Toxoplasma ROP18 Induces Neural Apoptosis During Toxoplasmic Encephalitis

Background:Toxoplasma gondii is an obligate intracellular parasite belonging to the protozoan phylum Apicomplexa with a complex lifecycle,which can spread among mammals including humans.The central nervous system?CNS?is the most common damaged organ during infection.Toxoplasma gondii tends to accumulate in the central nervous system and cause damage to the CNS.In patients with severe immune dysfunction,dormant cysts can be reactivated as fast-growing tachyzoites,causing damage to the brain.Our previous studies showed that the endoplasmic reticulum?ER?-associated protein RTN1-C is a substrate of Toxoplasma ROP18 kinase,and ROP18phosphorylated RTN1-C to increase apoptosis through ERS.In this experiment,it was demonstrated that Ser7/134 and Thr4/8/118 of RTN1-C are phosphorylation sites for ROP18.ROP18 phosphorylation of RTN1-C triggered ER stress-mediated apoptosis in neuralcells.Remarkably,ROP18phosphorylationofRTN1-Cenhanced glucose-regulated protein 78?GRP78?acetylation by attenuating the activity of histone deacetylase?HDAC?,and this event was associated with an increase of neural apoptosis.Objective:To explore the molecular mechanism of interaction between Toxoplasma gondii ROP18and host RTN1-C involved in neuronal apoptosis,which may provide new insights into the molecular mechanisms of neuropathogenesis during toxoplasmic encephalitis.Methods:Mass spectrometry,IP,pull-down,and cellular immunofluorescence were used to confirm the interaction between ROP18 and RTN1-C.phosphorylation assay,IP and bioinformatics analysis showed that Ser7/134 and Thr4/8/118 of RTN1-C are phosphorylation sites for ROP18.Flow cytometry and immunoblotting demonstrated that Toxoplasma ROP18 can promote neuronal apoptosis through ERS-assocaited apoptosis pathway.HDAC activity assays and IP demonstrated that phosphorylation of RTN1-C by ROP18 inhibited HDAC activity and enhanced the acetylation of GRP78.Results:1.Toxoplasma gondii ROP18 induces neuronal apoptosis in mice.?1?BALB/c mice were orally challenged with 20 tissue cysts of the PRU strain.After45 days,mice with latent infection were intraperitoneally given with cyclophosphamide to induce recurrence of toxoplasmosis.?2?Tissue H&E staining:necrosis and inflammation were present around Toxoplasma cysts?3?Immunofluorescence:a high degree of neural apoptosis was present in the cortex of immunosuppressed mice.?3?Immunofluorescence:Each individual in the group of BALB/c mice was infected i.p.with 1×10³ tachyzoites from ROP18-WT RH,ROP18-KD RH,?rop18 RH,or wild-type RH tachyzoites.Pathology in the brain tissues of animals was examined as soon as obvious clinical manifestations were observed.To detect the neuronal apoptosis in vivo,immunohistochemical staining of active caspase-3 was conducted on the brain sections.Active caspase-3 staining was more pronounced in the hippocampus and cerebral cortex of mice infected with the ROP18-WT RH or wildtype RH tachyzoites compared with the uninfected group.Remarkably,the active caspase-3 staining of the mice infected with the ROP 18-WT RH was significantly increased compared with the ROP18-KD RH or?rop18 RH-infected group.2.Identification of RTN1-C as a ROP18-Interacting Host Protein?1?Mass spectrometry analysis:To distinguish putative ROP18-binding proteins from nonspecific binding,ROP18-GST was used as an affinity matrix to isolate proteins interacting with active ROP18 from human SH-SY5Y neuroblastoma cell line extracts.Silver-stained bands that were enriched in the ROP18-GST pull-down were excised and subjected to protein identification using MALDI-TOF mass spectrometry.RTN1-C,an ER protein preferentially expressed in the CNS,was identified.?2?To determine whether ROP18 specifically interacts with RTN1-C,we constructed FLAG-tagged?20-RTN1-C?lacking amino acids 1–20 at the N terminus,which displays no sequence similarity to RTN1-A/B and other RTNs?and transfected it