TgROPs介导Raf/MEK/ERK信号通路诱导细胞自噬促进弓形虫增殖的分子机制

Toxoplasma gondii (T. gondii) is able to infect almost all nucleated cells and there is no effective therapy for toxoplasmosis right now. After being invaded, cell-autophagy would be induced to degrade pathogens and host immune responses could also be activated at the same time. However, T. gondii could escape cell-autophagy and even use the energy generated for self-proliferation. In previous study, we confirmed that T. gondii invasion could induce cell-autophagy through Raf/MEK/ERK pathway, in addition, after being treated with rapamycin and chloroquineor, the intracellular proliferation of T. gondii increased and decreased respectively. All these results indicated that the proliferation of T. gondii was closely related with the process of cell-autophagy. In this research, co-immunoprecipitation, western blot, protein spectrum analysis and knockdown-strain screening will be applied to verify the specific interaction between T. gondii ROPs and Raf/MEK/ERK pathway. Moreover, we will explore the influences of autolysosome on T. gondii proliferation and illustrate the role of Rabs on energy transportation of T. gondii through western blot, quantitative real-time PCR, immunofluorescence, electron microscope and so on. All together, we will try to paint a blueprint of the whole molecular mechanism of T. gondii proliferation regulated by Raf/MEK/ERK pathway. The results of our study will provide new thoughts for anti-T. gondii drug targets discovery and therapy development.

弓形虫可感染几乎所有有核细胞，其引起的弓形虫病尚无理想的治疗药物。宿主细胞被侵袭后能通过自噬降解病原并激活免疫反应。但弓形虫能够逃避细胞自噬甚至利用自噬产生的能量用于自身增殖，目前关于弓形虫与细胞自噬互作的分子机制仍不清楚。本团队前期研究发现弓形虫可激活细胞Raf/MEK/ERK信号通路进而引起自噬，雷帕霉素和氯喹分别对细胞进行处理后，弓形虫胞内增殖也相应地增加和减少，提示其胞内增殖与细胞自噬有密切联系。基于此，本研究拟通过免疫共沉淀、免疫印迹、蛋白质谱、基因条件性沉默等方法进一步研究弓形虫ROPs与Raf/MEK/ERK通路的互作方式，阐明ROPs诱导细胞自噬的信号通路，解释自噬溶酶体对弓形虫增殖的影响，验证Rabs蛋白的表达及定位对弓形虫转运能量的作用。研究结果不仅对探明Raf/MEK/ERK通路调控弓形虫胞内增殖的分子机制具有重要意义，也将为发现新抗虫药靶、研发创新药物等提供新思路。

弓形虫能感染包括人类在内的几乎所有温血动物，其引起的弓形虫病尚无理想的治疗药物。弓形虫的寄生方式为胞内寄生，研究发现某些活化的细胞能诱导产生IFN-γ控制弓形虫的增殖，甚至通过自噬清除弓形虫感染，另一方面弓形虫在长期进化过程中也获得了逃避宿主免疫的能力，甚至能够利用自噬产生的能量促进自身增殖。探明参与该机制的关键因子，对掌握弓形虫与宿主细胞的互作机制进而实现该病的防控有重大意义。本课题研究发现，弓形虫侵染后宿主细胞自噬标志物LC3-II蛋白水平显著上升，自噬流增强，电镜下自噬相关膜结构增多，明确弓形虫侵染引起宿主细胞发生自噬；利用基因编辑技术敲除宿主细胞自噬相关蛋白ATG5或ATG7，发现弓形虫在胞内的增殖变慢，证实弓形虫的增殖与宿主细胞自噬密切相关；进一步分析LC3蛋白水平与溶酶体信号，发现弓形虫感染引起宿主细胞LC3蛋白的表达增强，但溶酶体信号减弱，揭示弓形虫引起宿主细胞自噬并利用自噬产物；为明确弓形虫调控宿主细胞自噬的关键因子，通过在多种细胞系中过表达弓形虫毒力因子ROP5和ROP18，检测宿主细胞自噬水平，同时构建弓形虫ROP5和ROP18缺失株并进行表型分析，发现弓形虫引起未活化细胞的自噬与这两种毒力蛋白相关性不明显；本研究利用CRISPR/CAS9系统成功构建了多种弓形虫毒力因子缺失株，发现致密颗粒蛋白GRA2和GRA12缺失后弓形虫增殖变快，宿主细胞自噬水平显著增强，表明GRA2和GRA12参与调控宿主细胞自噬；同时为研究宿主细胞自噬的信号通路，建立慢病毒介导的敲除/敲降体系，探明Rab11蛋白参与弓形虫引起宿主细胞自噬。本项目立足于本领域国际前沿开展研究，研究结果不仅对阐释弓形虫感染与细胞自噬互作的分子机制提供了理论依据，也为弓形虫新药物靶标筛选和疫苗研发等提供了新思路。