iTRAQ-Based Phosphoproteomic Analysis Exposes Molecular Changes in the Small Intestinal Epithelia of Cats after Toxoplasma gondii Infection

Abstract

Toxoplasma gondii, an obligate intracellular parasite, has the ability to invade and proliferate within most nucleated cells. The invasion and destruction of host cells by T. gondii lead to significant changes in the cellular signal transduction network. One important post-translational modification (PTM) of proteins is phosphorylation/dephosphorylation, which plays a crucial role in cell signal transmission. In this study, we aimed to investigate how T. gondii regulates signal transduction in definitive host cells. We employed titanium dioxide (TiO₂) affinity chromatography to enrich phosphopeptides in the small intestinal epithelia of cats at 10 days post-infection with the T. gondii Prugniuad (Pru) strain and quantified them using iTRAQ technology. A total of 4998 phosphopeptides, 3497 phosphorylation sites, and 1805 phosphoproteins were identified. Among the 705 differentially expressed phosphoproteins (DEPs), 68 were down-regulated and 637 were up-regulated. The bioinformatics analysis revealed that the DE phosphoproteins were involved in various cellular processes, including actin cytoskeleton reorganization, cell necroptosis, and MHC immune processes. Our findings confirm that T. gondii infection leads to extensive changes in the phosphorylation of proteins in the cat intestinal epithelial cells. The results of this study provide a theoretical foundation for understanding the interaction between T. gondii and its definitive host.

Keywords: Toxoplasma gondii; cat; phosphoproteomics; post-translational modification.

Figure 1

The results of PCR for the samples. Identification of T. gondii infected cat small intestine samples at 10 DPI by T. gondii B1 gene. The order of the sample holes is M: Takara DNA marker (500 bp); E-group (lanes 2–4, 10 DPI_1, 10 DPI_2, 10 DPI_3), C-group (lanes 5–7, C_1, C_2, C_3), and P: T. gondii Pru strain PCR positive control; N: PCR negative control, E-group: experimental group, C-group: control group.

Figure 2

The properties of phosphopeptides of cat small intestine cells infected by Toxoplasma gondii. (A) The length distribution of phosphorylated peptides. (B) Polyphosphorylation distribution of phosphorylated peptides. (C) Distribution of phosphorylation on serine (pSer), threonine (pThr), and tyrosine (pTyr) for all phosphorylation sites. (D) Distribution of 1805 phosphoproteins based on identification of single or multiple phosphosites per protein.

Figure 3

The repeatability evaluation of phosphopeptide quantification based on the coefficient of variation (CV). The percentage on x-axis represents the values of CV. The left y-axis indicates the number of phosphopeptides and the right y-axis indicates the cumulative percentage of phosphopeptides.

Figure 4

Volcano map of the differential phosphoproteins. Significant DEPs are shown as a red (up) or green (down) dot. No significant difference between the expressions of phosphoproteins is shown as a brown dot.

Figure 5

Functional classification of phosphoproteins in feline intestine infected by Toxoplasma gondii. (A) Functional annotation of the DEPs based on COG. (B) GO functional classification of the up-regulated phosphoproteins based on gene ontology. (C) GO functional classification of the down-regulated phosphoproteins based on gene ontology.

Figure 6

GO enrichment analysis and KEGG pathway analysis of the differentially expressed phosphoproteins of feline small intestine infected with Toxoplasma gondii. (A) The 30 most enriched GO terms belonging to biological process, molecular function, and cellular component are shown. (B) The pathway obtained from KEGG pathway enrichment analysis. Rich factor represents the ratio of the DEP’s number and the number of proteins annotated in the pathway. The higher the rich factor is, the higher the degree of enrichment.

Figure 7

Protein–protein interaction (PPI) networks for the differential phosphoproteins. Protein interaction network of the differential phosphoproteins of feline small intestine. Nodes represent the phosphoproteins and edges represent interactors between phosphoproteins. Protein names are in red and green fonts, red indicates upregulation, and green indicates downregulation. The dotted circles represent six MCODE clusters (MCODE clusters 1: Ribosome biogenesis, MCODE clusters 2: spliceosome, MCODE clusters 3: Senine/threonine-protein kinase, MCODE clusters 4: Vial carcinogenesis, MCODE clusters 5: RNA transport, and MCODE clusters 6: Antigen processing and presentation).