into the cells.The GST pull-down assay revealed that FLAG-tagged full-length RTN1-C bound to ROP18 in vitro,whereas FLAG-tagged?20-RTN1-C did not.?3?Co-IP:ROP18 bind to the full length of RTN1-C but not to the RTN1-C deficient in20 N-terminal amino acids.?4?Immunofluorescence:RTN1-C was recruited around the parasites and co-distributed with ROP18,suggesting the colocalization of ROP18 with RTN1-C in the cytosol of the infectious primary neuron cells.?5?IP:SH-SY5Y cells were infected with the?ku80?hxgprt RH or?rop18 RH strain,and these cells were then immunoprecipitated for RTN-1C.The results indicated that the host RTN1-C is a binding protein of ROP18 during infection with the virulent type I3.ROP18 Phosphorylates RTN1-C at Ser7/134 and Thr4/8/118.?1?IP:Immunoprecipitation was performed using FLAG antibody followed by phosphorylated Ser/Thr immunoblotting analysis.The wild-type ROP18 phosphorylated RTN1-C at Ser/Thr residues,while the kinase-deficient ROP18 did not.?2?Bioinformatics analysis:Given the possible phosphorylated sites identified by bioinformatics,we focused on the five putative phosphorylated sites with the highest scores,that is,Ser7/134 and Thr4/8/118.?3?Phosphorylation assay in vitro:We generated the nonphosphorylatable RTN1-C-5A mutation and repeated the in vitro phosphorylation experiment.The result showed that Ser7/134 and Thr4/8/118 of RTN1-C are the main substrates for ROP18 kinase,while phosphorylation of the RTN1-C-5A mutant proteins by ROP18 was significantly reduced,as verified by ³²P incorporation in vitro.4.ROP18 Phosphorylation of RTN1-C Induces ER Stress-Associated Apoptosis.?1?WB:To explore whether ROP18 kinase activity is essential for activating ER stress-mediated apoptosis,Neuro2a cells transfected with ROP18-WT-GFP or ROP18-KD-GFP were treated with or without 5?mol/L Z-ATAD-FMK,a specific caspase-12 inhibitor,and immunoblotting analyses were performed.The expression levels of cleaved caspase-12,CHOP,and cleaved caspase-3 increased significantly in the ROP18-WT-GFP group compared with the ROP18-KD-GFP and GFP groups.After Z-ATAD-FMK pretreatment,the expression levels of these related proteins were notably decreased in the ROP18-WT-GFP group compared with the group that was not pretreated?2?Flow cytometry:RTN1-C-WT,RTN1-C-5D and RTN1-C-5A were transfected into Neuro2a cells.Flow cytometry quantitative analysis indicated that RTN1-C-WT and RTN1-C-5D significantly enhanced neural apoptosis.Importantly,the apoptosis level of nonphosphorylatable RTN1-C-5A–overexpressing cells was significantly decreased compared with RTN1-C-5D–overexpressing cells.5.ROP18-Dependent RTN1-C Phosphorylation Down-Regulates HDAC Activity to Induce Apoptosis.?1?RTN1-C-WT,RTN1-C-5D and RTN1-C-5A were transfected into Neuro2a cells,respectively,and HDAC activity was detected.We found that RTN1-C-WT and RTN1-C-5D reduced HDAC activity,and nonphosphorylatable RTN1-C-5A abolished the inhibition of HDAC activity?2?IP:RTN1-C-WT,RTN1-C-5D and RTN1-C-5A were transfected into Neuro2a cells,then these cell lysates were immunoprecipitated for GRP78,followed by immunoblotting with the antibody that can recognize antiacetylated lysine residues.Our results confirmed that HDAC3,a member of class I HDACs,interacted with GRP78 in vivo.Moreover,phospho-mimicking RTN1-C-5D overexpression compared with wild-type RTN1-C and control was found to lead to significant GRP78 acetylation.In contrast,nonphosphorylatable RTN1-C-5A dramatically reduced GRP78 acetylation,demonstrating that ROP18 phosphorylation of RTN1-C induces GRP78 acetylationConclusions:In this study,immunofluorescence,western blotting,flow cytometry,IP,pull-down,mass spectrometry and other experiments demonstrated that Toxoplasma ROP18interacted with and phosphorylated the host RTN1-C.ROP18 phosphorylation of RTN1-C enhanced GRP78 acetylation by attenuating the activity of histone deacetylase?HDAC?,and this event was associated with an increase of neural apoptosis.