| Toxoplasmosis is a food-borne zoonosis caused by Toxoplasma gondii(T.gondii).T.gondii is an obligate intracellular protozoa that can infect a wide range of hosts and has a complex life cycle.Although T.gondii typically causes asymptomatic toxoplasmosis in humans,as an opportunistic pathogen,infections may manifest as severe symptoms in immunosuppressed patients,such as those infected with HIV.Furthermore,primary infections during pregnancy can lead to miscarriage or congenital toxoplasmosis.T.gondii infections affect almost one-third of the world's population.T.gondii can also infect animals and cause death,and the mortality rate of infected pigs can be up to 60%.Therefore,toxoplasmosis seriously endangers the health of people and animals and leads to huge economic losses in the livestock industry.Toxoplasma gondii invasion-related proteins play key roles in the process of T.gondii invasion,which mainly involve in contacting the host cell,gliding motility,forming moving junction,forming and modifying a parasitophorous vacuole membrane(PVM),and regulating host transcription.Adhesion is the first step of T.gondii invasion,and T.gondii surface antigens and secreted proteins interact with host cell surface receptors to promote the parasite invasion in host cells.Heparan sulfate(HS)is widely distributed on the eukaryotic cell surfaces of vertebrates and can inhibit T.gondii invasion.There are few studies on the binding of T.gondii invasion-related proteins to heparin/HS.The molecular mechanism of T.gondii invasion in host cells is still unclear,and during T.gondii invasion,the functions of invasion-related heparin-binding proteins have not been fully revealed.Therefore,studying the function of these proteins will lay the foundation for further elucidating the molecular mechanism of T.gondii invasion in host cells.In this study,we investigated the function of the invasion-related heparin-binding proteins ROP9,MIC3,and SAG2,and the results not only laid a foundation for revealing the molecular mechanism of HS in the process of T.gondii invasion,but also provided theoretical support for developing new anti-toxoplasmosis vaccines and drug targets.In this study,we analyzed the bioinformatics of the amino acid sequences of coding ROP9,MIC3,and SAG2 proteins,and the results showed that these three proteins were highly conserved among different T.gondii strains and had good B cell epitopes,indicating that the proteins may play important role in T.gondii and are potential candidate antigens using vaccine and diagnosis.In order to analyze the functions of the proteins,Escherichia coli(E.coli)expression system was used to express His-tagged and GST-tagged recombinant proteins,respectively,and the specific polyclonal antibodies were successfully generated using purified His-tagged recombinant proteins.The transcription and expression levels of the target genes were analyzed by quantitative real-time PCR(RT-PCR)and Western blot.The results showed that in T.gondii RH strains,the transcription and expression levels of ROP9,MIC3,and SAG2 were higher in vitro than in vivo.The expression levels of the proteins were analyzed by indirect immunofluorescence assay(IFA),and the results found that during T.gondii invasion in host cells,the expression levels of ROP9,MIC3,and SAG2 were significantly higher than those of free parasites.The results of heparin-binding,competitive inhibition,and adhesion cell experiments demonstrated that ROP9,MIC3,and SAG2 proteins could specifically bind to heparin and adhere to the host cell surfaces.We further explored the effect of T.gondii invasion,when the invasive sites of the parasite or host cell surface were blocked.The results of T.gondii inhibition assays showed that the exogenous heparin could block T.gondii invasion,additionally,both the proteins and the specific polyclonal antibodies could inhibit T.gondii invasion.The immunorecognition characteristics were verified by the recognition of infected human serum and immunoprotection of the proteins.The results suggested that ROP9,MIC3,and SAG2 proteins could be specific recognized by infected human serum and had partial immunoprotection effects in mice.To further explore whether ROP9 protein is correlation with the invasiveness and virulence of T.gondii,CRISPR/Cas9 system was used to construct T.gondii ROP9 gene deletion strain(RH?ROP9)and recovery strain(RH-Re ROP9).Invasion rate,proliferation rate,egress time and pathogenicity in mice were analyzed,and the results demonstrated that the invasion rate of RH?ROP9 strain was significantly decreased and the pathogenicity of the deletion strain was weakened in mice.Currently,there are no vaccines and drugs that can completely prevent and control toxoplasmosis,hence,the development of a novel,effective,and safe vaccine against T.gondii is urgently required.In this study,the recombinant adenoviruses expressing the invasion-related heparin-binding proteins were constructed and their immunogenicity were analyzed.Firstly,ROP9,MIC3,and SAG2 genes were cloned into adenovirus shuttle vector,respectively,and then the recombinant shuttle vectors were co-transfected into HEK293 A cells with the skeleton vector to package the recombinant adenoviruses.The results of fluorescence microcopy observation and Western blot analysis showed recombinant adenoviruses expressing the target proteins were successfully constructed.BALB/c mice were immunized with the recombinant adenoviruses and the immunogenicity of the immunized mice were analyzed,and the results showed higher specific Ig G against T.gondii was generated in immunized mice,and the level of IFN-?,TNF-?,and IL-6 production secreted by Th1 lymphocytes from the recombinant adenoviruses groups of mice were significantly or extremely significantly higher(P<0.05,P<0.01,or P<0.001)compared to that of the control group.The percentages of the activated CD4+ T and activated CD8+ T lymphocytes in mice immunized with bivalent or trivalent adenoviruses were significantly or extremely significantly higher(P<0.05,P<0.01,or P<0.001)compared to that of the PBS group.The survival rate and survival time of the immunized mice were extremely significantly prolonged(P<0.05,P<0.01,or P<0.001).In summary,this study found that T.gondii invasion-related proteins ROP9,MIC3,and SAG2 could specifically bind to heparin and adhere to host cells.T.gondii ROP9 protein is involved in T.gondii invasion and is a virulence factor.The recombinant adenoviruses could stimulate humoral and cellular immune response to provide protective immunity against acute toxoplasmosis in mice.These results not only lay a foundation for elucidating the molecular mechanism of T.gondii invasion in host cells,but also provide new ideas for the development of new anti-toxoplasmosis vaccine. |
PDF Full Text Request