Study On Biological Functions And Immunoprotectivity Of Toxoplasma Gondii ROP54

Toxoplasma gondii is an obligatory intracellular opportunistic parasitic protozoan which can infect nearly all warm-blooded animals and humans with a worldwide distribution.T.gondii infection can cause serious encephalitis,ophthalmopathy,abortion,stillbirth,posing a serious threat to public health and causing huge economic loss to animal husbandry.T.gondii has two stages,namely tachyzoites and bradyzoites,in the intermediate host.At present,drug treatment has good effect on the tachyzoite stage,while it is less effective on the bradyzoite stage and the drug treatment has some severe side effects.In rencent years,anti-T.gondii vaccines have been considered as an optimal approach for controlling T.gondii infection.However,no effective vaccines are available currently for the prevention and control of this disease due to absence of suitable target genes.Previous studies have indicated that rhoptry proteins played important roles in host invasion and virulence of T.gondii to host cells.Rhoptry protein 54(ROP54)is a novel rhoptry pseudokinase effector which has a singificant effect on the regulation of GBP2 loading on parasitophorous vacuole and the virulence of T.gondii.However,its biological function and immunoprotective effect are still unknown.In the present study,the recombinant plasmid pVAX-ROP54 encoding Toxoplasma ROP54 gene was constructed and used to immunize Kunming mice three times.The levels of humoral and cellular immune responses of the immunized mice,as well as the proliferation ability of the spleen cells of the immunized mice,were measured.Meanwhile,immunized mice were challenged with different T.gondii strains with different virulence to evaluate the immune protective efficacy.The detection results of antibody levels indicated that the mice immunized with pVAX-ROP54 produced a high level of IgG antibody,compared with the control group.In addition,the level of IgG2 a was higher than that of IgG1,suggesting that a Th1 type humoral response was successfully elicited.The detection results of cytokine levels indicated that IFN-?,IL-2,IL-4,IL-10 and IL-12(p70)were significantly increased in the immunized mice compared with the control groups,and the levels of Th1-type cytokines were higher than those of Th2-type.These results revealed that vaccination with pVAX-ROP54 stimulated the immunized mice to produce a Th1 type cellular response.Challenge infection showed that the survival time of all mice vaccinated with pVAX-ROP54 was significantly prolonged for approximately 7 days and theaverage brain cyst number was reduced by 35.9% compared with those of the control group,which indicated that the recombinant ROP54 plasmid could provide partial protection against acute and chronic infections of T.gondii.Thereafter,the ROP54 gene of T.gondii PRU strain was disrupted using the CRISPR-Cas9 technology.The constructed gene-knockout strain was used to further study the biological functions of the ROP54 gene via investigating the effect of the ROP54 gene on the proliferative capacity in vitro,the virulence and cyst formation ability in mice.The plaque assay results showed that the replication ability of PRU-?ROP54 was significantly weakened in HFF cells in vitro compared with the wild-type strain.The virulence test showed that the survival rate of mice infected with the knockout strain was significantly higher than that infected with the wild-type strain.The results of cyst formation assay showed that the numbers of brain cysts in the PRU-?ROP54-infected mice group were significantly lower than that of the mice infected with the wild-type strain.Collectively,the recombinant plasmid pVAX-ROP54 and the PRU-?ROP54 deletion mutant were successfully constructed to evaluate the immunoprotectivity of Toxoplasma ROP54 gene,and the results indicated that ROP54 gene has partial immunogenicity.Immunization with pVAX-ROP54 can induce strong humoral and cellular immune responses.Compared with the T.gondii PRU wild-type strain,the proliferation,virulence and its ability of cyst formation of the PRU-?ROP54 strain were significantly reduced.However,neither the ROP54 DNA vaccine nor the deletion mutant could provide complete immune protection for mice,suggesting that they were not suitable candidate vaccines against T.gondii infection.