鹦鹉热衣原体质粒蛋白CPSIT_p8结构与功能研究

Chlamydia psittaci is an obligate intracellular zoonotic pathogen with a broad range of hosts including humans and animals. Studies have showed that plasmid proteins of Chlamydia are demonstrated to be key virulence factors, play an important role in maintaining intracellular growth and development and the pathogenesis. CPSIT_p8 is one of the proteins encoded by the C. psittaci plasmid, and we previously confirmed that CPSIT_p8 has been closely related to the pathogenicity, and induces relatively protective immunity against challenge with C. psittaci in mice. However, the structure and specific functions of C. psittaci plasmid-encoded CPSIT_p8 are still unclear. In this study, we focus on C. psittaci plasmid-encoded CPSIT_p8 structure, then we aim to employ heterologous secretion system and T3SS inhibitor to study the secretion pathway. Then both targeted proteomic and yeast two hybrid were used to screen host cell interaction partner; Then GST-pull down and Co-Immunoprecipitation technique are used to identify the interaction protein. We expect to adopt laser scanning confocal and cross-neutralization test to explore intracellular localization and the roles on C. psittaci infection of the interaction proteins. Then, detecting CPSIT_p8 induced inflammatory cytokines expression and cell apoptosis to study toxicity roles. After then, we set out to assess the protective capacity of vaccination with CPSIT_p8 against C. psittaci lung infection in a mouse model. Our study will display the strict functions and biological characteristics of plasmid-encoded CPSIT_p8, and provide novel and important information for new targeting the chlamydia drugs and chlamydial vaccines development.

鹦鹉热衣原体（Cps）是一种专性细胞内寄生的多宿主人兽共患病病原体。研究表明，衣原体质粒蛋白是其关键毒力因子，在维持衣原体胞内生长发育及致病过程中起重要作用。 CPSIT_p8是由Cps质粒编码的一种蛋白，我们的前期研究结果表明CPSIT_p8与Cps致病性密切相关，并具有良好的免疫保护作用，但其结构与具体功能尚未见报道。本课题以其结构为切入点，拟采用异源分泌系统及T3SS抑制剂鉴定其分泌途径，并通过靶向蛋白质组学和酵母双杂交技术等筛选宿主细胞互作蛋白，采用GST-pull down、免疫共沉淀技术进行验证，随后通过激光共聚焦、交叉中和实验等技术分析其定位及在Cps感染中的作用；并检测CPSIT_p8诱导炎性因子表达及宿主细胞凋亡，分析其毒力作用；以及评估其抗Cps感染免疫保护功能。本课题的开展可为明确质粒蛋白CPSIT_p8的生物学特性及功能、寻找新型靶向药物及研制Cps疫苗奠定基础。

鹦鹉热衣原体（Cps）是一种专性细胞内寄生的多宿主人兽共患病病原体。研究表明，衣原体质粒蛋白是其关键毒力因子，在维持衣原体胞内生长发育及致病过程中起重要作用。 CPSIT_p8是由Cps质粒编码的一种蛋白，但其具体功能尚未见报道。本课题通过体外基因克隆表达了CPSIT_p8，使用间接免疫荧光、Western Blot、RT-PCR、免疫组化和流式细胞术等技术，探究质粒蛋白CPSIT_p8抗Cps感染免疫保护功能，分析Cps在体外和体内持续性感染模型中的基因表达差异，并探究了Cps延缓人中性粒细胞凋亡的分子机制研究。研究结果表明：重组蛋白CPSIT_p8可诱导BALB/c小鼠产生高水平的Th1型细胞免疫应答，对BALB/c小鼠抗Cps感染具有良好的免疫保护作用，有望应用于诊断试剂盒和疫苗的研发。此外，成功构建由阿莫西林（Amox）诱导的Cps持续性感染BALB/c小鼠模型，Cps基因ahpC、euo、prmC表达上调，pbp3、sucB_1、oppA_4、pmpH、ligA表达下调；Cps激活P2X7受体的表达可延缓人中性粒细胞（hPMN）凋亡及CPSIT_0556可通过PI3K/Akt和NF-κB信号通路延缓hPMN凋亡。本项目的开展将进一步揭示衣原体和宿主细胞相互作用的分子机制，并通过评估质粒蛋白抗感染免疫保护作用，为寻找新型靶向药物及研制Cps疫苗奠定基础。