The Molecular Mechanism Of Toxoplasma Gondii Dense Granule Protein GRA14 On Down-regulating NF-?B Signaling Pathway

Background: Toxoplasma gondii(T.gondii)is an obligate intracellular parasite that causes irreversible damage to the fetus and poses a deadly threat to individuals with low immune function.According to statistics,about one-third of people worldwide were infected with T.gondii.The infection rate of T.gondii in China has reached 7.8%,and there is a rising trend.Toxoplasmosis is an important food-borne disease on a global scale.It not only poses a threat to people's health,but also causes serious medical and health pressures and social and economic burdens.Therefore,foodborne toxoplasmosis is an important public health problem affecting the health of people around the world.Purpose: T.gondii can invade all warm-blooded animals and activate the host immune system,but it can still survive in the host for a long time.NF-?B signaling pathway,the arm of the innate immune system,plays an important role in the clearance of T.gondii,but studies have shown that T.gondii can inhibit the activation of NF-?B signaling pathway and regulate host immunity.parasitophorous vacuole structure interfere with the signal transduction of host cells,protect them from host clearance,and play an irreplaceable role in the reproduction of T.gondii.The dense granule proteins are crucial for the stability and function of the parasitophorous vacuole structure.So far,The role of dense granule proteins in the immune escape of T.gondii is not well understood.Therefore,this study mainly studies the regulation of T.gondii compact granule protein on the NF-?B signaling pathway,and further explores the molecular mechanism of this regulation,which provides a basis for further elucidation of the relationship between dense granule protein and immune escape of T.gondii.Methods: 1.From the 33 dense granule proteins of T.gondii constructed in the laboratory,the dual luciferase assay was used to screen out candidate genes that significantly inhibited the NF-?B signaling pathway.2.Select the target gene with strong inhibition from the candidate genes,and further determine its specific inhibitory effect on NF-?B signaling pathway by using dual luciferase reporter gene assay.3.Selected target genes were detected by dual luciferase assay and real-time PCR to detect the expression of cytokines and chemokine downstream of NF-?B signaling pathways such as IL-6,IL-12,TNF-?,IFN-?,CXCL10 and CCL5.4.Using dual luciferase assay and western blot to explore the molecular targets of target gene on NF-?B signaling pathway.The effect of target gene on MyD88-mediated transcriptional expression was analyzed by RNA sequencing and verified by real-time PCR.Results: 1.By screening,GRA14 was found to have a significant inhibitory effect on NF-?B signaling pathway,and the higher the concentration,the more obvious the inhibitory effect.MyD88,cGAS-STING,MAVS,and TRIF-mediated NF-?B signaling pathways were all inhibited.2.In addition to inhibiting the NF-?B signaling pathway,GRA14 also has a significant inhibitory effect on the AP-1 signaling pathway,but the effect on the ISRE pathway is not very obvious.3.GRA14 can inhibit the expression of IL-6,IL-12,TNF-?,IFN-?,CXCL10 and CCL5 which were downstream of the NF-?B pathway.4.Overexpression of the key proteins on the NF-?B signaling pathway(such as TRAF2,TRAF6,TAB1,TAK1,IKK?,IKK?,p50,and p65),GRA14 can inhibit the activity of NF-?B promoter.It indicated that GRA14 inhibits NF-?B pathway may act on downstream of p65 or p65.5.Western blot results showed that GRA14 can inhibit the phosphorylation of p65 and promote the degradation of p65,both for endogenous and exogenous p65.The results of nuclear isolation showed that GRA14 could inhibit p65 entry into the nucleus and was mainly located in the host cytoplasm with a small amount of distribution in the nucleus.It suggested that GRA14 may regulate the host by promoting p65 degradation and inhibiting its entry into the nucleus,resulted in the activation of the NF-?B pathway prevented.Thereby T.gondii regulating host cell immune response and achieving immune escape.6.The results of RNA sequencing showed that GRA14 could regulate the expression of 49 genes in host cells,and QPCR confirmed that GRA14 could down-regulate HSPA1 A,HSPA6,and up-regulate PAK6.HSPA1 A and HSPA6 are downstream transcription factors of NF-?B signaling pathway,and their expression is down-regulated,further indicating the inhibitory effect of GRA14 on NF-?B signaling pathway.The relationship between PAK6 and NF-?B signaling pathway is unclear and needs further study.PAK6 is involved in the p38 MAPK pathway and may regulate NF-?B activity through the p38 MAPK pathway.Therefore,the relationship between PAK6 and p38 MAPK and NF-?B needs to be further explored.Conclusion: Toxoplasma gondii GRA14 inhibits NF-?B and AP-1 signaling pathways,and inhibits the transcriptional expression of cytokines downstream of the pathway;T.gondii GRA14 can negatively regulate NF-?B signaling pathway by promoting p65 degradation and inhibiting phosphorylation of p65 into the nucleus.In conclusion,T.gondii GRA14 can regulate host immunity by negatively regulating the NF-?B signaling pathway and assisting immune evasion.