Apoptotic Mechanism Of JEG-3 Cells Induced By Dense Granule Protein 15 Of Chinese Isolates Of Toxoplasma Gondii

Background:Toxoplasma gondii(T.gondii,Tg)is an obligate intracellular parasitic and opportunistic protozoan which infects most warm-blooded mammals worldwide,threatning human health and cattle husbandry.There are different clinical manifestations of toxoplasmosis,determined by the strain virulence and host species.Curently,congenital vertical toxoplasmosis is one of the major causes of adverse pregnancy.Tg can infect the fetus via transplacental transmission.Although birth defects caused by T.gondii could be attributed to structural damage,endocrine disorders and increased cell apoptosis of placental tissue,but the exactmechanisms and key events underlying congenital toxoplasmosis remain unclear.T.gondii has a fascinating dual involvement in host cell apoptosis.On the one hand,T.gondii-infected cells became relatively resistant to some apoptotic stimuli.On the other hand,it has been observed that T.gondii infection can induce apoptosis,as with splenocytes,eye cells,neural stem cells,trophoblastic cells,et cl.High levels of apoptosis and an increased mortality rate have been proven to be associated with T.gondii infection with high-virulence strains.Therefore,the initiation and the degree of cell apoptosis may play crucial roles in the pathogenesis and outcomes of toxoplasmosis.The virulence of Tg is closely correlated to polymorphic proteins originated from different genotypes.Strains of Tg collected from North America and Europe were mainly categorized into three genotypes,namely Type I,II and III.Chinese 1(Toxo DB#9)is the dominant genotype in China.Polymorphic proteins that released directly into the host cell include: rhoptry proteins(ROPs),such as ROP16,ROP18;dense-granule proteins(GRAs),such as GRA15,GRA3.It is reported that parasitic expressing ROP16I/III activates the M? to the alternatively activated phenotype(AAM,M2)by phosphorylating STAT3 and STAT6 in M?,resulting in mass production of interleukin-10(IL-10),arginase and down-expressing interleukin-12(IL-12);GRA15II activates M? to the classically activated macrophage phenotype(CAM,M1)by phosphorylating of NF-?B p65,resulting in massive release of pro-inflammatory factors,such as IL-12?IL-23 a,IL-6,as well as activation of T helper 17(TH17)and natural killer(NK)cells.The endoplasmic reticulum(ER)is the primary intracellular organelle for proper protein synthesis,folding and assembly.The accumulation of unfolded or misfolded proteins in the lumen of the ER,which induces an adaptive program called the unfolded protein response(UPR),leads to ER stress(ERS).Increasing evidence shows that ERS plays a key role in the regulation of apoptosis caused by a variety of toxic insults,infecton and so on.Alterations of apoptosis in trophoblastic cells can result in pregnancy loss or embryonic damages.It has been observed that the incidence of apoptosis was higher in ME49-infected cells than RH-infected cells.The reason for the two strains of Tg causing the difference of the host cell apoptosis may be associated with their virulence factor polymorphism.ME49 strain,as a representative of the type II Tg strain,carrys GRA15 II genes and GRA15 possesses kinase activity.However,GRA15 involved molecular mechanism of apoptosis induced by toxoplasma needs to be clarified.Objective:To define the role of dense granule protein 15(GRA15)of Wh3,a strain of Chinese 1 type which predominantly prevalent in China in cell apoptosis,to clarify the exact mechanisms and key events underlying congenital toxoplasmosis and T.gondii-triggered apoptosis.Methods:The recombinant eukaryotic expression vector p EGFP-GRA15 II was transfected into JEG-3 cells.Cell viability was measured by MTS assay.Apoptosis of transfection cells were detected by DAPI staining.The transcription of apoptosis-associated gene was detected by q PCR method.The flow cytometry and western blotting was used to detect the apoptosis level and the expression of apoptosis related proteins respectively.To detect the apoptosis level and the expression of related proteins involved in the endoplasmic reticulum stress pathway after inhibitors treatment,p EGFP-GRA15 II transfected cells were treated by 4?8C and SP600125,then apoptosis level was detected by flow cytometry and related protein expression involved in ERS by western blotting.Results:The recombinant eukaryotic expression vector p EGFP-GRA15 II was constructed successfully.The results of MTS assay show that cell viability of transfection cells were decreased obviously.The results of DAPI staining revealed obvious apoptosis of transfection cells.Apoptosis level of transfection cells were increased obviously noted by flow cytometry.The transcription of apoptosis-associated gene and the expression of apoptosis related proteins were upregulated respectively.After p EGFP-GRA15 II transfected cells were treated by the inhibitors such as 4?8C and SP600125,cell viability was increased and apoptosis level was decreased.In treated cells,the expression of apoptosis related proteins were down-expressed compared with the untreated cells.Conclusions:The recombinant eukaryotic expression vector p EGFP-GRA15 II was constructed successfully.The gene sequence of GRA15 II is completely homologous to that of GRA15 II.GRA15 II induced JEG-3 cells apoptosis by IRE1 pathway.