Glycosylation Analysis of Feline Small Intestine Following Toxoplasma gondii Infection- raw datas 1
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 PRU strain that were collected from the infected mice. The cat's intestinal epithelial tissue was harvested at 10 days post-infection (DPI), and then sent for protein glycosylation analysis. High-performance liquid and mass spectrometry were used 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 in the feline small intestine infected by T. gondii. Additionally, we also identified 8 N-glycosylated proteins of T. gondii, including 8 N-glycopeptides and 8 N-glycosylation sites. The protein A0A086JND6_TOXGO (eEF2) and its corresponding peptide sequence were identified in T. gondii infection. During the process of glycosylation of T. gondii oocysts, some special GO terms (cellular process and metabolic process, cell and cell part, catalytic activity) were significantly enriched, and the COG function prediction results showed that posttranslational modification, protein turnover, and chaperones (11%) had the highest enrichment for T. gondii. Interestingly, eEF2 is also involved in the significantly enriched T. gondii MAPK pathway. Protein–protein interaction networks were analyzed by MCODE_Cluster of Cytoscaype for the differentially expressed peptides/proteins. 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 and eEF2 is involved, which provides a new target for anti-T. gondii therapy to prevent the spread of T. gondii oocysts in the future.
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The extracted peptide samples were redissolved in mobile phase A (2% ACN, 0.1% FA) and centrifuged at 20,000 g for 10 min, and then the supernatant was used for gradient separation by UHPLC (UltiMate 3,000, Thermo). The separated peptides were ionized by the nanoESI source and transferred to the tandem mass spectrometer Q-Exactive HF (Thermo Fisher Scientific, San Jose, CA) for DDA (data-dependent acquisition) mode detection. Main parameter settings: MS1 scan range is 350~1500 m/z, and the resolution is set to 120,000. The MS2 starting at 100 m/z, the resolution was 30,000. The ion fragmentation mode is HCD. The FFPE sample data generated by the high-resolution mass spectrometer were identified by MaxQuant (Andromeda, v.188.8.131.52), fixed modifications was carbamidomethyl (C), and variable modifications were Oxidation (M), Acetyl (Protein N-term), and Asn->Asp (N). Complete the filtration with PSM level FDR < =1% at the spectrum level, and filter with protein level FDR < =1% at the protein level. At the same time, filter with 1% FDR for the modified sites, so as to obtain significant modification results. MaxQuant was used to perform quantitative analysis based on the peak strength, peak area, retention time and other information about the peptide segment (peptide mass tolerance 20 ppm, fragment mass tolerance 0.05 Da) related to the primary mass spectrometry, extracting the peptide segment related to the target modification, and conducting a series of statistical analyses and quality controls. Welch’s t-test (p value) was used to test the significance, and the modification quantification was screened according to fold change ≥ 2 and p value ≤ 0.05 as the criterion of significant difference.