| Toxoplasma gondii,a common opportunistic pathogenic protozoan,is a leading cause of illness and mortality among immunocomprimised or defective individuals.It is very regrettable that current therapeutic strategies for toxoplasmosis are not fully effective at curtailing disease progression in these cases.T.gondii has multiple genotypes and the dominant genotype prevailing in China is Chinese I(ToxoDB #9).Although the DNA sequence difference among different T.gondii genotypic strains is only 1-2%,there is a significant difference in acute virulence in mice.However,the mechanism underlying the virulence difference among different T.gondii genotypic strains is not fully clear.Therefore,this study attempted to explore the underlying mechanism of virulence differences among different T.gondii genotypic strains from the genomic and transcriptomic perspectives.Secondly,type II strains are the most widespread in humans and animals.Next we used liquid chromatography-mass spectrometry(LC-MS/MS)-based non-targeted metabolomics approaches to study the metabolic changes in the livers and spleens of T.gondii-infected mice.The main findings are as follows.(1)Different T.gondii genotypic strains(RH,C7,PYS,Pru,VEG;tachyzoites)were cultured on HFF cells,and a large number of T.gondii tachyzoites were harvested and purified.Restriction fragment polymorphism experiments and microscopic observations confirmed that the purification effects of different genotypes of T.gondii were very good.In addition,animal experiments verified that the order of virulence of different genotypic strains was RH> C7> PYS> Pru> VEG.(2)Whole genome sequencing of different T.gondii genotypic strains showed that there were a large number of genetic variations among the examined strains.The total number of SNPs was 443748,14611,290899,287317 and 274787,respectively.The total number of InDels was 37313,23988,28118,18591 and 17524,respectively.The total number of SVs was 1155,860,904,1126 and 1028,respectively.The total number of CNVs was 818,1468,752,2629 and 2240,respectively.From SNP,InDel,SV and CNV numbers of the five T.gondii strains,we drew a conclusion that SNPs and InDels were the main sources of T.gondii genome diversity.The difference between RH and Pru was the most obvious,while the difference between PYS and C7 was the least obvious.There were less SVs and CNVs between RH and VEG.SVs and CNVs of Pru,PYS and C7 were relatively more,indicating a certain degree of similarity.The unique SNP and InDel in different genotypic strains were mainly distributed in exons,introns and intergenic regions.The most variations were in introns,while less in exons.We compared and analyzed the pleomorphism of T.gondii virulence-related genes,and found that different genotypic strains had variations in reported virulence genes,in which the rhoptry neck proteins had the most genetic variations.(3)Using RNA-Seq technology,we completed the transcriptome sequencing of different T.gondii genotypes,and identified a large number of differentially expressed genes among different genotypic strains.The difference in the transcriptional levels of RH and other strains were significant,and the difference in gene expression between RH and PYS was the most significant.In contrast,the differential expression genes between PYS with C7 were minimal,indicating that there was a positive correlation between transcriptional levels with genome levels.The difference in T.gondii virulence-related gene expression between RH and PYS was the most significant,and the difference in gene expression between PYS with C7 was the most non-significant.The functional annotation and pathway analysis of differential genes revealed that many differentially expressed genes are involved in biological processes such as replication,translocation,and amino acid synthesis pathways.These genes may be related to the virulence of T.gondii and can be used as candidate genes for studying the mechanism of the differential formation of T.gondii virulence.(4)First,we successfully constructed a mouse model with distinctly acute and chronic infection phases.Next,metabolomic analysis of liver and spleen of mice infected with T.gondii was performed by LC-MS/MS.We detected a large number of differentially expressed metabolites and perturbed metabolic pathways in the livers and spleens during infection phases.In the livers and spleens of T.gondii-infected mice,the perturbed metabolic pathways mainly included primary bile acid biosynthesis,sphingolipid metabolism,steroid biosynthesis,steroid hormone biosynthesis,arachidonic acid metabolism,etc.Comparing the acutely and chronicially infected phases,we found that arachidonic acid metabolic pathway in the two infection phases showed an opposite expressing trends,which was due to the immunomodulatory and inflammatory response of the host,and was associated with the presence of T.gondii in both infection phases.These findings indicated that T.gondii infection induced changes in the liver and spleen metabolism of mice and provided new insights into the mechanism of metabolic changes during different T.gondii infection phases.In summary,the genomic and transcriptomic differences among different genotypic T.gondii strains were investigated and a large number of gene mutations were identified between different genotypes,for which many variations occur in virulence-related genes.At the transcriptional level,there were also a large number of differentially expressed genes among T.gondii different genotypes.Some of these differentially expressed genes were virulence-related genes,and some were involved in pathways related to virulence regulation.These findings provided a theoretical basis for exploring the underlying mechanism of virulence differences among different genotypic strains.From the metabolomics study of the livers and spleens of T.gondii-infected mice,we identified a large number of differentially expressed metabolites in the infected livers and spleens,and these differential metabolites were involved in many important biological activities.The analysis of metabolic pathways showed that the main pathways of metabolism in T.gondii infection could help us to deepen our understanding of the molecular mechanisms of T.gondii-infected pathophysiology.Meanwhile,it could help us to better understand the molecular changes associated with T.gondii infection and the disease it causes,which is crucial to explore new ways to improve treatment and diagnosis of toxoplasmosis. |
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