The Effect Of IMP1 Deletion And Autophagy On The Proliferation Of Toxoplasma Gondii

Toxoplasma gondii(T.gondii)is spread worldwide as an intracellular protozoan parasite,is able to infect almost all worm blood animals and causes toxoplasmosis which is lethal to immunocompromised individuals.T.gondii has the property to infect all of the nucleated cells with a complex life cycle and several infectious form make toxoplasmosis a big challenge for public health,thus develop safe and effective vaccines are the key the prevention and control of the disease.Immune mapped protein1(IMP1)is a novel discovered protein located on the surface of T.gondii,it is considered to have strong immunogenicity and can be used as candidate vaccine.In this study,gene engineering,cellular molecular biology and immunobiology were applied to research on the function of imp1 on intracellular proliferation of T.gondii and the interaction with autophagy,to provide a new strategy for Toxoplasmosis control and prevention.1.Construction of imp1 knockout and complement plasmidsIn order to study the function of imp1 of T.gondii,we plan to construct knockout and complement plasmids of imp1 and screen of gene knockout and complement strains.DNA and cDNA of T.gondii were used as templates to amply impl 5' UTR,imp1 3' UTR,imp1 CDS and promoter of sag1,and the promoter of sag1 were linked with imp1 CDS through fusion PCR.The fragments of imp1 5' UTR,imp1 3' UTR and ble were inserted into pBSK(+)plasmid through enzyme digestion,the knockout plasmid pBSK-imp1 5' UTR-ble-imp1 3'UTR was constructed and identified by PCR.Then the fragments of imp1 5' UTR?imp1 3'UTR and psagl-imp1 CDS were inserted into pTCY plasmid through enzyme digestion,and the complement plasmid pTCY-imp1 5' UTR-CAT-psagl-imp1 CDS-imp1 3' UTR was constructed and identified by PCR.2.Screen of imp1 gene knockout and complement T.gondii strains and the research of biological characteristicsPlasmids were linearized by enzyme digestion and transferred into T.gondii Aku80 RH strain with the technique of electroporation and recombined into the genome through homologous recombination,gene knockout and complement strains were screened by the addition of phleomycin or chloromycetin,methods of PCR,western blot and indirect immunofluorescent assay(IFA)were applied to identify these strains.IFA and qRT-PCR was used to study the adhesion(15 min,30 min,45 min,1 h,2 h,4 h),invasion(45 min,1 h,2 h,4 h),proliferation(24 h,48 h,72 h)and toxicity of the knockout mutant,the results showed that the adhesion,invasion and proliferation were significantly reduced,the toxicity to mice was notably decreased due to the fact that mice this group prolonged the survival time to 26.8±2.6 days while the mice infected with wild type or complement mutant were all dead within 9 days,the parasite burdens of lung,liver and spleen tissue were significantly less than control group.3.Autophagy in Vero cells induced by T.gondiiIn order to study the interaction between intracellular proliferation of T.gondii and autophagy,?ku80 RH strain were incubated with Vero cells,and the methods of transmission electron microscope,IF A,western blot and qRT-PCR were used to study.The results showed that when infected with T.gondii,autophagosome and a few autolysosome in Vero cells were observed,and the protein LC3 were gathered and surround with T.gondii,co-location of LC3 and lysosome were also observed through IFA.When applied with western blot,we found LC3-II in T.gondii-infected cells increased significantly indicating that autophagy was induced.3-MA and rapamycin were used as autophagy inhibitor and promoter,proliferation of T.gondii were found significantly decreased or increased in 3-MA or rapamycin treated cells compared to control group.4.Autophagy signal pathways in Vero cells induced by T.gondiiIn order to study which signal pathway play major role on autophagy induced by T.gondii,several proteins from different classic signal pathways were detected by western blot.The results showed that Raf1 and ERK1/2 from Raf1-MEK-ERK pathway were increased notably in T.gondii-infected cells.In addition,mRNA levels of Rafl,MEK and ERK were also increased significantly detected by qRT-PCR compared with control group.After incubated with inhibitors(ERK1/2:FR180204,Raf1:sorafenib,MEK:U0126-EtoH),we found the expression of Rafl and ERK1/2 in Vero cells were all decreased compared to control group,and when the inhibitors were removed and infected with T.gondii,the expression of Rafl and ERK 1/2 were recovered.Furthermore,the proliferation of T.gondii analyzed in inhibitor-treated cells through qRT-PCR were all significantly decreased compared to control group,and the mRNA levels of imp1,rop18 were decreased while that of gra7 was increased.These results indicated that autophagy induced by T.gondii were Rafl-MEK-ERK pathway-depended.5.Interaction between TgIMP1 and autophagyIMP 1 is considered to have strong immunogenicity and the vaccine based on IMP 1 could provide effective protection against T.gondii.In order to study the interaction of IMP 1 with autophagy,we constructed the pcDNA-pSAG1-IMP1 plasmid to express IMP 1 protein in Vero cells.After transferred into cells,the expression of IMP1 was comfirmed by western blot.After detection of several proteins from different classic signal pathways by western blot,we found the Rafl-MEK-ERK and AMPK-TSC2-mTOR pathways may have involved in IMP1 induced autophagy.Then co-immunoprecipitation assay was used to study the interaction between IMP1 and Rafl-MEK-ERK pathway and we found the interaction was indirect.In conclusion,was obtained through homologous recombination.The ability of Aku80 Aimpl RH strain on adhesion,invasion and proliferation were all reduced when infected with Vero cells.In addition,the toxicity of Aku80 Aimpl RH strain to mice was significantly reduced.Autophagy was induced trhough Rafl-MEK-ERK pathway via the infection of T.gondii and was related with the prolification of T.gondii.