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[目的]微孢子虫是一类专性细胞内寄生的真核病原微生物,能够感染人类和几乎所有的动物.本课题以海伦脑炎微孢子虫(Encephalitozoon hellem)为研究对象,探讨其极管蛋白4(EhPTP4)作为一个潜在的分泌性毒力因子在宿主细胞内的定位和功能.[方法]制备EhPTP4的鼠源多克隆抗体,利用间接免疫荧光分析和Western blotting确定EhPTP4在感染细胞中的亚细胞定位;基于序列特征,在HEK293细胞中转染野生型和突变体EhPTP4,分析该蛋白的定位及其对病原增殖的作用;利用RNA-seq对转染EhPTP4的HEK293细胞进行转录组测序,分析EhPTP4引起的宿主基因表达和通路的变化;进一步通过RNAi和细胞转染分析差异表达基因的调控作用,利用RT-qPCR和Western blotting验证调控效果.[结果]EhPTP4的N端具有信号肽,C端具有富含组氨酸的结构域(HRD)和核定位信号序列(NLS).蛋白定位分析显示,在感染和转染细胞中,EhPTP4均被分泌至宿主细胞核内.在HEK293细胞中过表达EhPTP4显著促进了病原的增殖.RNA-seq和蛋白泛素化分析发现,EhPTP4显著上调了宿主内质网蛋白降解通路(endoplasmic reticulum-associated degradation,ERAD)关键基因(PDIA4、HERP、HSPA5和 Derlin3)的表达和宿主蛋白的泛素化修饰,表明EhPTP4具有调控宿主内质网蛋白降解的功能.在HEK293细胞中过表达PDIA4发现,蛋白泛素化修饰明显上调,而RNA干扰PDIA44则导致HERP基因表达下调,表明PDIA4在病原调控宿主ERAD过程中发挥着关键作用.进而,对EhPTP4的HRD进行了缺失突变(EhPTP4ΔHRD)并转染细胞,发现此缺失突变体并未引起PDIA4和HERP的表达发生显著上调,表明HRD对EhPTP4的毒力功能起到关键作用.[结论]首次报道了微孢子虫利用分泌蛋白调控宿主的ERAD和蛋白泛素化,为深入解析该病原的致病机理提供了重要参考.“,”[Objective]Microsporidia are a group of obligate intracellular parasites that can infect humans and nearly all animals.Here,we studied the polar tube protein 4 of Encephalitozoon hellem(EhPTP4)on its subcellular localization and functions as a potential secretory virulence factor in host cells.[Methods]A polyclonal antibody against EhPTP4 was produced to verify the protein subcellular localization in E.hellem-infected cells using indirect immunofluorescence assay(IFA)and Western blotting.HEK293 cells were transfected with wild-type or mutant EhPTP4 fused with HA-EGFP,and the impacts on pathogen proliferation,protein subcellular localization and sequence functions were assessed.RNA sequencing of EhPTP4-transfected cells was conducted to identify differentially expressed genes(DEGs)and pathway responses.The regulatory effects of candidate DEGs were analyzed via RNAi and cell transfection,and the effects were determined with RT-qPCR and Western blotting.[Results]EhPTP4 contains a signal peptide at the N-terminal,a nuclear localization sequence(NLS)and a histidine-rich domain(HRD)at the C-terminal.In the infected and transfected cells,EhPTP4 was secreted into the host nucleus.Transfection and overexpression of EhPTP4 in HEK293 cells significantly promoted pathogen proliferation.RNA-seq of the transfected cells showed that genes involved in endoplasmic reticulum(ER)-associated degradation(ERAD),a quality control mechanism that allows for the targeted degradation of proteins in the ER,were prominently upregulated.Upregulation of the ERAD genes PDIA4,HERP,HSPA5 and Derlin3 determined by RNA-seq data was verified using RT-qPCR and Western blotting.Protein ubiquitination of the transfected cells was then assayed and found to be markedly increased,confirming the activation of ERAD.PDIA4 knockdown with RNAi significantly suppressed the expression of HERP,indicating that PDIA4 is vital for the modulation by EhPTP4.Moreover,EhPTP4ΔHRD,a deletion mutant lacking the HRD,could not cause the upregulation of ERAD genes,indicating that the HRD is essential for the function of EhPTP4.[Conclusion]This study is the first report on a microsporidian secretory protein that targets the host nucleus to upregulate the ERAD pathway and subsequently promote protein ubiquitination.Our work provides new insights into microsporidia-host interactions.