Study On The Virulence Relationship And Mechanism Between TgMIC4 And Chinese 1 Toxoplasma

Background: Infection of host cells by Toxoplasma gondii is an active process,which is regulated by secretion of microneme proteins(MICs)and rhoptry proteins(ROPs and RONs)from specialized organelles in the apical pole of the parasite.According to the transcriptome sequencing and related experimental verification in the early stage of our laboratory,the transcriptome levels of ROP18 and ROP5,which are recognized as important virulence factors in the international classic Type Ⅰ,Ⅱ,and Ⅲ,are no significant difference in the Chinese 1 dominant genotype virulent strain WH3 and the attenuated strain WH6 in China,and the WH3 strain with knockout of ROP16 has no weakened virulence,while the transcriptome levels of MIC1,4,and 6 proteins are significantly different between the two strains.These results suggest that Chinese 1 dominant genotype Toxoplasma gondii strain has unique virulence characteristics and pathogenic mechanisms.MIC1,MIC4 and MIC6 assemble into an adhesin complex secreted on the parasite surface that functions to promote infection competency.In addition,the A5 domain of MIC4 has the properties of galectin,which can physically bind to the N-glycans of TLR2 and TLR4 on the surface of dendritic cells and macrophages to stimulate the production of pro-inflammatory cytokines,and playing an important role in vivo by changing the infection ability of Toxoplasma gondii and the pathogenesis of mice.Interestingly,in addition to inducing proinflammatory cytokine release by macrophages,MIC1 and MIC4 also trigger secretion of the anti-inflammatory cytokine IL-10 through an unknown mechanism in a TLR4 internalization-dependent manner.Meanwhile,the fact that the stimulated macrophages acquired transient tolerance to LPS provides a possible mechanism for evasion of the host inflammatory response by T.gondii Therefore,we speculated that MIC4 might be a factor for the difference in virulence between the two strains of Chinese 1.Objective: To explore the virulence relationship and mechanism between TgMIC4 and Chinese1 dominant genotype Toxoplasma gondii.Methods:(1)Verification of transcriptome data of WH3 and WH6 strains: Online protein analysis software Bepipred 1.0 Server was used to analyze the antigen epitopes of MIC4 protein,and relatively specific TgMIC4 peptides in the binding region of TgMIC1 and TgMIC4-A5 peptides in the binding region of TLR were selected respectively,which were sent to the company for synthesis and immunization of New Zealand rabbits to obtain the corresponding specific polyclonal antibodies.Opti GeneTM codon analysis platform was used to optimize MIC4 gene codon and construct TgMIC4 and TgMIC4-A5 protein expression vectors,which were transformed into capable cell BL21(DE3)for inducing expression and purified by affinity chromatography.The mRNA and protein expression levels of TgMIC4 between WH3 and WH6 strains were verified by q PCR and Western Blot,respectively.(2)Construction and validation of the WH3Δmic4 strain: The WH3Δmic4 knockout strain was constructed using CRISPR/Cas9 targeted gene knockout technology,and the deletion of the MIC4 gene and protein in the knockout strain was identified by PCR and Western Blot,respectively.(3)Invasion and proliferation of WH3-WT,WH3Δmic4 and WH6: immunofluorescence,Giemsa staining and crystal violet staining were used to evaluate the invasion and proliferation of tachyzoites in HFF,respectively.(4)Virulence of WH3-WT,WH3Δ MIC4 and WH6 in mice: 1000 tachyzoites in each group were intraperitoneally injected into Kunming mice,respectively.The survival and mortality of mice in each group were observed within 15 days.The expression levels of IL-12,IFN-γ,TNF-α,IL-6 and i NOS in each group were detected by ELISA on day 5.(5)Validation of lectin properties of MIC4 and MIC4-A5 recombinant proteins(rMIC4 and rMIC4-A5): Seven monosaccharides(L-Fucose),sialic acid(NANA),mannose(D-mannose),acetylglucosamine(D-Glc NAC),acetylgalactosamine(D-Gal NAC),glucose(D-GLC)and galactose(D-GAL)were used to perform hemagglutination inhibition experiments on rMIC4 and rMIC4-A5 to verify their galectin properties.(6)Effects of WH3-WT,WH3Δmic4 and WH6 on the secretion of cytokines in mouse peritoneal macrophages(RAW264.7): According to MOI of 3,WH3-WT,WH6,WH3Δmic4,WH3Δmic4+rMIC4 and WH3Δmic4+rMIC-A5 were infected with RAW264.7,uninfected cell culture supernatant as negative control,LPS as the positive control,the expression of IL-6 and TNF-α was detected by ELISA after 24 hours of infection.Results:(1)TgMIC4-PET30 a and TgMIC4-A5-PET30 a expression vectors were successfully constructed,and after induction and expression,rMIC4 and rMIC4-A5 proteins were purified by Ni column.Polyclonal antibodies against TgMIC4 and TgMIC4-A5 peptides were successfully obtained.The results of q PCR and Western Blot showed that the MIC4 mRNA and protein expression of the strong strain WH3 were significantly higher than that of the weak strain WH6.(2)PCR and Western Blot identification results show that the WH3Δmic4 strain was successfully constructed using CRISPR/Cas9 targeted gene knockout technology.(3)Compared with the WH3-WT strain,the invasion,proliferation and macrophage abilities of WH3Δmic4 were significantly decreased,but still slightly higher than that of WH6.(4)The virulence test of WH3-WT,WH3Δmic4 and WH6 in mice showed that the mice infected with 1000 WH3-WT,WH3Δmic4 tachyzoites all died within 10 days,and compared with WH3-WT group infected mice mortality,WH3Δmic4 group was slightly but significantly reduced,while the mortality rate of WH6 group was less than 50%.Serological tests on the fifth day of infected mice showed that WH6 strain induced stronger IFN-γ and IL-12 production than WH3,but there was no statistically significant difference in the expression levels of TNF-α,IL-6 and i NOS,in addition.Compared with the WH3-WT group,the expression levels of IFN-γ and IL-12 in the WH3Δmic4 group were significantly increased,while TNF-α and IL-6 were slightly decreased.(5)The results of hemagglutination inhibition experiments showed that among the seven selected monosaccharides,only D-Gal Nac and D-Gal can effectively inhibit the hemagglutination of rMIC4 and rMIC4-A5 proteins.(6)Effects of WH3-WT,WH3Δmic4 and WH6 on the secretion of cytokines in mouse RAW264.7: Compared with the control group,the expression of IL-6 and TNF-α increased in the infected group,moreover the expression in WH3 group obviously higher than that of WH6,and the expression of IL-6 and TNF-α in WH3Δmic4 group was significantly reduced.Both rMIC4 and rMIC4-A5 proteins could restore the the expression of IL-6 and TNF-α to the level of the WH3-WT group.Conclusions: The MIC4 of WH3 is significantly higher than WH6 in mRNA and protein expression levels.WH3-WT shows stronger invasion and proliferation ability than WH3Δmic4 and WH6,and has stronger ability to induce IL-6 and TNF-α production on RAW264.7,suggesting that the differential expression of MIC4 in Chinese 1 dominant genotype strains WH3 and WH6 is related to the difference in virulence.