宿主细胞信号水平弓形虫感染机制的研究

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。

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.