The Regulatory Effect And Mechanism Of TgMIC1 On Toxoplasma Gondii Chinese 1 Virulence

Background:Toxoplasmosis is a global zoonosis.As the causative agent of Toxoplasma gondii,it has complex population structure and genetic diversity.There are many typing methods for Toxoplasma gondii,the most commonly used typing method was cleaved amplification polymorphism sequence-tagged sites(PCR-RFLP),and based on this typing method the population structure of T.gondii was divided into three types:Type Ⅰ,Type Ⅱ and Type Ⅲ,which called archetypal clonal lineage.The dominant genotype in our country is completely different from the archetypal clonal lineage and is named Chinese 1.Different genotypes lead to differences in the expression of effectors among different strains,in this case,different strains have different virulence.The differences in virulence of Type Ⅰ,Ⅱ and Ⅲ were thought to be related to the key virulence factors ROP18/ROP5,ROP16 and GRA15.Although according to the results of early transcriptome sequencing and whole genome sequencing in our laboratory,no significant differences have been found between these classical virulence factors in representative strains Wh3(strong strain)and Wh6(weak strain)of dominant genotypes in our country,mRNA and protein expression levels of Toxoplasma microneme proteins(TgMIC1)were significantly different between the two strains of Toxoplasma genotype 1 in China.Whether these differentially expressed genes are involved in the regulation of virulence of different strains and their specific mechanisms remain unclear.Previous functional studies on TgMICs mainly focus on their role in toxoplasma invasion and adhesion.For example,TgMIC 1/4/6,TgMIC3/8 and TgMIC2/M2AP are important complexes that play a role in toxoplasma invasion of the host.However,recent studies have shown that some microneme proteins are also involved in immune regulation during Toxoplasma infection and associated with different outcomes after infection.The aim of this study was to investigate the regulatory effect and mechanism of TgMIC1 on Toxoplasma gondii Chinese 1 virulence,which is of great significance for further revealing virulence characteristics and pathogenic mechanisms of representative Chinese 1 dominant genotypes Toxoplasma gondii.Purpose:The micl knockout strain Wh3Δmicl was constructed based on Wh3 by using CRISPR/Cas9 technology,and investigate the regulatory effect and mechanism of TgMIC1 on Toxoplasma gondii Chinese 1 virulence.Method:(1)Construction and verification of Wh3Δmic1 Strain:pSAG1::CAS9-U6::sgMIC1 plasmid and MIC1-donor DNA were constructed.The mixed clones were obtained by electric transfer,and pyrimidine was screened.After five times of screening,monoclonal selection was performed.The Wh3Δmic1 strain was identified by PCR and Western Blot(WB).(2)Detection of in vitro invasion,proliferation and plaque phagocytosis:Vero cells were used for planking,and there were three groups of Wh3,Wh6 and Wh3Δmic1 with three multiple pores in each group.Invasion assay,two hours after infection,tachyzoids were labeled with rabbit anti-TgGAP45(1:1000)polyclonal antibody and goat anti-rabbit conjugated Alexa594(1:1000)antibody(red).Tachyzoite was labeled with a rabbit antiTgGAP45(1:1000)polyclonal antibody and a goat anti-rabbit conjugated Alexa488(1:1000)antibody after permeabilization with 0.1%Triton X-100(green).The non-invaded tachyzoites located on the outer membrane and were labeled with both red and green fluorescence(yellow).The tachyzoites that successfully invaded were located within the mold and could only be labeled with green fluorescence and appeared green.We arrive at the invasion efficiency by calculating the ratio of successful invasion to all tachyzoites on 100 cells.For the proliferation experiment,each T.gondii strain was incubated in Vero for 36h at 37℃,fixed with 4%paraformaldehyde,and stained with Giemsa staining solution at room temperature.After mounting,50 nanovesicles were randomly selected under a high-power microscope to observe the tachyzoite numbers of T.gondii in the nanovesicles.For plaque assay,1000 tachyzoites of each parasite strain were used to infect Vero for 12 days,and crystal violet staining was used to calculate the number of plaques in each well of each group.The plaque formation ability of each parasite strain was evaluated according to the number of plaques formed.(3)Virulence test in mice:Six five-week-old C57BL/6 female rats in each group were intraperitoneal injected with 1000 tachyzoites of Wh3,Wh6 and Wh3Δmic1.The death time was recorded.(4)Detection of Toxoplasma gondii load:Total Wh3,Wh6 and Wh3Δmic1 four groups,each group take 5 five-week-old female C57BL/6 mice,each mice intraperitoneal injection 1000 tachyzoites of Wh3,Wh6 or Wh3Δmic1.The intestinal,brain,spleen and blood samples were collected on the 5th day to detect the load of T.gondii in each organ and blood.(5)Changes of serum IL-1β,IL-18,IFN-γ and IL-10 in mice:Five 5-week-old female C57BL/6 mice in each group were intraperitoneally injected with 1000 tachyzoites of Wh3,Wh6 and Wh3Δmic1,respectively.Serum samples were collected on the 5th day to detect the changes in serum levels of IL-1β,IL-18,IFN-γ and IL-10.