Construction And Biological Function Research Of TR And ROPs Double Gene Deletion Strains Of Toxoplasma Gondii

Toxoplasma gondii(T.gondii)is a parasitic protozoan of the apicomplex family that specializes in the host cell.The invasion of T.gondii into host triggers the oxidative stress response.It has been demonstrated that thioredoxin reductase(TR)of T.gondii plays important roles in resisting host reactive oxygen species(ROS)damage,while the rhoptry protein16(ROP16),rhoptry protein18(ROP18)and rhoptry protein5(ROP5)of T.gondii play key roles in immune evasion by downregulating the host innate immunity to suppress excessive inflammation.However,whether TR/ROP16,TR/ROP18 and TR/ROP5 of T.gondii have synergistic effects in resisting ROS damage of host immune cells and their mechanisms remains unclear.Our laboratory has constructed the TR/ROP18 double gene deletion strain(TR-ROP18-KO)of T.gondii,and found that the biological activity of TR-ROP18-KO strain was reduced.In this study,we used CRISPR/Cas9 gene knockout technology to successfully obtain the TR/ROP16 double gene knockout strain(TR-ROP16-KO),TR/ROP5 double gene deletion strain(TR-ROP5-KO).By biological activity analysis,we discovered that the invasion,proliferation ability in vitro,and virulence in vivo of TR-ROP16-KO strain were lower than those of RH strain,TR gene deletion strain(TR-KO)and ROP16 gene deletion strain(ROP16-KO).Similarly,the invasion,proliferation ability in vitro,and virulence in vivo of TR-ROP5-KO strain were lower than those of RH strain,TR-KO strain and ROP5 gene deletion strain(ROP5-KO).These results indicated that the simultaneous deletion of TR/ROP16 and TR/ROP5 in T.gondii not only lead to a decrease in the viability of the parasites in vitro cells,but also reduce the virulence of toxoplasma in the host.Secondly,this study detected the oxidative stress level of the single and double gene deletion strains of TR/ROP16,TR/ROP18 and TR/ROP5 of T.gondii and their effects on the ROS level of mouse macrophages(RAW264.7).The results showed that the TR-KO and ROP16-KO strains significantly higher than the RH strain,and the TR-ROP16-KO strain was significantly higher than the RH,TR-KO and ROP16-KO strains,indicating that TR/ROP16 deficiency could synergically induce increases of oxidative stress level in parasites and ROS level in RAW264.7 cells.In addition,although the TR-ROP18-KO and TR-ROP5-KO strains were significantly higher than the RH strain,they were not significantly different from the TR-KO strain,indicating that the oxidative stress level of TR-ROP18-KO and TR-ROP5-KO strains and the ROS level of infected RAW264.7 cells mainly depended on the deletion of TR gene.The results suggested that TR/ROP16 of T.gondii exerts a synergistic effect in resisting host ROS damage,but TR/ROP18 and TR/ROP5 have no synergistic effect in resisting host ROS damage.Finally,in order to analyze the reasons for the changes in oxidative stress levels of TR-ROP16-KO,TR-ROP18-KO and TR-ROP5-KO strains,the IFN-γ m RNA levels produced by TR/ROP16,TR/ROP18,TR/ROP5 single and double gene deletion strains infected with RAW264.7 cells were detected by RT-PCR,and the levels of IL-12 in serum of infected mice was also detected by ELISA.The results showed that the IFN-γ m RNA level of infected RAW264.7 cells by ROP16-KO strain and the level of IL-12 in mouse serum infected by ROP16-KO strain were significantly higher than the RH strain at 4 or 6 h after infection,indicating that ROP16 could down-regulate the ROS level of host immune cells through IL-12-IFN-γ-ROS pathway.Moreover,The IFN-γ m RNA level of RAW264.7cells infected with TR-ROP16-KO strain for 12 h and the level of IL-12 in mouse serum infected with TR-ROP16-KO strain at different time points was greatly higher than infected with RH,TR-KO and ROP16-KO strains.Based on the results,we concluded that TR/ROP16 of T.gondii have a synergistic effect in resisting host ROS damage from the level of cytokines.In addition,according to the changes in cytokine levels,we also found that the TR/ROP18 and TR/ROP5 of T.gondii have no synergistic effect in resisting host ROS damage.By using TR/ROP16,TR/ROP18 and TR/ROP5 double gene deletion strains to explore the effects of TR and ROPs gene deletion of T.gondii on oxidative stress levels and analyze the reasons for the changes,this study can clearly resolve the confusion of synergistic effect on resisting the ROS damage of host,and may lay foundation for exploring the synergistic effect of TR and ROPs in the immune escape pathway of T.gondii type I,which also provided new insights for understanding synergistic functions of double genes in T.gondii.