Glycosylation Analysis of Feline Small Intestine Following Toxoplasma gondii Infection

Abstract

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.

Keywords: N-glycosylation; Toxoplasma gondii; glycosylation; oocysts.

Figure 1

Differential quantitative analysis of the modified peptides/proteins. (A) The mass distribution of the modified proteins. The x-axis represents the protein amount interval (kilodalton), and the y-axis is the number of modified proteins. (B) The unique peptide distribution of the target-modified proteins. The x-axis represents the number of unique matching peptides per protein, and the y-axis represents the number of modified proteins. (C) The motif distribution of the posttranslational modification sites of all the modified peptides. The x-axis represents the base number, and the y-axis represents the corrected score. The higher the base height, the higher the probability of the base appearing in the motif.

Figure 2

Volcano of the up/downregulated differentially modified peptides. Red indicates upregulation, blue indicates downregulation, and gray indicates no significant difference. The first two significantly enriched upregulated and downregulated modified peptides are marked with horizontal lines. The Toxoplasma gondii-modified proteins A0A086JND6_TOXGO (putative translation elongation factor 2 family) and A0A086L1S1_TOXGO (condensin complex subunit 1) were not significantly different.

Figure 3

GO term distribution of the proteins sequence corresponding to the modified peptides in the small intestine of cats. Red indicates upregulation, and green indicates downregulation. The bar graph shows the number of modified peptides enriched in GO terms belonging to the three GO categories, biological process (BP), cellular component (CC), and molecular function (MF), at 10 DPI. The x-axis represents the GO terms, and the y-axis represents the number of upregulated and downregulated modified peptides in the different GO terms. The five-pointed star (★) represents that some Toxoplasma-related modified peptides were also involved in the host’s GO pathway. One five-pointed star (★) indicates that less than 4 were involved, and two five-pointed stars (★★) indicate that more than 7 peptides were involved.

Figure 4

COG term distribution of the modified peptides for feline small intestine. The x-axis is the number of proteins corresponding to functional categories, and the y-axis is the COG category entries. The graph represents the statistical number of proteins with different functions in the sample. In the COG classification of the 24 host-related modified peptides, 8 (O, G, S, T, P, P, J, L, C)-related modified peptides of T. gondii were also involved.

Figure 5

KEGG pathway annotation for the small intestine of cats. The pathways that were significantly enriched for proteins corresponding to the differentially modified peptides. The x-axis represents the number of differentially modified peptides in the corresponding KEGG pathways in each KEGG subsystem. The y-axis represents the main clusters of the KEGG pathways.

Figure 6

KEGG pathway enrichment of the host. The top 30 enriched KEGG pathways of the identified modified peptides in this study. The y-axis represents the distinct KEGG pathways, and the x-axis represents the Rich factor. The Rich factor refers to the ratio of modified peptides annotated in the pathway to the total number of modified peptides annotated in the pathway. The greater the Rich factor, the greater the degree of pathway enrichment. The dot size represents the number of modified peptides (bigger dots denote a large number of modified peptides and vice versa). The colors of the dots represent the p-values of enrichment: red indicates high enrichment, blue indicates low enrichment.

Figure 7

Protein–protein interaction (PPI) networks for the differentially modified peptides/proteins of the host. Red indicates upregulation, and green indicates downregulation. The filled circle represents protein, and the direction of the arrow indicates regulation. The dotted circles represent three MCODE clusters (MCODE clusters 1: viral myocarditis, MCODE clusters 2: fatty acid elongation, and MCODE clusters 3: protein processing in endoplasmic reticulum).