Functional Study On Moving Junction Complex Member Rhoptry Neck Protein 4 Of Toxoplamsa Gondii

Toxoplasma gondii is an obligate intracellular parasite,which belongs to Apicomplexa,is responsible for important disease affecting humans and animals,Toxoplasmosis.The distribution of the disease is all over the world.Data published in 2013 in the United States from CDC showed that,the infection rate of toxoplasmosis in USA arrived 22.5%and approximately 33%global human beings infected with Toxoplasma gondii.The infection rate in China is approximately 5?10%.Although the infection of Toxoplasma gondii usually present mild clinical symptoms similar to flu,it is very dangerous for person who with immune deficiency.20%?80%of the patients with immunodeficiency virus?HIV?infection also infected with Toxoplasma gondii.Toxoplasma gondii is one of the important agent lead to death in AIDS.Proteins secreted from micronemes and rhoptries formation of a ring-shaped tight junction interface between parasite and host plasma memebranes called moving junction?MJ?,including the apical membrane antigen 1?AMA1?,rhoptry neck protein 2,4,5,8.While much attention has been directed toward function dissected of AMA1,RON2,RON5 and RON8,little is known about the function of another MJ members,TgRON4.Several attempt to generate RON4-deficient parasites in both Plasmodium falciparum and Toxoplasma gondii had been defeated in previous study,however,RON4 was considered as an essential factors during invasion in Apicomplexans for a long time and the accurate function of RON4 is unknown.In this study,according to genome sequence information of TgRON4?accession number:TGGT1₂29010?and TgRON5?accession number:583.m00636?from ToxoDB and NCBI,signal peptide,transmembrane domain and hydrophilic were analyzed,we designed primers for truncated gene of TgRON4 and TgRON5,and then performed PCR,the amplicons were cloned into pGEX-4T-3 by BamHI and xhoI.Recombinant proteins fused with GST tag were expressed in E.coli BL21DE3.Purified proteins were subjected to SDS-PAGE,a 30kDa and a 70kDa proteins were obtained,consistent with the theoretical molecular weight of TgRON4t and TgRON5t.Then 100 ?g recombinant proteins were injected into ICR,produced anti-sera.The anti-sera could recognized the native TgRON4 and TgRON5 in both western blot analysis and immunofluorescence assays.Indicated the polyclonal antibody preparation in this study has favorable antigenicity.In this study,we generated TgRON4 deficient parasites using the CRISPR-Cas9 system.The TgRON4 targeting sequence is designed and amplified by overlap PCR,then cloned into pSAG1::Cas9-U6::sgUPRT using pmeI.RH?? HxGPRT?were transfected with the CRISPR plasmids by electroporation and then selection with pyrimethamine and limited dilution method.Two stable clones?? TgRON4-2D10 and ? TgRON4-2G8?were isolated.Then we performed PCR to amplify the target RON4 locus,we found both sgRNA targeting sites were edited in RON4 locus:an approximately 5700bp fragment was inserted into the first targeting site and a 5 base pairs were deleted in the second targeting site.The depletion of TgRON4 in the knockout mutants was also confirmed by western blot and IFAs.All above results indicated we obtained the TgRON4 deficient strain successfully.Next,we explored the expression and location of another MJ member,TgRON5 by western blot and IFAs.Results showed that the localization of TgRON5 in TgRON4-deficient parasites was similar to Cas9-conrol parasites,both located to the neck of rhoptries.But the expression of TgRON5 in TgRON4-deficient parasites was obviously decreased in western blot analysis.Indicated loss of TgRON4 affected the stability of endogenous TgRON5.In order to investigate the function of TgRON4 during invasion,we analyzed the ability of attachment,invasion,replication and egress.The invasion rate were measured with flow cytometry,results showed the invasion rate of TgRON4-deficient parasites were significantly lower than of Cas9-control parasites.The results of invasion assays were confirmed by immunofluorenscence analysis,both TgRON4-deficient strains exhibited a significant decrease in the invasion rate,but didn't affect the ability of attachment.On the other hand,loss of TgRON4 didn't affect growth of parasites in host cells.After incubated with 3?M calcium ionophore A23187,both TgRON4-deficient strains and Cas9-control parasites could egress from host cells.Finally,full-length of TgRON4 was amplified from DNA extracted from Toxopalsma gondii tachyzoite.The sequence of target gRNA was synonymous mutanted and fused to a HA epitope tag,then cloned into pBluescript.Plasmid pBluescript-synoTgRON4-HA was transfect into ? TgRON4-2G8 by electroporation and then selected monoclonal strain.Then measure invasion ability of complement strain,Cas9-control strain and TgRON4-deficient parasites by Red/Green assays,result show that compensation of synoTgRON4 restored invasion ability of TgRON4-deficient parasites,similar to Cas9-control strain.In this study,function analysis of TgRON4,one of the MJ complex members,improved our knowledge about molecular mechanism of MJ formation,supplied reliable theory base of development of method to block invasion of Toxoplasma gondii and other members in Apicomplexa.