Glycosylation Analysis of Feline Small Intestine Following Toxoplasma gondii Infection

Simple Summary Toxoplasma gondii has a serious impact on public health and the economic development of animal husbandry. Glycosylation, especially N-glycosylation, the pattern modification of proteins, is closely related to the biological functions of proteins, and our study used it to analyze glycosylation alterations in the small intestine of cats infected with T. gondii. The results of the present study showed that 56 glycosylated peptides were upregulated and 37 glycosylated peptides were downregulated. Additionally, we also identified eight N-glycosylated proteins of T. gondii including eight N-glycopeptides and eight N-glycosylation sites. Moreover, the protein eEF2 and its corresponding peptide sequence were identified, with GO terms (i.e., cellular process and metabolic process, cell and cell part, and catalytic activity) that were significantly enriched in the T. gondii MAPK pathway. In addition, the Clusters of Orthologous Groups of proteins (COG) function prediction results showed that posttranslational modification, protein turnover, and chaperones (11%) had the highest enrichment for T. gondii. The host proteins ICAM-1 and PPT1 and the endoplasmic reticulum stress pathway may play an important role in the glycosylation of T. gondii-infected hosts. Our study may provide a new target for T. gondii detection to prevent the spread of T. gondii oocysts in the future. Toxoplasma gondii (T. gondii) is responsible for severe human and livestock diseases, huge economic losses, and adversely affects the health of the public and the development of animal husbandry. Glycosylation is a common posttranslational modification of proteins in eukaryotes, and N-glycosylation is closely related to the biological functions of proteins. However, glycosylation alterations in the feline small intestine following T. gondii infection have not been reported. In this study, the experimental group was intragastrically challenged with 600 brain cysts of the Prugniuad (Pru) strain that were collected from infected mice. The cats' intestinal epithelial tissues were harvested at 10 days post-infection and then sent for protein glycosylation analysis. High-performance liquid chromatography coupled to tandem mass spectrometry was used to analyze the glycosylation alterations in the small intestine of cats infected with T. gondii. The results of the present study showed that 56 glycosylated peptides were upregulated and 37 glycosylated peptides were downregulated in the feline small intestine infected by T. gondii. Additionally, we also identified eight N-glycosylated proteins of T. gondii including eight N-glycopeptides and eight N-glycosylation sites. The protein A0A086JND6_TOXGO (eEF2) and its corresponding peptide sequence were identified in T. gondii infection. Some special GO terms (i.e., cellular process and metabolic process, cell and cell part, and catalytic activity) were significantly enriched, and the Clusters of Orthologous Groups of proteins (COG) function prediction results showed that posttranslational modification, protein turnover, and chaperones (11%) had the highest enrichment for T. gondii. Interestingly, eEF2, a protein of T. gondii, is also involved in the significantly enriched T. gondii MAPK pathway. The host proteins ICAM-1 and PPT1 and the endoplasmic reticulum stress pathway may play an important role in the glycosylation of Toxoplasma-infected hosts. This is the first report showing that T. gondii oocysts can undergo N-glycosylation in the definitive host and that eEF2 is involved, which may provide a new target for T. gondii detection to prevent the spread of T. gondii oocysts in the future.