Mechanism Study On Regulating Host Immune Response Of Toxoplasma Gondii Wx2 Gene Via Inhibiting Pyroptosis

Background:Toxoplasma gondii is an opportunistic protozoan that infects about 1/3 of the population in the world,with acute consequences especially in high-risk groups such as immunocompromised or immunodeficient individuals and pregnant women.Despite the fact that Toxoplasma gondii has been discovered more than 100 years,the pathogenic mechanisms,including invasion and immune evasion in the host,remain unclear.Drug treatment for Toxoplasmosis has disadvantages such as long treatment period and side effects.There were no effective drugs can kill the cysts in tissues.And there was no safe and effective vaccine against Toxoplasmosis for clinic yet.Therefore,to elucidate the pathogenic mechanisms and develop effective vaccine were still the hot and difficulty spots in the field of Toxoplasma research.Previous studies in our group has found that the wx2 gene was associated with virulence of Toxoplasma gondii and involved in the regulation of host immunity.Objective:The aim of this study was to investigate the mechanism of regulation of host immune response by Toxoplasma gondii virulence gene wx2 through in vivo and vitro experiments.Methods:Construction of Toxoplasma gondii wx2 gene knockout strain(RH^wx2-/-strain)and complemented strain(RH^wx2+/+strain)using CRISPR/Cas9 technology;the effects of wx2 gene on virulence of Toxoplasma gondii and host cell pyroptosis pathway were observed by in vivo and vitro experiments.Results:（1）Mice have significantly longer survival time after infection with the RH^wx2-/-strain compared to the wild-type strain;however,the survival time of mice was significantly shorter with wx2 gene restoration(RH^wx2+/+),suggesting that the virulence of knockout strain RH^wx2-/-was restored after wx2 gene reversion.（2）In vitro experiments,the transcript levels of pyroptosis associated molecules NLRP3,GSDMD and IL-1βwere significantly increased in RH^wx2-/-strain compared to wild and RH^wx2+/+strains after 24 h and 48 h.The protein expression levels of NLRP3,caspase-1,caspase-1p20,GSDMD and GSDMD-N were significantly increasing in RH^wx2-/-strain at 24 hours and 48 hours,and the phosphorylation level NF-κB were significantly elevated at 48 hours.（3）In vivo experiments,compared with the wild strain and RH^wx2+/+strain,the protein expression levels of NLRP3,caspase-1,caspase-1p20,GSDMD-N,IL-1βand IL-1βp17 in RH^wx2-/-strain of infected mouse lymphocytes were increased in various degrees,while the m RNA levels of NLRP3,pro caspase-1,GSDMD and ASC were also increased.It is suggested that wx2may regulate the host immune response through the classical pathway of pyroptosis.Conclusion:The wx2 gene is probably involved in host immune regulation by inhibiting the classical pathway of pyroptosis.11 figures,11 tables,51 references...