Studies On The Molecular Mechanism That Toxoplasma Gondii ROP18 Suppressed IFN-β Production

Toxoplasma gondii,as a kind of strictly obligate parasitic protozoa,can infect the nucleated cells of almost all warm-blooded animals,including humans.The invasion of host cells by T.gondii can induce the production of type I interferon,and there are related mechanisms to inhibit the production of type I interferon.Existing studies have shown that type I interferon plays an important role in the control of T.gondii infection,but there are relatively few studies on the related mechanisms to inhibit the production of type I interferon.During the process of infection,T.gondii will secrete effector proteins to participate in immune evasion to facilitate the growth of T.gondii in the host cells.When T.gondii invasions the host cells,it will secrete a class of rhoptry proteins with kinase/kinase-like domain,most of which are involved in physiological activities such as invasion replication virulence and immune avoidance.Therefore,these proteins are most likely effector proteins that inhibit the production of type I interferon in the hosts.In this study,the rhoptry protein ROP18 with more effective inhibition of SEV-induced IFN-βpromoter activity was screened out from the 17 successfully constructed rhoptry protein recombinant plasmids using dual luciferase detection system,and the specific mechanism of ROP18 inhibiting IFN-β production was further explored.The main results are as follows:(1)Screening of rhopty proteins that inhibit SEV-induced IFN-β productionAfter data analysis and screening,the 17 kinds of rhopty protein eukaryotic recombinant plasmids were successfully constructed and transfected into the HEK293 T cells,and the activity of IFN-β promoter were detected by the dual luciferase system after SEV infection.The results showed that the overexpression of the two rhopty proteins,TGGT1＿291960 and TGGT1＿205250(ROP18),significantly inhibited the SEV-induced activation of IFN-β promoter,the inhibition effect of TGGT1＿205250(ROP18)was more significant.(2)ROP18 inhibites the RLR signaling pathway mediated by TBK-1NF-κB and IRF3 are two important transcription factors that regulate IFN-βproduction.To investigate whether ROP18 inhibits the activation of NF-κB and IRF3 promoters.The HEK-293 T cells were transfected with ROP18 eukaryotic recombinant plasmid,and the activity of NF-κB promoter and IRF3 promoter were detected by the dual luciferase system after SEV infection.The results showed that ROP18 inhibited the activity of SEV-induced IRF3 promoter,but did not inhibit the activity of SEV-induced NF-κB promoter.SEV is a stimulant that strongly induces RLR mediated IFN-β production.ROP18 inhibits SEV-induced activation of IFN-β and IRF3 promoters,possibly because ROP18 targets one or more adaptor molecules in the RLR signaling pathway.To investigate whether ROP18 can inhibit the production of IFN-β by interacting with related proteins in the RLR signaling pathway,the eukaryotic expression plasmid ROP18 was co-transfected into HEK-293 T cells with the eukaryotic plasmid RIG-IN,MDA-5,MAVS,TBK1,IKKε,IRF3-5D which are the RLR signaling pathway key molecules.The dual-luciferase assay showed that ROP18 inhibited IFN-β promoter activation induced by RIG-IN,MDA-5,MAVS,TBK-1,and but did not inhibit IKKε and IRF3-5D-induced IFN-β promoter activation,suggesting that TBK-1 may be a potential target of ROP18.The western blot results showed that the protein amount of TBK-1 was significantly reduced after the co-transfection of ROP18 and TBK-1 eukaryotic plasmid compared with the control group.At the same time,the co-immunoprecipitation(Co-IP)test found no direct effect between ROP18 and TBK-1.(3)The mechanism of ROP18 and TBK-1In order to study the effect of ROP18 on the amount of TBK-1 protein,the HEK-293 T cells were first transfected with pc DNA3.1-ROP18-3HA or pc DNA3.1-3HA.The q RT-PCR analysis showed that ROP18 did not inhibit the transcription of TBK-1,so it was speculated that ROP18 influenced the protein level of TBK-1 by inducing degradation.ROP18 was divided into two truncated mutants based on the ROP18 structure published in the literature(ROP18-N:43-244 aa ROP18-C:245-554aa).The two truncated ROP18 eukaryotic expression plasmids were co-transfected with TBK-1,the western blot results showed that ROP18-N truncated fragment was the key region for degradation of TBK-1.When using proteasome inhibitor MG-132,the western blot analysis showed that the protein amount of TBK-1 did not increase significantly after the treatment of MG-132.These results suggest that ROP18 may degrade TBK-1 by other pathways,thereby inhibiting the activation of IFN-β promoter.In this study,TGGT1＿291960 and TGGT1＿205250(ROP18)significantly inhibited SEV-induced activation of IFN-β promoter,and the inhibition effect of TGGT1＿205250(ROP18)was more significant.ROP18 inhibits the production of IFN-βin RLR signaling pathway,and further study of the mechanism revealed that TBK-1 may be a potential target of ROP18,and there is no direct interaction between the two.ROP18 affects the protein level of TBK-1 by degrading rather than inhibiting transcription.The ROP18-N truncated fragment is the key region to induce degradation of TBK-1,but the degradation pathway may not be ubiquitin-proteasome.The above studies provide the ideas for finding other target proteins that inhibit the production of type I interferon in the hosts,and provide theoretical support for the prevention and control of T.gondii infection.