Studies On Screening And Function Of Proteins Interacting With Theileria Annulata SVSPs

Tropical theileriosis is the disease caused by tick-transmitted apicomplexan parasite-Theileria annulata,which has ability to transform bovine B cells,macrophage cells and dendritic cells.T.annulata infected cells was confirmed having some cancer hallmarks,including uncontrolled proliferation,metastasis,genomic instability.Many studies were focused on host cell signaling pathways affected by the molecules of T.annulata,yet the key proteins of T.annulata directly responsible for host cell transformation remain unknown.Previous studies have proposed that SVSPs family,the largest multigene family in T.annulata,maybe associated with host cell transformation,invasion and immune evasion.However,the roles of SVSPs proteins during host-parasite interactions are unclear.Therefore,to explor the host cell molecules interacting with SVSPs will provide a new sight to illustrat the molecular mechanism of Theileria-induced cancer features.In the present study,some genes of SVSPs were used as the target genes and screened its interacting molecules of the host cells using Yeast two hybrid system,and to further confirm the interactions of the host cell proteins with SVSPs by using Co-IP and Bi FC assays.Moreover,the effects of SVSP family molecules on host cell signals were detected,which will pave the way tounderstand the pathogenesis of T.annulata.The main findings of this study are as following:1.The recombinant prokaryotic expression plasmids(SVSP452,SVSP453 and SVSP453)were constructed.The recombinant proteins with a concentration of 0.4 mg/L were obtained after expression and purification.Together with the previous expressed SVSP450 and SVSP454 proteins,these five proteins were used to immunize New Zealand rabbits respectively,for acquiring polyclonal antibodies.From the cellular localization results,these proteins were distributed mainly in cytoplasm and nucleus,but SVSP455 was located in host cell cytoplasm and surface of T.annulata schizont.2.The recombinant bait plasmids were successfully constructed,and the autoactivation and toxicity of the plasmids were tested.The results demonstrated that these 7 bait plasmids showed no characteristics of autoactivation and toxicity,which can be used for Y2 H system screening.3.The expression profiles of SVSP449 in different life cycle stages and passages of T.annulata-infected cells were detected using qPCR and western blotting.The SVSP449 was served as a bait plasmid and RBMX2-like and UBQLN4 of host cells were screened out as its interacting molecules-by using Y2 H system.The results of Co-IP and Bi FC assays confirmed that SVSP449 only bound to RBMX2-like.4.The transcription levels of SVSP454 in different life cycle stages and passages of T.annulata-infected cells were identified using qPCR.The potential interact proteins of host cells with SVSP454 were screened by Y2 H system,which were CCDC181 and MRPL30.Co-IP and Bi FC assays were carried out and demonstrated that SVSP454 could interact with both CCDC181 and MRPL30.5.The expression levels of SVSP455 in different life cycle stages and cell passages of T.annulata-infected cells were identified using qPCR.The possible interact proteins of host cells with SVSP455 were screened by Y2 H system,which were HSP60 and POMP.With the Co-IP,Bi FC and confocal microscopy assays results,SVSP455 was only interacted with HSP60.After RNAi for HSP60 and SVSP455 overexpression,SVSP455 could partially rescue the expression levels of molecules related with mitochondrial apoptotic pathway to further regulate host cell apoptosis.