Comparative Proteomics Of Different Developmental Stages And Different Strain Sporulated Oocysts,and Comparative Proteomics Of Post-translational Modification Between Different Strain Tachyzoites Of Toxoplasma Gondii

Toxoplasma gondii is an obligate intracellular protozoa which has a complex two-host life-cycle between intermediate host and definitive host.Tachyzoites and cysts are two forms in intermediate hosts and oocysts is the form in definitive host.T.gondii was divided into four major genotypes(Type I,II,III and 12)and Chinese 1(ToxoDB#9)is the dominant genotype in mainland China.Pathogenicity and distribution of different genotypes of T.gondii are quite different.In this study,the quantitative proteomics technology and quantitative proteomics techniques of post-translational modification were used to study the proteomes of different developmental stages and different genotypic sporulated oocysts of T.gondii,and phosphoproteome and acetylome of different genotypic tachyzoites of T.gondii.We utilized the iTRAQ labeling technology to identify differentially expressed proteins(DEPs)specific to tachyzoite(T),bradyzoites-containing cyst(C)and sporulated oocyst(O)stages of the cyst-forming T.gondii Prugniuad(PRU)strain.Some virulence-related factors and ribosomal proteins exhibited distinct expression patterns across the life-cycle stages.The virulence factors expressed in sporulated oocysts and the number of up-regulated virulence factors in the cyst stage were about twice as many as in tachyzoites.Of the 79 ribosomal proteins identified in T.gondii,the number of up-regulated ribosomal proteins was 33 and 46 in sporulated oocysts and cysts,respectively,compared with tachyzoites.These results support the hypothesis that oocyst and cystic stages are able to adapt to adverse environmental conditions and selection pressures induced by the host’s immune response,respectively.We used the iTRAQ approach to study the proteomic differences between oocysts of the virulent PYS(Toxo DB#9)strain vs the less virulent PRU(Type II,Toxo DB #1)strain.Gene Ontology and KEGG pathway analyses revealed a large number of DEPs enriched in various metabolic processes.Protein interaction network analysis using STRING identified inosine monophosphate dehydrogenase(IMPDH),bifunctional GMP synthase/glutamine amidotransferase protein,Glucose-6-phosphate 1-dehydrogenase,and citrate synthase as the top four hubs.Of the 22 virulence proteins commonly expressed in the oocysts of the two strains,13 and 2 proteins were increased in PYS strain and PRU strain,respectively.Also,10 and 3 of the 22 identified oocyst wall proteins showed higher expression in oocysts of the PRU strain and PYS strain,respectively.The quantitative phosphoproteomic approach—iTRAQ technology combined with titanium dioxide(TiO2)affinity chromatography was firstly applied to investigate the phosphoproteomic difference between different genotypic tachyzoites of T.gondii.We obtained 392,298 and 436 differentially expressed phosphoproteins(DEPs)in group RH vs PRU,group PRU vs PYS and group PYS vs RH,respectively.Functional properties of DEPs were clarified by GO analysis,KEGG analysis and STRING analysis.Motif analysis revealed three motifs,three motifs and five motifs abundant in DEPs from group RH vs PRU,group PRU vs PYS and group PYS vs RH.Those differentially expressedmotifs shed light on difference of the kinase categories expressed in tachyzoites of different genotypes of T.gondii and reflect their substrate-recognition properties on a certain extent.The label-free quantitative acetylated proteomic approach was applied to investigate the acetylated proteomic difference between groups of different genotypic tachyzoites of T.gondii.A total of 566 acetylated proteins were obtained and 13,2 and 26 acetylated proteins were differentially expressed in group RH vs PRU,group PRU vs PYS and group PYS vs RH.Compared with PYS strain and PRU strain,histone acetyltransferase and glycyl-t RNA synthase have higher expression level in the RH strain.This may reflects the difference of resistance against external pressure and development between different genotypes of T.gondii.In summary,we firstly studied the comparative proteomes of different developmental stages of the Type II(ToxoDB#1)PRU strain,the comparative proteomes of sporulated oocysts between the Type II(ToxoDB#1)PRU strain and ToxoDB#9 PYS strain,and the phosphoproteomic and acetylated proteomic differences between the Type I(ToxoDB#10)RH strain,Type II(Toxo DB#1)PRU strain and Toxo DB#9 PYS strain.These results revealed the proteomic differences between different developmental stages and different genotypic sporulated oocysts of T.gondii,and features of post-translational modification between different genotypic tachyzoites of T.gondii.These findings will lay foundation for further studies of the biology and genotypic diversity of T.gondii.