pORF5与Pin1相互作用对沙眼衣原体生长发育的影响及其机制研究

Growth of Chlamydia trachomatis(Ct) depends on the complex cross-talk between the Chlamydial secreted proteins and host cell. Our previous work has demonstrated that pORF5 encoded by chlamydial plasmid was a secreted protein and mainly located in cytoplasm of Ct-infected host cells for the first time. Pin1, a peptidyl-prolyl isomerase was screened to be up-regulated in pORF5-tranfected cells by using proteomic technique, we further confirmed that the two proteins interacted with each other by co-immunoprecipitation. However, the effect and mechanism of interaction between pORF5 and Pin1 proteins on Ct growth are still kept unkown. Based on our fingdings, this project first intends to apply gene transfection, antibody blocking and siRNA to identify the effect of interaction between pORF5 and Pin1 proteins on Ct growth in cell and animal models. Then tandem affinity purification combined with mass spectrometry and bioinformatic technique will be used to screen Pin1-interacting proteins and map the protein interaction network in pORF5-transfected cells, and also to elucidate the roles of key target proteins and KEGG/BIOCARTA signal pathways for Ct growth. This work will reveal the molecular mechanism and regulatory network responsible for Chlamydial growth mediated by interaction between pORF5 and Pin1, which not only enriches the understanding of Ct pathogenesis, but also provides a basis for its prevention and treatment.

沙眼衣原体（Ct）的生长发育依赖于其分泌蛋白与宿主细胞之间复杂的cross-talk。pORF5是申请者首次发现由Ct质粒基因编码并主要定位于宿主细胞胞浆的一种分泌蛋白。前期研究采用蛋白质组学技术发现pORF5上调宿主肽基脯氨酰顺反异构酶Pin1的表达，免疫共沉淀证实二者为相互作用蛋白，但两者相互作用对Ct生长发育的影响及机制尚不明确。本项目在此基础上，首先采用基因转染、抗体阻断和siRNA等从细胞和动物水平上研究pORF5与Pin1相互作用对Ct生长发育的影响，然后采用串联亲和纯化结合质谱技术、生物信息学技术等筛选pORF5功能相关的Pin1蛋白复合物，构建蛋白相互作用网络，确认与Ct生长发育相关的关键蛋白和KEGG/BIOCARTA信号通路并探讨其生物学功能，揭示Ct通过pORF5与Pin1相互作用促进Ct生长发育的网络调控机制。本研究能丰富对Ct致病机制的认识，也为其防治提供依据。

沙眼衣原体（Ct）发育周期的顺利完成需要依赖于分泌蛋白与宿主细胞的相互作用。前期研究已证实pORF5分泌蛋白与宿主Pin1蛋白能发生相互作用，为进一步探讨pORF5 与Pin1互作后对Ct生长发育的作用及其机制，本研究首先利用慢病毒载体系统构建稳定表达pORF5基因的HeLa细胞株和对照细胞株，通过对细胞自噬和凋亡相关指标的检测，发现 pORF5可调控细胞自噬和细胞凋亡等生物学行为。应用iTRAQ标记结合2D LC-MS/MS质谱技术，构建了pORF5、Pin1功能相关的宿主蛋白数据库，共筛选到314个差异表达蛋白, 其中有159个蛋白表达上调，155个蛋白表达下调；通过GO注释对差异表达蛋白质功能进行聚类分析，发现免疫反应相关蛋白质以及细胞粘附、基因表达翻译和增殖相关的蛋白质在pORF5基因转染的HeLa细胞中显著富集；KEGG通路主要涉及了核糖体通路、MAPK通路、p53通路等；STRING软件分析发现各差异表达蛋白质之间存在直接和间接的相互作用，提示pORF5可能通过改变宿主蛋白质的表达，影响宿主细胞生物学行为以促进Ct的生长发育。进一步利用RNA干扰原理，建立干扰HMGB1表达的HeLa细胞株和对照细胞株，发现HMGB1干扰后，LC3、SQSTM1/p62的表达显著降低，parkin蛋白线粒体转位减少，细胞色素c释放增加，线粒体膜电位下降，同时显著增加Bax表达水平、降低Bcl-2表达水平。证实pORF5质粒蛋白通过上调宿主HMGB1的表达诱导线粒体自噬和抑制细胞凋亡，pORF5质粒蛋白介导宿主细胞自噬和凋亡的对话影响Ct的生长发育。本项目可为衣原体疾病的诊断和防治提供重要的理论和实验依据。