(6)Purification of rMIC1 and detection of its lectin properties:pET30a-MIC1 induced expression of recombinant proteins by Isopropyl-β-D-thiogalactopyranoside(IPTG)and purified by affinity chromatography.Detect the lectin properties of rMIC1 by inhibition of rMIC1 hemagglutination by seven monosaccharides(D-Gal,NANA,D-GalNac,D,GlcNac,D-Glc,D-mannose,L-Fucose).(7)Pathway enrichment and pyroptosis-related gene analysis:Spleen cells from Wh3infected and non-infected mice were collected,and genes were annotated according to Kyoto Encyclopedia of Genes and Genomes(KEGG)after sequencing.The annotated genes were matched with KEGG pathway database,and the statistically significant and over-expressed gene pathways were found.In addition,genes related to pyroptosis,such as IL1β,NLRP3,and GSDMD,were annotated and enriched.(8)The effects of Wh3,Wh6 and Wh3Δmic1 on BMDM:After BMDM was extracted,MOI was used as 3 and infected with Wh3,Wh6 and Wh3Δmic1 respectively.After 24 hours of infection,the expression level of IL1β was detected by qRT-PCR and ELISA,and the expression of GSDMD,NLRP3,IL1β,Caspase1 in cell lysates was detected by WB.Results:(1)Wh3Δmic1 strain was successfully constructed by CRISPR-Cas9 after PCR and WB analysis.(2)Results of invasion,proliferation and plaque capacity:The invasion abilities of Wh3,Wh6 and Wh3Δmic1 strains were about 70%,20%and 25%,respectively.Compared with Wh3 strains,the invasion abilities of Wh6 and Wh3Δmic1 strains were decreased by about 70%and 65%,respectively,with significant statistical differences(P<0.05).However,there was no significant difference in invasion ability between Wh6 and Wh3Δmic1 strains.There was no significant difference in the proliferation ability of Wh3Δmic1 strain compared with Wh3 strain.The proportion of T.gondii tachyzoids in the two strains was less than 16,equal to 16,and greater than 16,respectively,about 30%,50%,and 20%,respectively.The proliferation ability of the Wh6 strain was significantly decreased(P<0.05),and the proportion of T.gondii tachyzoites in the Wh6 strain was about 60%,30%and 10%,respectively.The number of plaques formed in Vero cells infected with Wh3 strain was about 60,while no obvious plaques were found in Vero cells infected with Wh6 strain.The number of plaques in the Wh3Δmic1 infected group was about 5,and the Wh6 and Wh3Δmic1 strains were more than Wh3 strains.The plaque capacity was significantly decreased(P<0.05).(3)Virulence test in mice:The mice infected with Wh3Δmic1 tachyzoite survived for 9 days,the mice infected with Wh3 tachyzoite survived for 7 days,and the mice infected with Wh6 tachyzoite did not die after 12 days,with a mortality rate of about 50%.The survival time of mice infected with Wh3Δmic1 tachyzoites was 2 days longer than that of mice infected with Wh3 tachyzoites(P<0.05),but the survival time of mice infected with Wh6 tachyzoites was still shorter than that of mice infected with Wh3 tachyzoites.(4)T.gondii loads in all organs of mice infected with the Wh3Δmic1 strain were significantly lower than those of mice infected with the Wh3 strain,but T.gondii loads in the spleen and blood of mice infected with the Wh3Δmic1 strain were significantly higher than those of mice infected with the Wh6 strain.(5)Changes of IL1-β,IL-18,IFN-γ and IL-10 in serum of mice:The expression levels of IL1-β,IL-18,IFN-γ and IL-10 in Wh3Δmic1 infected mice were lower than those in Wh3 infected mice,but the expression levels of IL-1β,IL-18,IFN-γ and IL-10 in Wh6 infected mice were similar.(6)After purification,the hemagglutination inhibition test showed that sialic acid inhibited the hemagglutination of rMIC1 in the concentration range of 12-100mM,and rMIC1 had sialic acid agglutinin activity.(7)By comparing the transcrip tome sequencing of spleen cells from uninfected mice and Wh3-infected mice,it was found that pyroptosis-related genes such as IL-1β,NLRP3 and GSDMD were enriched in the Wh3-infected group,and the Wh3 strain of T.gondii Chinesel had a regulatory effect on pyroptosis of host cells.(8)Expression and activation of NLRP3 signaling pathway related molecules after BMDM infection with different T.gondii strains in vitro:The expression and activation of NLRP3 signaling pathway related molecules were detected by qRT-PCR,ELISA and WB.By qRT-PCR and ELISA,the mRNA and protein expression of IL1β in Wh3Δmic1 infected BMDM were significantly lower than those in Wh3 infected BMDM.The protein expression levels of NLRP3,Pro-Caspasel,Cleaved-Caspase1,GSDMD and NGSDMD in Wh3Δmic1 infected group were significantly lower than those in Wh3 infected group,and there was no significant difference between the Wh6 infected group and the WH6 infected group.The expression levels of Pro-Caspase1 and CleavedCaspase1 in the Wh6 infected group were slightly lower than those in the WH6 infected group.Conclusion:T.gondii Wh3 strain showed stronger invasion and in vivo proliferation ability than Wh3Δmic1 and Wh6 strain.TgMIC1 can effectively activate the NLRP3 signaling pathway in BMDM and participate in the immune regulation of the body.The differential expression of TgMIC1 in Wh3 and Wh6 strains may be involved in the regulation of the virulence of TgMIC1 in mice.