鹦鹉热衣原体质粒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 animals, including humans, while little is known about its virulence factors. Plasmid encoded proteins of C. trachomatis and C. muridarum are demonstrated to be key virulence factors, and we previously confirmed that CPSIT_p8 shows relatively protective immunity when immunised mice. However, the pathogenic roles and biological characteristics of C. psittaci plasmid-encoded proteins are not clear. In this work, we focus on all the C. psittaci plasmid-encoded proteins. First, we expect to adopt indirect immunofluorescence, laser scanning confocal and heterologous secretion system to study their time course, intracellular localization and secretion pathway. Then, detecting C. psittaci plasmid proteins induced inflammatory cytokines expression, cell apoptosis as well as pathological injury degree of lungs to analyze their toxicity. After then, immunising mice to evaluate their protective immunity. By chlamydia transformation, we further generate plasmid free C. psittaci strain and individual plasmid gene deletion strain to immunise mice to verify their pathogenicity and protection function. Our study will reveal the strict roles and biological characteristics of C. psittaci plasmid-encoded proteins, and thus for further improvement knowledge of C. psittaci pathogenic mechanism and vaccine development.

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，目前对其毒力因子了解甚少。沙眼衣原体与鼠衣原体质粒蛋白是关键致病因子，我们前期研究结果证实Cps质粒蛋白（CPSIT_p8）具有较好的免疫保护性，但其生物学功能及致病作用尚未见报道。本课题以Cps所有质粒蛋白为切入点，拟采用间接免疫荧光、激光共聚焦和异源分泌系统等方法分析其表达时相、细胞内定位及分泌途径等生物学特性。检测Cps质粒蛋白诱导炎性因子表达、宿主细胞凋亡及肺组织的病理损伤程度，分析其毒力作用。质粒蛋白免疫小鼠，评价抗Cps感染免疫保护功能。通过衣原体转化技术构建质粒缺失株与单一质粒蛋白基因缺失株，并建立小鼠感染模型，验证质粒蛋白的致病性和保护作用。本项目的开展将明确Cps质粒蛋白的生物学特性及功能，为进一步揭示Cps致病机制，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。质粒编码的质粒蛋白是衣原体重要的毒力因子，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质粒编码蛋白鲜有报道，其生物学特性和功能仍然不清楚。本项目通过体外基因克隆表达了Cps质粒蛋白，采用间接免疫荧光、RT-PCR、Western Blot、蛋白芯片、RNA干扰、免疫磁珠、免疫组化和流式细胞术等技术确定Cps关键质粒蛋白，探究Cps质粒蛋白在抗感染免疫、诱导宿主细胞自噬和炎症中的作用及机制。研究结果表明：质粒蛋白CPSIT_p7是Pgp3同源物，是Cps唯一分泌质粒蛋白；基于质粒蛋白CPSIT_p7和CPSIT_p8设计的多种疫苗均产生了良好的抗感染免疫保护作用，CPSIT_p7DNA疫苗还可抑制Cps向远端器官扩散；此外，基于质粒蛋白CPSIT_p6和MOMP设计构建了多表位融合疫苗并对免疫策略和佐剂进行了优化；我们进一步研究发现质粒蛋白CPSIT_P7可经TLR4和TLR6途径诱导THP-1细胞表达炎症因子；质粒蛋白CPSIT_p7可诱导RAW264.7细胞自噬且通过TLR2介导的ERK信号通路诱导。本项目的开展鉴定了Cps质粒蛋白并评估质粒蛋白免疫原性以及抗感染保护作用，探究了Cps质粒蛋白诱导细胞自噬和炎症及相关信号通路，为阐明Cps致病机制及疫苗研发提供重要的实验依据。