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

SIMPLE SUMMARY: Toxoplasma gondii is a parasite that invades nucleated cells and causes changes in the cellular signal transduction network. This study aimed to perform a comprehensive analysis of the phosphorylated proteome in the small intestine cells of the definitive host following T. gondii infection. We used titanium dioxide affinity chromatography to enrich phosphopeptides in cat small intestinal epithelia infected with T. gondii and then used iTRAQ technology to quantify the phosphopeptides. A total of 4998 phosphopeptides, 3497 phosphorylation sites, and 1805 phosphoproteins were identified. Among the differentially expressed phosphoproteins, 68 were down-regulated and 637 were up-regulated. The bioinformatics analysis revealed that these phosphoproteins were involved in various cellular processes such as actin cytoskeleton reorganization, cell necroptosis, and the MHC immune process. The study confirmed that T. gondii infection leads to extensive changes in the phosphorylation of cat intestinal proteins. This is the first study to report global protein phosphorylation alterations in feline small intestinal epithelia following T. gondii infection. These findings provide a theoretical basis for understanding the interaction between T. gondii and its definitive host, which could have implications for the development of treatments or preventive measures. 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 (TiO(2)) 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.