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. 2022 Feb 2;27(3):1022.
doi: 10.3390/molecules27031022.

Comparative Phosphoproteomic Analysis of Sporulated Oocysts and Tachyzoites of Toxoplasma gondii Reveals Stage-Specific Patterns

Ze-Xiang Wang  1 , Liang Che  1 , Rui-Si Hu  2 , Xiao-Lin Sun  1
Affiliations

Comparative Phosphoproteomic Analysis of Sporulated Oocysts and Tachyzoites of Toxoplasma gondii Reveals Stage-Specific Patterns

Ze-Xiang Wang et al. Molecules. .

Abstract

Toxoplasma gondii is an obligate intracellular protozoan of severe threat to humans and livestock, whose life history harbors both gamic and apogamic stages. Chinese 1 (ToxoDB#9) was a preponderant genotype epidemic in food-derived animals and humans in China, with a different pathogenesis from the strains from the other nations of the world. Posttranslational modifications (PTMs) of proteins were critical mediators of the biology, developmental transforms, and pathogenesis of protozoan parasites. The phosphoprotein profiling and the difference between the developmental phases of T. gondii, contributing to development and infectivity, remain unknown. A quantitative phosphoproteomic approach using IBT integrated with TiO2 affinity chromatography was applied to identify and analyze the difference in the phosphoproteomes between the sporulated oocysts and the tachyzoites of the virulent ToxoDB#9 (PYS) strain of T. gondii. A total of 4058 differential phosphopeptides, consisting of 2597 upregulated and 1461 downregulated phosphopeptides, were characterized between sporulated the oocysts and tachyzoites. Twenty-one motifs extracted from the upregulated phosphopeptides contained 19 serine motifs and 2 threonine motifs (GxxTP and TP), whereas 16 motifs identified from downregulated phosphopeptides included 13 serine motifs and 3 threonine motifs (KxxT, RxxT, and TP). Beyond the traditional kinases, some infrequent classes of kinases, including Ab1, EGFR, INSR, Jak, Src and Syk, were found to be corresponding to motifs from the upregulated and downregulated phosphopeptides. Remarkable functional properties of the differentially expressed phosphoproteins were discovered by GO analysis, KEGG pathway analysis, and STRING analysis. S8GFS8 (DNMT1-RFD domain-containing protein) and S8F5G5 (Histone kinase SNF1) were the two most connected peptides in the kinase-associated network. Out of these, phosphorylated modifications in histone kinase SNF1 have functioned in mitosis and interphase of T. gondii, as well as in the regulation of gene expression relevant to differentiation. Our study discovered a remarkable difference in the abundance of phosphopeptides between the sporulated oocysts and tachyzoites of the virulent ToxoDB#9 (PYS) strain of T. gondii, which may provide a new resource for understanding stage-specific differences in PTMs and may enhance the illustration of the regulatory mechanisms contributing to the development and infectivity of T. gondii.

Keywords: IBT; Toxoplasma gondii; life cycle; phosphoproteomics.

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Conflict of interest statement

The authors confirm that the research was performed without any mercantile or monetary relations and that no potential controversy exists in the research.

Figures

Figure 1
Figure 1
Large-scale information regarding the phosphoproteomic research between sporulated oocysts and tachyzoites of the T. gondii PYS strain. (A) The distribution of phosphopeptides on different peptide lengths. (B) The proportion of mono-phosphosites and multi-phosphosites among total phosphopeptides showed that a large part of the phosphopeptides have single phosphosites. (C) Proportion of the pSer/pThr/pTyr phosphoproteome. Phospho-pSer, phospho-pThr, and phospho-pTyr accounted for 86.27% of all phosphosites, 9.28% of all phosphosites, and 0.42% of all phosphosites, respectively. (D) Distribution of phosphosites on phosphoproteins. Most phosphoproteins included single phosphosites.
Figure 2
Figure 2
Repeatability analysis of the phosphopeptide quantification founded on the value of CV (coefficient of variation). The x-axis represents the percentage values of CV. The quantities of phosphopeptides and the accumulative proportion of phosphopeptides are indicated by the left and right y-axis, respectively.
Figure 3
Figure 3
Quantitative analysis of characterized phosphopeptides. (A) Scatter plot of quantified phosphopeptides. The log2 fold change value and −log10 p value of phosphopeptides are indicated by the x-axis and the y-axis, respectively. The red circles and green circles denote upregulated and downregulated DEPs, respectively. (B) Distribution of upregulated DEPs and downregulated DEPs between the sporulated oocysts and tachyzoites on different levels. The x-axis and y-axis indicate the values of changes and the quantity of DEPs. The red color and green color represent upregulated DEPs and downregulated DEPs, respectively.
Figure 4
Figure 4
Hierarchical clustering analysis of DEPs. Log1.5 fold change value of DEPs is denoted by different colors.
Figure 5
Figure 5
Phosphorylation site motif analysis of the upregulated DEPs using Motif-X. The degree of amino acids emerging at specific locations is represented by the elevation of the symbols.
Figure 6
Figure 6
Phosphorylation site motif analysis of the downregulated DEPs using Motif-X. The degree of amino acids emerging at specific locations is represented by the elevation of the symbols.
Figure 7
Figure 7
GO enrichment analysis of DEPs. (A) GO enrichment analysis of upregulated DEPs. (B) GO enrichment analysis of downregulated DEPs. The number of DEPs and GO terms are displayed on the horizontal axis and vertical axis, respectively.
Figure 8
Figure 8
KEGG pathway enrichment analysis of DEPs between sporulated oocysts and tachyzoites. The y-axis indicates the KEGG pathways DEPs significantly enriched and the x-axis denotes the rich factors of the pathways. Rich factor implies the proportion of DEPs among total phosphoproteins in the pathway. Greater degrees of enrichment are indicated by higher rich factors. The color and size of the node represent the p value and quantity of phosphoproteins of the corresponding pathways.
Figure 9
Figure 9
Protein–protein interaction (PPI, combined score ≥ 0.9) network of the DEPs between sporulated oocysts and tachyzoites. The nodes indicate DEPs and the lines between the nodes refer to the interactors between DEPs. The color of the edge symbolizes the combined score of the interacting partners.
Figure 10
Figure 10
Network of kinase-phosphopeptide partners and potential phosphor- reliant interactions determined by correlation analysis. Light red nodes denoted phosphoproteins and edges represented interactors between phosphoproteins. The gray arrow denotes protein interactions which are presumed to be activated by the phosphorylation and green arrows represents protein interactions that are presumed to be inactivated by the phosphorylation.
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