梅毒螺旋体膜脂蛋白Tpn32相互作用宿主蛋白的筛选及功能研究

Syphilis is a chronic sexually transmitted infection caused by the bacterium Treponema pallidum subsp. pallidum (T. pallidum, Tp). Membrane lipoproteins of Tp have been found to play important roles in the pathogenesis of syphilis. We also demonstrated previously that Tpn32, a membrane lipoprotein encoded by Tp, may have good immunogenicity and play an important role in triggering inflammation. However, the mechanism that the protein interacts with the host cells has not been identified. In this work, we will firstly determine the sub cellular location of Tpn32, and then produce recombinant protein Tpn32 in E. coli. The target proteins that interact with Tpn32 were identified by the membrane yeast two-hybrid system (MYTH), Far western blotting, GST pull down, Co-immunoprecipitation, mass spectrometry, and other methods. After silencing the interaction proteins by RNA interference, RT-PCR, ELISA, indirect immunofluorescence and flow cytometry were used to explore the pathogenicity of Tpn32 in Tp infection. These results of our study will be conducive to investigating the pathogenic mechanism of Tpn32, and laying a solid foundation for targeting the Tp drugs and engineering vaccine development.

梅毒螺旋体(Treponema pallidum, Tp)是人类性传播疾病梅毒的病原体。既往研究认为Tp具有很强的侵袭力，而且Tp膜脂蛋白在其致病中具有重要作用。本课题组前期研究表明Tp主要膜脂蛋白Tpn32不仅具有良好的免疫原性，还具有很强的致炎作用，但其与宿主细胞相互作用的机制尚不清楚。本项目拟首先采用伯氏疏螺旋体异源表达系统研究Tpn32膜脂蛋白的亚细胞定位，然后采用基因重组技术获得膜脂蛋白Tpn32，运用膜酵母双杂交、Far western blotting、GST pull down、免疫共沉淀以及质谱技术筛选和验证与Tpn32相互作用的宿主蛋白。RNA干扰技术沉默靶蛋白后以RT-PCR和ELISA检测促炎细胞因子表达水平，间接免疫荧光和流式细胞技术等检测细胞凋亡率，进一步研究Tpn32的致病作用。本课题的完成将为揭示Tpn32的致病机制及其作为干预靶点提供重要的理论和实验依据。

梅毒螺旋体（Treponema pallidum, Tp）是人类性传播疾病梅毒的病原体。既往研究认为Tp具有很强的侵袭力，而且Tp膜脂蛋白在其致病中具有重要作用。近年来我国梅毒发病率呈逐年上升趋势,但其致病机制目前尚不清楚。本课题组首先在体外纯化Tpn32膜脂蛋白，随后发现其可通过ERK1/2、p38和NF-κB途径诱导THP-1细胞表达促炎细胞因子。Hoechst染色结果表明Tpn32膜脂蛋白还可诱导THP-1细胞凋亡。此外，本课题组还发现Tpn32膜脂蛋白可抑制Tp在新西兰兔体内扩散，具有良好的抗感染免疫保护作用。本项目的开展阐明Tpn32膜脂蛋白在梅毒螺旋体所致炎症反应与免疫病理损伤中的作用机制，为梅毒防控及疫苗研发提供新的思路与依据。