The Molecule Mechanism Of How Toxoplasma Gondii Antigens Modulateing The NF-κB Signaling Pathway

As an important zoonotic pathogen at worldwide, Toxoplasma gondii(T.gondii) is an obligate intercellular protozoan. The life cycle of this parasite is very complex, and its host range is widely. Almost all warm-blooded animals including humans can be infected. T.gondii has been an important model organism for understanding how the immune system promotes resistance to intracellular pathogens. The NF-κB signaling pathway have an important role in the immune response to T.gondii, which is expected considering its evolutionarily conserved role in the regulation of many aspects of innate and adaptive immune functions. In recent years, a series of T.gondii antigens have been found to be able to modulate the host NF-κB signaling pathways, but their specific mechanism are still unclear.In this study, the effects on the NF-κB signaling pathway were tested by 5 strains T.gondii tachyzoites. Then 22 T.gondii antigens were used dual luciferase system to test whether can activate the NF-κB signaling pathway. The NF-κB signaling pathway could be activated by GRA15, and the specific molecule mechanism how GRA15 activated the NF-κB signaling pathway were explored. 1) The effects on the NF-κB signaling pathway by different strains of T.gondiitachyzoitesHEK293T cells were infected by RH, Me49, VEG, Type I and Type II strains of T.gondii tachyzoites and the effect of different infection abundance on NF-κB signaling pathway was detected by dual luciferase reporter assay system were used dual luciferase assay system.After infected, the NF-κB signaling pathway could be activated at different degrees by Me49, VEG, Type I and Type II. The NF-κB signaling pathway could be obviously inhibited by RH.The abilities of these tachyzoites infecting cells were evaluated by IFA. Under the similar amount of invasion, the effects on the NF-κB signaling pathway by different strains T.gondii tachyzoites were still different. 2) Screening of T.gondii antigens which activate the NF-κB signaling pathwayT.gondii antigens eukaryotic expression plasmids were transfected into HEK293 T cells. The NF-κB signaling pathway could be activated by pCMV- GRA15(II) and pCMV- GRA15(I).After analyzed the host cells expressing GRA15 I protein by Western blot and IFA, phosphorylated p65 was significantly increased and p65 was transferred into nuclear. It verified the NF-κB signaling pathway could be activated by GRA15 I. 3) The molecular mechanism GRA15 activate the NF-κB signaling pathwayFull- length GRA15 had two kinds of domains, transmembrane domain and nuclear localization signal. So 7 GRA15 truncated eukaryotic expression plasmids(pCMV-GRA15-1, pCMV-GRA15-2, pCMV-GRA15-3, pCMV-GRA15-4、pCMV-GRA15-5, pCMV-GRA15-6 and pCMV-GRA15-7) were constructed. After tranfected into HEK293 T cells and used dual luciferase system, pCMV-GRA15-4、pCMV-GRA15-5, pCMV-GRA15-6 and pCMV-GRA15-7 could activate the NF-κB signaling pathway. The 194-518 amino acid region in GRA15 I might be the key domain activating NF-κB signaling pathway.Finally IκB and phosphorylated IκB in the GRA15 expressed cells cytoplasm were detected using western blot. IκB expression was significantly reduced by GRA15, but phospho- IκB expression was also significantly reduced. The results indicated GRA15 might activated the NF-κB signaling pathway by depredating IκB.In this study, a variety of techniques were used to explore how T.gondii modulated the host NF-κB signaling pathway. It not only help us better understanding the interaction between T.gondii and host immune system, but also provide the theoretical basis for the development of anti-Toxoplasma new drugs and vaccines.