宿主细胞信号水平弓形虫感染机制的研究

Toxoplasma gondii is an intracellular protozoan parasite that has a broad host spectrum. Nearly one-third of the world's population is infected by this parasite. Toxoplasma can cause severe and life-threatening disease in immune-compromised patients and in developing fetuses when mothers are infected during pregnancy. Interaction between T. gondii and its host cell is the foundation of infection occuring. T. gondii infection has broad impact on host cell signaling, affecting mRNA levels of some cell signaling protein genes and causing extensive phosphorylation/dephosphorylation of phosphoproteins.In our former research, we found great changes in the transcription level of some PI3K-AKT pathway signal proteins, and in the 2-D electorphoresis profile of the host cell phosphoproteins.We will use Cell Signal Pathway PCR chips to obtain mRNA profiles of different proteins in various signaling pathways pre- and post- infection. To examine the phosphorylation status of cell signaling proteins, we will use Stable Isotope Labeling with Amino Acids in Cell Culture (SILAC) in combination with enrichment of phosphoproteins and mass spectrometry. This approach will provide information regarding phosphoprotein profile, Gene Ontology classes of the proteins (based on biological process, molecular function, and cellular component databases), phosphorylation level, phosphorylation sites, and protein metabolism pathway of the host cell pre- and post-infection. Systematic analysis of host cell signaling during T. gondii infection will advance our understanding of the interaction between T. gondii and its host cell, and shed light on the regulatory mechanisms controlling defense reactions of the host cell. This research will provide a solid basis for diagnosis, prognosis and therapy of intracellular pathogen infection.

弓形虫为细胞内寄生原虫，具有广泛的宿主群，全球约有1/3人口受感染。弓形虫和宿主细胞之间复杂的相互作用是弓形虫感染的基础，弓形虫感染使宿主细胞信号发生广泛的变化：信号蛋白基因表达水平变化及磷酸化蛋白广泛的磷酸化/去磷酸化。我们前期研究发现弓形虫感染宿主细胞后，磷酸化蛋白2D电泳图谱及细胞信号蛋白的基因表达水平发生了显著的变化。本研究拟采用信号转导PCR芯片技术检测并揭示弓形虫感染前、后，宿主细胞信号通路蛋白基因表达模式的变化；结合SILAC、磷酸化蛋白纯化与质谱技术，分析弓形虫感染前后宿主细胞磷酸化蛋白质谱及蛋白质Gene Ontology、磷酸化水平模式、磷酸化位点信息、及蛋白质代谢通路，从而在细胞信号水平说明弓形虫感染的机制，对探讨弓形虫与宿主细胞相互作用的分子机制，阐明宿主的抵抗策略，揭示其应答线索和调节机制具有重要的意义；为弓形虫感染的诊断、治疗和预后等临床实践提供重要的理论依据。

为了更好地了解弓形虫感染对宿主细胞信号转导的调控作用，我们对用SILAC标记的HFF细胞的磷酸化蛋白质组进行分析。感染弓形虫2h和6h时间段内，弓形虫调控细胞周期和细胞骨架的重组是被感染的宿主细胞的主要反应，为弓形虫有效入侵和细胞内增殖所必需。磷酸化蛋白谱聚类分析显示弓形虫感染对宿主细胞进程的影响并不显著，但在感染2h及6h时间，影响了特定的不同的细胞进程。一些被显著调节的磷酸化位点的磷酸化蛋白的Western blot验证，结果与质谱数据一致。发现以下与ROP18互作的宿主蛋白及其影响的细胞信号通路：.1. 发现弓形虫感染可引起宿主细胞Vimentin的重排及其可溶性、表达水平的变化。弓形虫的入侵及从宿主细胞释放受宿主细胞Vimentin调节,Vimentin与弓形虫棒状体蛋白ROP18存在相互作用,ROP18能够调节宿主Vimentin的可溶性，表达水平。.2. 宿主细胞Rac1 GTP酶被抑制之后弓形虫的入侵效率显著下降；抑制剂处理宿主细胞后弓形虫入侵率下降可能是由于影响宿主细胞骨架蛋白重排引起；弓形虫入侵的早期阶段宿主细胞Rac1被招募到PVM上，随着弓形虫入侵时间的延长Rac1磷酸化水平上升。.3. 通过FRET、及Co-IP实验证实了NMI是ROP18在人细胞内的作用靶点。确定人源HEK293T作为细胞模型，证实了ROP18与细胞内源性NMI蛋白的相互作用。确定ROP18可影响NMI的基因转录水平及蛋白表达水平。细胞核/浆蛋白含量分析提示ROP18影响NMI的核转移过程。Co-IP及磷酸化抗体检测结果表明ROP18可能使NMI发生磷酸化。.4. FRE及co-IP验证ROP18与IL20RB的相互作用，且是与该蛋白的胞外段相互作用，而与胞内段的互作效果较弱。间接免疫荧光实验检验COS-7细胞中过表达人蛋白IL20RB，弓形虫RH株入侵宿主细胞蛋白IL20RB的分布。荧光定量PCR和免疫印迹验证人HFF细胞内源性表达IL20RB..5.FRET与co-IP实验确认ROP18与P2X1的相互作用。选用COS-7为细胞模型，分别过表达ROP18以及P2X1，当P2X1表达72小时后，能够诱导细胞凋亡。本实验选择内源表达P2X1的SF268作为细胞模型，通过荧光定量PCR以及免疫印迹验证SF268细胞能够表达P2X1蛋白，验证内源ROP18与P2X1的相互作用。