Research On The Role Of Toxoplasma Gondii Type Ⅰ ROP18 In Resisting Host Ubiquitin-mediated Immunity

Background:The host ubiquitin system plays an important role in resisting the invasion of pathogens.Therefore,many pathogens manipulate the host ubiquitin system via their effectors to evade immune clearance.It has been found that pathogens such as Salmonella typhimurium infection can cause dynamic changes in the global ubiquitinome of host epithelial cells.However,it’s still unclear whether infection can manipulate the ubiquitin system of host cells,especially immune cells,to achieve successful infection and parasitism,and change the ubiquitinome of host cells.T.gondii main virulence factor ROP18 plays an important role in resisting host immune clearance,but its role in manipulating the host ubiquitin system remains unclear.Herein,we analyzed the differential ubiquitinomes of host cells infected with different T.gondii strains and explored the role of TgROP18 in manipulating host ubiquitin system.Methods:THP-1 cells were infected with T.gondii RH and ME49 tachyzoites or without infection and labeled as "RH","ME49"and "N" respectively.The ubiquitinated proteins from each group were enriched by using anti-ubiquitin antibody,and identified by mass spectrometry.Gene Ontology(GO)analysis of differentially ubiquitinated proteins(DUP)was performed by DAVID Bioinformatics Resources 6.8.The pathway enrichment of DUPs was analyzed with Kyoto Encyclopedia of Genes and Genomes(KEGG)analysis.The protein-protein interaction(PPI)network was described with STRING 11.1.The ubiquitinated level of CDC42 was verified by immunoprecipitation assay.Human cells(HFF,HUVEC and THP-1 cells)were induced with IFN-γ and infected with CEP(type Ⅲ T.gondii strain),CEP+ROP18Ⅰ(CEP strain expressing type I ROP18)and CEP+ROP18Ⅱ(CEP strain expressing type Ⅱ ROP18).Immunofluorescence assay was performed to observe the binding of Ub to the PV membrane.The Lyso-tracker was used to label the acidified PV and the acidification of PV was observed under a fluorescence microscope.HFF cells were treated with E1 inhibitor PYR-41 and infected with the above three strains.The proportion of PVM labeled by Ub and acidified PV were calculated.Result:Compared with the control group,the differentially ubiquitinated protein(DUP)analysis showed that there were 194 DUPs in "RH" group,47 DUPs in "ME49"group,and 31 DUPs in both groups.Functional analysis of DUP in different infection groups showed that DUP in RH infection group was mainly involved in energy metabolism,protein synthesis and processing,GTPase activity,ubiquitin ligase and other pathways,while DUP in ME49 infection group was mainly involved in actin cytoskeleton regulation and other pathways.Immunoprecipitation assay confirmed that the ubiquitination level of host CDC42 was increased significantly after T.gondii infection.Compared with the wild-type CEP and CEP+ROP18Ⅱinfection groups,the proportion of Ub labeled PV in CEP+ROP18Ⅱ infection group was significantly lower(P<0.05),as well as the proportion of acidified PVs in CEP+ROP18Ⅰ infection group was significantly decreased(P<0.05).Furthermore,the proportion of acidified PVs was significantly decreased in the HFF cells infected with these three T.gondii strains treated with PYR-41 compared with their untreated counterparts.These results showed that TgROP18Ⅰ could prevent the acidification of T.gondii PV through inhibiting the binding of Ub to the PVM.Conclusion:T.gondii infection resulted in significant changes in the ubiquitinated protein spectrum of host cells,which is related to T.gondii virulence.The DUPs were involved in the pathways related to cytoskeleton,ribosome and ubiquitin proteasome,which indicated that T.gondii could utilize the host ubiquitin system for successful invasion,immune evasion and dissemination.