842定位于包涵体膜，且CPSIT_0556为Cps中期表达蛋白，CPSIT_0842为Cps中期表达蛋白，且CPSIT_0556/CPSIT_0842可经TLR4/MyD88依赖的MAPKs和NF-κB途径诱导THP-1细胞中IL-6和IL-8的表达；此外，CPSIT_0844也可经TLR2/TLR4/MyD88依赖途径激活MAPK和NF-κB信号通路诱导THP-1细胞表达IL-6和IL-8；我们进一步研究发现，CPSIT_0842可通过MAPK/ERK-mTOR途径诱导THP-1细胞不完全自噬进而促进巨噬细胞凋亡；Cps可在hPMN中存活及增殖，并通过CPSIT_0556激活PI3K/AKT和NF-κB信号通路抑制hPMN凋亡。本项目的开展筛选、鉴定了Cps关键包涵体蛋白并评估了其生物学功能，探究了包涵体膜蛋白诱导细胞炎症、自噬、凋亡及相关信号通路，为阐明Cps致病及免疫逃逸机制提供重要的实验依据。