Identification And Function Study Of Parasitophorous Vacuole Membrane Key Proteins In Neospora Caninum

Neosporosis is widely distributed all over the world.It brings serious damage to the breeding’s development.The pathogeny of neosporosis is Neospora caninum,which belongs to Apicomplexa.The clinlcal sysptoms include pregnancy cow abortion,newborn fetal bovine death,canine nervous system dysfunction,etc.The invasion process of Apicomplexa is relatively conservative,and mainly divided into the following stages: recognition the surface of cell,adhesion to the cell,invasion into the cells,and formation parasitophorous vacuole(PV).After Neospora caninum invasion,the host cells discharge many cell membrane proteins.Also,some rhoptry proteins and proteins secreted by dense granules organelles combining membrane proteins form a non-fusion chamber-PV.PV shares the function of resistance of the host cells acidification and lysosomal enzymes zymohydrolysis,which prevent host cleans exogenous parasites.Moreover,the formation of PV is accompanied by much microtubules network system,which carry nutrients from PV membrane or PV cavity to the tachyzoites.On the contrast,the metabolites of tachyzoites are also eliminated through microtubules network system.The study through the iTRAQ quantitative protein group technical appraisal in new spores insects infected host enrichment of a large number of protein components.By immune electron microscope and immunofluorescence subcellular localization technology to screen new spores in insect insect vesicles secreted protein components,and through the infected cell component separation experiments secreted into form of protein components in insect bubble,determine the membrane protein components.Using precipitation,immune coprecipitation and GST-pulldown technology research the relation of interaction between the membrane protein components,preliminarily determine the key protein molecules.Application CRISPR gene knock out newspores editing system bug,bubble film consisting of key protein molecules,studies the key protein molecules to new sporozoan speed son of invasion,escape,and the influence of the biological functions such as intracellular value-added,determine the key protein molecules,for its pathogenicity to host and the study of the theoretical basis for new spores of insect disease prevention and control.The composition quantitative analysis of Neospora caninum’s PV: The study uses the method of iTRAQ to quantify proteins and identified 228 up-regulated proteins,which may involve in the camping parasitism of Neospora caninum.A total of 48 secreted antigens were detected 1.5 times expression among infected models comparing with the control group.This illustrated that these proteins were secreted into the PV or host cytoplasm and involved in the nutrition metabolism,stable internal environment,immune escaption and other life activities.These proteins may be the key molecules of the host cells.After infection 48 h,the dense granules organelles(GRA)proteins’ levels were obviously increased and much higher that other secreted antigens.This indicated that those GRAs play important function in the growth of Neospora caninum.In the study of Toxoplasma gondii,GRAs involves in the process of PV modification,and provides nutrients and stable internal environment for tachyzoites growth.The present study identified a total of 12 higly expressed GRAs in the PV,and most expressed 4 times than the cntrol group.The implies that these GRAs take part in the formation of PV.However,where these GRAs locate and what specific biology function do GRAs have is still need further researched.The location and morphology study of Neospora caninum’s GRAs: The study uses the method of immune electron microscope and immunofluorescence to study the parasites location after invasion.Results showed that a total of seven GRAs were secereted into the net structure.After analysing the infected cell components isolation,we found four transmembrane proteins and four annexins,and NcGRA2 both exists in two forms.And the seven secreted GRAs in the PV network structure has any relations with each other? Do they interact with each other or form into complex? These need further research to affirm.The interaction study of Neospora caninum’s GRAs: The study obtained a complex relatedto the network structure of PV,which enriched by immunoprecipitation.This complex is composed of NcGRA2,Nc GRA4,NcGRA6 and NcGRA9.For further research,we used immunoprecipitation to affirm the interact relationships between any two proteins.In this complex,NcGRA2 and NcGRA6 were identified transmembrane proteins and both located in the center of the complex.If Nc GRA2 and NcGRA6 are the core of this complex? What kind of biology function do the complex have during the invasion process?The function study of Nc GRA2 and NcGRA6: The study research the biology function of NcGRA2 and NcGRA6,our study firstly established the CRISPR gene editing system of Neospora caninum.We constructed the NcGRA2 and NcGRA6 gene deletion strains: △GRA2and △ GRA6.By subcellular co-localization,we found that the NcGRA2 deletion strain influenced the distribution of NcGRA4 and NcGRA6,however,the NcGRA6 deletion strain had no influence on the distribution of NcGRA2 and NcGRA9.This indicated that NcGRA2 was the key protein of the complex,and the NcGRA2 deletion would destroy the structure of the complex.We also measured the △GRA2 and △GRA6’s invasion ability,escaption ability and growth ability in the cells.Comparing with the wild Nc-1 strain,△GRA2 and △GRA6 had no influence on the invasion,escaption ability,however,NcGRA2 and NcGRA6 gene deletion would effect the growth ability.NcGRA2 and Nc GRA6 may affect the network structure of PV and reduce the parasites’ growth by destroying or influencing on the complex.In vivo experimental results in mice showed that the absence of NcGRA2 and NcGRA6 would affect the virulence of Neospora caninum,indicating that NcGRA2 and NcGRA6 were one of the key virulence factors of Neospora caninum.The present study identified 48 secreted antigens,which shared over 1.5 times in infected models than control groups using the protein quantitative method of iTRAQ.Furthermore,a total of 7 GRAs were confirmed to be distributed in the PV network structure by subcellular localization and morphological analysis.Four proteins,including NcGRA2,NcGRA4,NcGRA6 and NcGRA9,were proved to form a complex existed in the PV network structure after immunoprecipitation(IP)and co-IP analysis.With CRISPR gene editing system,the present studyconstructed the NcGRA2 deletion strain and the NcGRA6 deletion strain,respectively.Results indicated that the NcGRA2 gene is the key compotent in forming PV and the NcGRA2 deletion strain would affect other compotents of the complex distributed in the PV network structure.And the complex was the core compotents of forming Neospora caninum PV.The growth speed would reduce if destroying the structure of the complex.