Function Analysis Of Encephalitozoon Hellem Secreted Protein EH7108 In Host Cell

Microsporidia is an obligate intracellular opportunistic parasite,which can infect almost all animals from invertebrates to vertebrates,including humans.It is listed as a Class B priority pathogen by the United States CDC.Encephalitozoon hellem（E.hellem）is one of the main microsporidians that infect humans,and it is a potential threat to human health.However,the mechanism of its pathogenicity remains unclear.Secreted proteins are important virulence factors of pathogens and play an important role in the process of infection.In the early stage,we analyzed secreted proteins of E.hellem.Among those,we screened a protein EH7108 which was secreted and located in the host cell nucleus.Domain prediction analysis revealed that EH7108 has a signal peptide（SP）and nuclear location sequence（NLS）,but no any known functional domain.Phylogenetic analysis showed that the protein is widely present in microsporidia and may play an important role.Therefore,in this study,we investigated localization characteristics of EH7108,its roles in the process of pathogen infection and proliferation,its transcriptional regulation of host cell,and its interacting proteins.The main results are as following:1.Subcellular localization characteristics of EH7108 in host cellRT-qPCR study showed that EH7108 had high transcriptional level from 36 hpi to72 hpi,suggesting that EH7108 may play an important role during pathogen growth.To confirm NLS has an impact on localization of EH7108 at host cell,we constructed EH7108,EH7108^ΔSPand EH7108^ΔNLS fusing HA-tag and EGFP-tag plasmids and they were overexpressed in HEK293.Further,indirect immunofluorescence assay（IFA）showed that EH7108 located in host cell nucleus,while lacking of NLS prevented EH7108 from entering host cell nucleus.Meanwhile,the nuclear protein of EH7108-overexpressed cells was extracted and detected by western blot,the result also showed EH7108 was located in nucleus.In addition,the IFA analysis of the infected cells using the prepared polyclonal antibody of EH7108 showed that EH7108 was located in meronts of E.hellem and in the nucleus of HFF cells.These experimental results indicate that EH7108 is a secreted protein that can be targeted to the host nucleus and play a regulatory role.2.The role of EH7108 in pathogen proliferationThe transcription and localization characteristics of EH7108 in infected cells suggest that this protein may have an important role in the proliferation of microsporidia.Therefore,we conducted infection experiments on host cells transfected with EH7108,and analyzed them using absolute quantitative PCR（qPCR）and fluorescence in situ hybridization（FISH）.qPCR showed that number of spores significantly increased in 48 hpi,compared to control.Meanwhile,FISH indicated that parasitophorous vacuole（PV）was observed earlier in host cells obtaining EH7108than control group.Collectively,these data demonstrated that EH7108 is benefited for microsporidia proliferation and parasitophorous vacuole formation.3.Regulation of host gene transcription by EH7108The cells expressing HA-EGFP and EH7108-HA-EGFP,respectively,were used to RNA sequencing analysis.The result showed that 34 different expression genes（DEG）have a significant change,including 22 upregulated genes and 12downregulated genes.Further,different expression genes were enriched for KEGG pathway,showing that endoplasmic reticulum associated protein degradation（ERAD）pathway and thyroid hormone synthesis pathway had been changed.ERAD pathway enrichment ratio is higher,and the 2 differential genes in thyroid hormone signaling pathways were enriched in ERAD.Additionally,Gene Ontology clustering analysis revealed that there were six biological processes that changed,four of which were related to ER function,including response to endoplasmic reticulum stress and ER-associated ubiquitin-dependent protein catabolic process.RNA-seq analysis showed that EH7108 has the function of regulating the ERAD pathway in host cells,that is,E.hellem may use EH7108 to regulate the degradation of misfolded proteins in host endoplasmic reticulum.Furthermore,RT-qPCR was performed to analyze specific genes,revealing that ER-associated degradation（ERAD）genes,including PDIA4,HSPA5 and HERPUD1,which is consistent with RNA-seq results,further indicating that EH7108 has a role in regulating the degradation of misfolded proteins in host ER.Likewise,western blot also found that the PDIA4 protein in transgenic cells was significantly up-regulated,and the ubiquitination level of the proteins was also up-regulated.Using RNAi to knock down the expression of PDIA4,it was found that the expression of HERPUDI was significantly down-regulated,indicating that PDIA4 is a key regulatory gene in the ERAD pathway.The above results indicated that EH7108 upregulated the expression of PDIA4 and activated the ERAD pathway by entering the host nucleus,thus regulating the degradation of some proteins and leading to changes in the function of related pathways.Taken together,EH7108 is a secreted protein of E.hellem,which can target the nucleus of the host cell.It may have a role in regulating the degradation of glycoproteins in the endoplasmic reticulum of the host cell.It is an important virulence factor for pathogens.This study has important value for revealing the pathogenic mechanism of microsporidian infection.