The Mechanism About Activation Of DNA Receptor Signaling Pathway In Innate Immune By Toxoplasma Gondii

Objective:Toxoplasma gondii can infect almost all of the nucleated cells, about a third ofthe world’s population have ever been infected with Toxoplasma. Toxoplasma caninfect the host cells and form the parasitophorous vacuole in host cell. On the onehand，Toxoplasma regulated the expression and secretion of immunomodulatorycytokines; on the other hand, the growth of T. gondii in vacuoles interfering thesignal transduction of host cell, thereby inhibiting the host cell response againstToxoplasma gondii infection. In the process of organism infections or tissue damage,exogenous DNA accumulated in the cytoplasm or their own DNA can trigger a strongimmune response, inducing product a series of cytokines, such as IFN-beta.Identification of exogenous DNA by receptor DNA is a key link in the process of thebody against bacteria and viruses. Studies have reported that the worm cavity ofToxoplasma can be broken under the action of IFN-gamma, after a series of immunereaction Toxoplasma removal by the body, at the same time toxoplasma DNA can bedetected in the cytoplasm. Based on the above research background, this experimentwas to study after the bubble bursting, the toxoplasma DNA got into the hostcytoplasm could be recognized by DNA receptor and activation downstream signalingpathways, induced the type I interferon and whether activation of the pathwaydependent on the STING, cGAS and TBK1key genes, thus showing innate immunemechanism of toxoplasma infection, to understand the intracellular pathogens innateimmune mechanism provides new content.Methods:1. Using human foreskin fibroblasts (HFF) to cultivate Toxoplasma gondiiME49-PTG strain, extraction of toxoplasma DNA. HEK293cells were transfected byToxoplasma DNA, and then detected related genes IFN-beta, ISRE, NF-kappa B, AP-1promoter expression by luciferase report gene technology.2. Using2FTGH cells which were stable expression of the ISRE luciferasereporter gene to detect the type I IFNs. 3. Using the native polypropylene amide gel electrophoresis and western blottingreaction detection the formation of transcription factor IRF-3dimers.4. Through overexpression or gene silencing of Sting and cGAS to analysis therole of Sting and cGAS gene in Toxoplasma DNA activation pathways.5. The tachyzoite of Toxoplasma gondii ME49-PTG strain infected HEK293cells, the luciferase report gene technology was used to inspect IFN-beta, ISRE,NF-kappa B, AP-1gene promoter expression level.6. Western Blot detected whether TBK1protein expression in TBK1knockout(TBK1-/-) mice fibroblast cells. Toxoplasma tachyzoite infected TBK1-/-cells,q-PCR detected the early changes in mRNA’s level of IL-6, IL-18, STING, ISG15,IRF3, SAG1gene, analysis the role of TBK1gene in Toxoplasma activate innateimmune.Results:1. Toxoplasma’s DNA can promote the expression of IFN-beta and ISREpromoter. ISRE is the activation element for interferon response, it can support theexpression of IFN-beta. At the same time, express the plasmid of cGAS and STINGfound, IFN-beta expression have further increases. For further validation to revealthe mechanism of action of Toxoplasma’s DNA, we tested the upstream signal path ofthe IFN-beta, which is the interferon regulatory factor3(IRF3), the results confirmedthat the Toxoplasma’s DNA could make IRF-3become dimerization, the formation ofIRF3dimers showed the IRF3protein was activated. It also confirmed theToxoplasma’s DNA activated IFN-beta signaling pathways through IRF3.2. Western Blot results confirmed TBK1protein was not expression in MEF cellswhich were knocked out TBK1gene. Then Toxoplasma infected TBK1-/-cells, theresults of q-PCR showed that the Toxoplasma surface antigen protein1(SAG1)mRNA transcription increases, the indirect proved the growth state of Toxoplasma inmouse embryonic fibroblast. Toxoplasma can inhibit the mRNA transcription of IL6,IL-18, ISG15and STING. In TBK1-/-cells infected with Toxoplasma results foundthat IL–18’s mRNA level is more significant decline. Under Toxoplasma infectioncondition TBK1gene plays a protective role for the mRNA transcription of IL-18, but there were not related report about TBK1and IL-18. The same experiment, ISG15’smRNA level dropped significantly. In the stage of infection, a large number ISG15expression reflects this protein play an important role in innate immunity. Thedecreased of ISG15’s mRNA indirect illustrates the escape of Toxoplasma in innateimmune. In addition, after TBK1knockout, ISG15mRNA is more inhibited, explainTBK1plays a important role in inhibition transcription of ISG15in Toxoplasmainfection.Conclusion:1. Toxoplasma’s DNA can activate IRF3, and make IRF3to be dimers, inducing theproduction of IFN-beta, and then promote the expression of ISRE, the active waypossible by cGAS-STING signaling pathways.2. Toxoplasma’s tachyzoite can promote the expression of NF-kappa B promoter, andthat may activate the NF-kappa B signaling pathway.3. Toxoplasma’s tachyzoite can inhibit the mRNA transcription of IL-6, IL-18, ISG15and STING. TBK1genes plays a important role in inhibit the mRNA transcription ofIL-18and ISG15in Toxoplasma infection.