Transcriptional Analysis Of Porcine Macrophages Infected With Different Genotypes Of Toxoplasma Gondii

Toxoplasma gondii is an obligate intracellular parasite that infects almost all warm-blooded animals,it regulates the expression of host genes through its own proteins.Most isolates of Toxoplasma from Europe and North America fall into one of three genetically distinct clonal lineages,the type I,II and III lineages.In recent years,as the economy has grown and people's living standards have continued to increase,the demand for pork has risen sharply.However,swine toxoplasmosis has not only caused major economic losses to the breeding industry,but also caused toxoplasmosis in our country's residents.Due to the lack of basic research on the prevention and control of toxoplasmosis,the molecular mechanism of the interaction between T.gondii and host is not clear.Therefore,many researchers have established animal models for the study of the molecular mechanism of interaction between T.gondii and host.In this study,we used RNA-Seq high-throughput sequencing to study the transcriptome changes of porcine macrophages infected with different genotypes of T.gondii,identified differentially expressed genes and immune pathways after infection with T.gondii,and we found the differences of different genotypes T.gondii,which regulated Type I Interferon and NF-?B Signaling Pathways.BALB/c mice were infected with T.gondii and the cytokine in the spleen were detected by Real-time PCR.Then Western blot was used to study the molecular mechanism of GRA15 regulating NF-?B signaling pathway.The plasmid GRA15 was electroporated into RAW264.7 cells to study its effect on immune factors.(1)Transcriptome analysis of 3D4/21 cells infected with different genotypes of T.gondii tachyzoites3D4/21 cells were infected with RH,HB1 and ME49.RNA-Seq was used to detect the transcriptome changes at 6h,12 h and 24 h time points.It was found that differentially expressed genes increased with the extension of infection time.The differentially expressed genes induced by type II strains were significantly higher than those of type I strains.GO enrichment and KEGG analysis revealed that the three genotypes T.gondii differed in their immune or disease-related pathways involving in macrophages.(2)Effects of different T.gondii strains on mouse spleen immune factorsRH,HB1 and Me49 were infected in BLAB/c mice and the relative expression levels of congenital immune cytokines IFN-?,IFN-?,IFN-?,IL-10 and PD-1 were detected by Real-time PCR.The results showed that in the three genotypes T.gondii infection,the expression of IFN-? and IL-10 was very obvious,and both had high expression levels in the late stage of infection.In contrast,the expression level of PD-1 induced by T.gondii infection was low in all genotypes,and it was less obvious with time.(3)Molecular mechanism of NF-?B activation by GRA15The GRA15 plasmid was transfected into HEK293 T cells and the total amount of I?B and p-I?B was detected by Western blot.It was found that GRA15 can significantly increase the expression levels of p-I?B.After using I?B kinase inhibitor,the expression levers of p-I?B was significantly reduced.The results showed that GRA15 may activate NF-?B by degrading I?B.(4)Effect of GRA15 on RAW 264.7 cytokinesThe electroporation method was used to transfect GRA15 plasmid into RAW 264.7 cells,and the expression levers of cytokine gene was detected by Real-time PCR.The results showed that GRA15(I),GRA15(II)and GRA15(-4)can all increase the expression levers of IFN-? in cells,and the induction effect of GRA15(-4)was stronger;GRA15(I)and GRA15(II)can activate and induce cells to secrete IFN-?;while GRA15(I),GRA15(II)and GRA15(-4)have no significant effect on IL-12,TNF-?,i NOS and PD1.In this study,the transcriptomes of porcine alveolar macrophages after infection with T.gondii were studied and analyzed for the first time,and the expression changes of differentially expressed genes were described.At the same time,functional differentially expressed genes and KEGG pathway analysis at different time points contributed to a better understanding of the host immune and defense mechanisms,and promoted the prevention and control of T.gondii infections,understanding this information may be critical to the development of Toxoplasma therapeutics and vaccines.