目的 探究刚地弓形虫不同毒力虫株感染后宿主细胞泛素化蛋白谱变化的特点。方法 将人外周血单核细胞诱导成巨噬细胞后分为3组,分别为未感染组、RH感染组、ME49感染组。2个感染组分别用弓形虫不同毒力株（RH株与ME49株）速殖子感染4 h后,收集3组细胞的总蛋白,应用泛素抗体富集泛素化蛋白,十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）分离蛋白后进行蛋白定性质谱鉴定。检测完成后,搜索UniProt数据库中的人类数据库,利用Mascot鉴定蛋白质种类。蛋白质免疫印迹（Western blotting）分析蛋白CDC42的泛素化水平。利用DAVID数据库对感染组与对照组、不同毒力株感染组之间的显著差异性泛素化蛋白（DUP）进行基因本体（GO）注释和富集分析,利用京都基因与基因组百科全书（KEGG）在线网站对DUP进行信号通路富集分析,利用STRING 11.0在线网站对DUP进行蛋白-蛋白相互作用网络分析。结果 质谱筛选结果显示, RH感染组的特异性泛素化蛋白有194种,ME49感染组的有47种,两个感染组均筛选到的宿主泛素化蛋白有31种。Western blotting检测结果显示,宿主CDC42在弓形虫感染后泛素化水平显著升高。GO聚类分析显示,细胞组分聚类中,RH感染组的DUP主要涉及能量代谢以及蛋白质合成、加工的细胞器,有67个蛋白;ME49感染组的DUP则主要涉及细胞骨架结构,有11个蛋白;在生物进程聚类中两组差异不大,不同的是,ME49感染组的DUP富集到肝配蛋白受体信号通路（3个）和网格蛋白介导的内吞作用蛋白（2个）;在分子功能聚类中,RH感染组的DUP主要富集在与RNA转录/加工、GTPase活性、泛素连接酶相关的聚类项,分别有47、7和11个蛋白;ME49感染组的DUP则主要富集在肌动蛋白丝结合（3个）等GO项上。KEGG信号通路富集显示,RH感染组的DUP主要富集在核糖体（12个）、泛素介导的蛋白水解（8个）、吞噬体（7个）、核苷酸切除修复（4个）、致病性大肠埃希菌感染（4个）等通路;ME49感染组的DUP则富集在核糖体（3个）、肌动蛋白细胞骨架调节（3个）和致病性大肠埃希菌感染（2个）的通路上。RH感染组DUP相互作用网络图主要的节点蛋白包括60S核糖体蛋白L9、蛋白质转运蛋白SEC61亚单位α亚型1以及RAS相关C3肉毒毒素底物1等。RH感染组与ME49感染组共有泛素化蛋白的相互作用网络图主要节点蛋白则有核糖体蛋白以及细胞分裂周期蛋白42等。结论 弓形虫感染引起宿主细胞的泛素化蛋白谱发生显著变化,与感染虫株的毒力相关;DUP包括细胞骨架、核糖体、泛素蛋白酶体途径的主要蛋白。

Objective To explore the changes of ubiquitinated protein profile in host cells infected with Toxoplasma gondii. Methods After induced into macrophages, the human acute monocytic leukemia cell line (THP-1) cells were assigned into three groups: uninfected group, RH infected group, and ME49 infected group. Cells in the infection groups were infected with strain RH and strain ME49 T. gondii tachyzoites with difference virulence, respectively, for 4 h. Then total proteins were extracted from the cells, and the ubiquitinated proteins were enriched using anti-ubiquitin antibody FK-2, followed by protein separation by sodium lauryl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) for qualitative mass spectrometry identification. Human source information was searched from UniProt databse, and the protein species were identified using Mascot. The ubiquitination level of CDC42 was verified by Western blotting. The proteins were digested into peptides and underwent liquid chromatography-tandem mass spectrometry (LC-MS/MS). Using the DAVID Bioinformatics Resources 6.8, Gene Ontology (GO) annotation and enrichment analysis was performed for significantly differentially ubiquitinated proteins (DUPs) between the infection and control groups and between the infection groups with different virulence. The pathway enrichment of DUPs was analyzed with KEGG online tool. The protein-protein interaction (PPI) network was analyzed with online STRING 11.1. Results LC-MS/MS results showed that compared with the control group, there were 194 DUPs in the RH infected group, 47 DUPs in the ME49 infected group, and 31 DUPs in both groups. Western blotting assay confirmed that the ubiquitination level of host CDC42 was increased significantly after T. gondii infection. GO clustering analysis showed that the DUPs in the RH infected group were mainly involved in energy metabolism and organelles of protein synthesis and processing (67 proteins), while the DUPs in the ME49 infected group were mainly involved in cytoskeleton structure (11 proteins). With regard to biological process clustering, no significant difference was found between the RH and ME49 infected groups, however, the DUPs of the ME49 infected group were enriched in Ephrin receptor signaling pathway (3 proteins) and clathrin-mediated endocytosis proteins (2 proteins). With regard to molecular function clustering, the DUPs of the RH infected group were significantly enriched in GO terms of RNA transcription/processing (47 proteins), GTPase activity (7 proteins) and ubiquitin ligase (11 proteins), while the DUPs of the ME49 infected group were significantly enriched in actin filament binding (3 proteins) and other GO terms. The enrichment of KEGG signaling pathway showed that DUPs in the RH infected group were mainly enriched in ribosome (12 proteins), ubiquitin-mediated proteolysis (8 proteins), phagosome (7 proteins), nucleotide excision repair (4 proteins) and pathogenic E. coli infection (4 proteins); while the DUPs in the ME49 infected group were mainly enriched in ribosome (3 proteins), actin cytoskeleton regulation (3 proteins) and pathogenic E. coli infection (2 proteins). The main node proteins of DUP interaction network in RH infection group included 60S ribosomal protein L9, protein transporter SEC61 subunit α subtype 1 and RAS related C3 botulinum toxin substrate 1. And in the interaction network of ubiquitinated proteins shared by RH infection group and ME49 infection group, the main node proteins were a cluster of ribosomal proteins and cell division control protein 42. Conclusion T. gondii infection results in significant changes in the ubiquitinated protein spectrum of host cells, which is related to virulence of the parasite strain. The DUPs involve in cytoskeleton, ribosome and ubiquitin proteasome pathway.