Research On The Phosphoproteome Of Host Cell Infected With T.Gondii And The Mechanism Of Host Vimentin For T.Gondii Infection

Background:Protein phosphorylation,one of the important post-translational regulatiory protein modifications,plays key roles in protein turnover,cell signaling,transcription and intracellular transport.Previous reports have shown that protein phosphorylation occurs extensively and plays vital roles in the process of T.gondii-host cell interaction.As an intracellular protozoan,the process of T.gondii infection is complex and progressive.However,phosphoproteome analysis with respect to the host cell at different infection stages of T.gondii have little been identified and characterized.Vimentin,a type III intermediate filament(IF)cytoskeletal protein,has been reported to play important roles in the interaction between the host cell and the intracellular pathogens,such as regulating the invasion and multiplication of both bacteria and virus.However,the role of host cell vimentin in T.gondii infection has not been reported till now.Herein,we analyzed the phosphoproteome of the host cells infected by T.gondii for different time and explored the role of host vimentin during T.gondii infection.Methods:HFF cells were infected T.gondii RH tachyzoites harvested by mechanical lysis with a MOI of 3 for 30 min(EI),28 h(LI)or left uninfected(UI),which were then collected separately.Total protein from different groups was extracted and further digested for further phosphoproteomic analysis with iTRAQ.Several identified and quantified phosphoproteins were then chosen for identification with western blot.Immunofluorescence and western blot were performed to analyze the vimentin’s localization and dynamic patterns including solubility,phosphorylation,expression level in T.gondii infected host cells at different time points.Human brain microvessel endothelial cell(HBMEC)and HBMEC AVim were then infected with RH-GFP to evaluate the effect of host vimentin on the invasion,proliferation and egress of T.gondii.Moreover,the interaction between host vimentin and TgROP18 were demonstrated with FRET and Co-IP,respectively.Host cells were then infected with recombinant T.gondii RH-Arop18 and RH wild type to further compare the vimentin’s solubility,phosphorylation and expression level under the condition of having ropl8 and having not ropl8.Additionally,over-expression of TgROP18 in host cells and in vitro kinase assay was adopted to evaluate the effect of TgROP18 on the solubility,phosphorylation and expression level of host cell vimentin.Results:In this research,a total of 3692 peptides matching to 1541 proteins and 1207 phosphopeptides,with 1031 phosphorylation sites,matching to 665 phosphoproteins were identified.Among these phosphorylation sites,the most abundant phosphorylated residue was phosphor-Serine(pS,880),followed by phospho-Threonine(pT,138)and then phospho-Tyrosine(pY,13).Proteins with hosphorylation level fold change>and p values<0.05 were considered as significant.A total of 105 significantly regulated phosphoproteins were identified in our study,and the number of which in comparison goups EI/UI,LI/UI,LI/EI were 47,67,40,respectively.Further function analysis suggested that at the infection phase of T.gondii perior to egress,phosphorylation involved in the regulation of host cell,apoptosis,immunity and metobism to favor its proliferation,while in the infection phase of T.gondii just invading,T.gondii can utilize host energy and membrane and reorganize host cytoskeleton to contribute parasite invading.Bioinformatics analysis on these significant regulated phosphorylated proteins in the comparison groups,EI/UI and LI/UI,revealed that host cell infected with T.gondii for 30 min emphasized on cytoskeleton changes,while host cell with T.gondii infection for 28 h was dominant on regulation of cell cycle.T.gondii infection leading to the reorganization of host cell vimentin and its change of solubility,phosphorylation and protein level.The invasion efficiency of T.gondii into HBMEC △Vim was significantly higher comparing to that into HBMEC(p<0.05),while there was no significant difference of the proliferation and egress efficiency between these two comparison groups(p>0.05).Additionally,host cell vimentin was demonstrated to interact with TgROP 18,and its solubility and phosphorylation were regulated by TgROP18 partically.Conclusion:Different infection phases of host cell were emphasized with different and specific cellular processes.T.gondii infection induced the host cell vimentin reorganization and its change of solubility,phosphorylation and protein,and among which,the vimentin’s solubility and phosphorylation were partially regulated by TgROP18.Moreover,host cell vimentin was demonstrated to inhibit the invasion of T.gondii,but have on significant effect on its proliferation and egress.