鹦鹉热嗜衣原体蛋白CPSIT_0271通过激活MAPK信号通路诱导宿主细胞炎症反应及机制研究

Chlamydophila psittaci(Cps), an obligate intracellular bacterial parasite, can lead to many diseases such as pneumonia, rthritis, lymphoma and so on. Chlamydial infection is characterized by inflammation which, exacerbated by reinfection, ultimately leads to tissue damage and scarring. Chlamydial interaction with the cytokine system is thus likely to be central to disease, while the molecular mechanism is not well-documented. Our previous results showed that CPSIT_0271 protein could induce the expression of interleukin-1 beta (IL-1β), interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-α) and interleukin-8 (IL-8), and upregulate the phosphorylated p38 protein. According to the academic reports or thesis both at home and abroad, we postulate that effector protein CPSIT_0271 induce inflammatory response by TLR2-mediated MAPK signaling pathway. To prove this thesis statement, cell transfection about related receptor and key molecules of cell signaling pathways should be performed. The cells will be pretreated with MyD88 RNAi or specific chemical inhibitors of T3SS (INF007), P38 MAPK (SB203580), ERK (U0126), and JNK (SP600125), followed by CPSIT_0271 protein treatment. The levels of inflammatory factor, including IL-1β, IL-6, TNF-α and IL-8, will be detected with quantitative real time polymerase chain reaction (QPCR) and ELISA. The aim is to clarify the molecular mechanism of inflammation induced by CPSIT_0271 protein. These results would not only enrich research theory about inflammatory response induced by Cps infection, but also provide new ideas for clinical care of Cps infection.

鹦鹉热嗜衣原体（Cps）感染可引起肺炎、淋巴瘤等多种疾病，炎症反应过度导致免疫性损伤是Cps致病的关键，但机制尚未明确。本课题组的前期研究结果表明CPSIT_0271能诱导Hela细胞产生IL-1β等炎症因子，并上调磷酸化p38蛋白。结合国内外文献报道，我们提出如下科学假说：Cps通过T3SS分泌CPSIT_0271，激活MAPK信号通路，发挥促炎症作用。本研究拟建立相关受体和信号通路关键分子的细胞转染模型，采用RNA干扰技术沉默MyD88基因和特异性化学抑制剂INF007、SB203580、U0126、SP600125阻断T3SS、p38、ERK、JNK信号分子，研究MAPK信号通路对CPSIT_0271诱导炎症因子的影响，阐明CPSIT_0271致炎症反应的信号转导与调控的分子机制。本项目的完成不仅能丰富Cps致炎症反应的新理论和新知识，而且能为Cps临床新的抗炎药物的研制提供新思路。

鹦鹉热嗜衣原体是一种严格胞内寄生的人兽共患病病原体，通常是由鸟类传染给人，导致呼吸道感染等多种疾病。研究鹦鹉热嗜衣原体的致病作用及其机制,将为衣原体的防治提供重要的实验依据。本项目在国家自然科学基金的资助下，进行了以下实验研究：.（一）鹦鹉热嗜衣原体T3SS激活JNK/ERK信号通路诱导宿主细胞炎症反应研究.T3SS抑制剂INP0007抑制鹦鹉热嗜衣原体的生长，降低THP-1细胞 IL-6、IL-8、TNF-α 和IL-1β炎症因子的产生，而外源性铁可逆转INP0007对鹦鹉热嗜衣原体生长的抑制作用，但是并不能恢复INP0007对炎症因子的抑制作用；ERK和JNK抑制剂能减少鹦鹉热嗜衣原体诱导炎症因子的产生，但是p38抑制剂不能减少鹦鹉热嗜衣原体诱导炎症因子的产生。因此推测：鹦鹉热嗜衣原体诱导炎症因子的产生与T3SS有关，且鹦鹉热嗜衣原体T3SS通过激活JNK/ERK信号通路诱导宿主细胞炎症反应。.（二）鹦鹉热嗜衣原体T3SS效应蛋白的筛选与鉴定研究.利用生物信息学的方法，初步预测了CPSIT_0217、CPSIT_0018、CPSIT_0531、CPSIT_p7四种鹦鹉热嗜衣原体T3SS效应蛋白编码基因。并克隆、表达、鉴定其免疫原性，明确其是否是三型分泌系统效应蛋白。结果发现：这四种蛋白均可刺激小鼠产生特定的免疫反应，其中CPSIT_p7 免疫原性最强，是一种鹦鹉热嗜衣原体 T3SS效应蛋白； CPSIT_0217、CPSIT_0018 和 CPSIT_0531 蛋白可能是鹦鹉热嗜衣原体菌体上的结构蛋白。.（三） CPSIT_p7激活JNK/ERK信号通路诱导宿主细胞炎症反应研究.CPSIT_p7蛋白能诱导PMA处理的 THP-1 细胞IL-6、IL-1β、IL-8和TNF-α 的含量呈剂量依赖性增加，且细胞ERK 和 JNK 磷酸化水平显著升高，p38 磷酸化水平变化不明显；JNK 和 ERK 阻断剂能明显降低 CPSIT_p7 蛋白诱导的IL-6、IL-1β、IL-8 和 TNF-α 表达。因此我们推测：CPSIT_p7通过 JNK/ MAPKs和 ERK/MAPKs 信号传导途径诱导THP-1细胞产生IL-1β、IL-6、IL-8和TNF-α 炎症因子，可能与p38/ MAPKs 信号传导通路无关。