Phosphoproteome of Toxoplasma gondii Infected Host Cells Reveals Specific Cellular Processes Predominating in Different Phases of Infection

Abstract

The invasion of Toxoplasma gondii tachyzoites into the host cell results in extensive host cell signaling activation/deactivation that is usually regulated by the phosphorylation/dephosphorylation. To elucidate how T. gondii regulates host cell signal transduction, the comparative phosphoproteome of stable isotope labeling with amino acids in cell culture-labeled human foreskin fibroblast cells was analyzed. The cells were grouped (Light [L], Medium [M], and Heavy [H] groups) based on the labeling isotope weight and were infected with T. gondii for different lengths of time (L: 0 hour; M: 2 hours; and H: 6 hours). A total of 892 phosphoproteins were identified with 1,872 phosphopeptides and 1,619 phosphorylation sites. The M versus L comparison revealed 694 significantly regulated phosphopeptides (436 upregulated and 258 downregulated). The H versus L comparison revealed 592 significantly regulated phosphopeptides (146 upregulated and 446 downregulated). The H versus M comparison revealed 794 significantly regulated phosphopeptides (149 upregulated and 645 downregulated). At 2 and 6 hours post-T. gondii infection, the most predominant host cell reactions were cell cycle regulation and cytoskeletal reorganization, which might be required for the efficient invasion and multiplication of T. gondii. Similar biological process profiles but different molecular function categories of host cells infected with T. gondii for 2 and 6 hours, which suggested that the host cell processes were not affected significantly by T. gondii infection but emphasized some differences in specific cellular processes at this two time points. Western blotting verification of some significantly regulated phosphoprotein phosphorylation sites was consistent with the mass spectra data. This study provided new insights into and further understanding of pathogen-host interactions from the host cell perspective.

Figure 1.

Processes used to generate phosphoproteomic data for human foreskin fibroblasts (HFFs) infected with Toxoplasma gondii RH tachyzoites. HFF cells were grown in Dulbecco's modified Eagle's medium supplemented with one of the three different forms of lysine and arginine to label almost all the phosphoproteins with the stable N and C isotopes via the incorporation of the labeled lysine or arginine. The H, M, and L groups were infected with T. gondii at a multiplicity of infection of 10 for 6, 2, and 0 hours (uninfected), respectively. The cells were harvested and lysed, followed by alkylation with Iiodoacetamide and trypsin digestion. The phosphopeptides were separated with strong cation-exchange chromatography and enriched with a TiO₂ column. Then spectrometric analysis was performed. This figure appears in color at www.ajtmh.org.

Figure 2.

Large-scale mass spectra of the phosphopeptides. (A) Identification of total proteins, phosphoproteins, total peptides, phosphopeptides, and phosphosites. A total of 1,375 proteins were identified, among which 892 phosphoproteins and 1,619 phosphorylation sites were found. (B) Distribution of the pS/pT/pY phosphoproteome. Phospho-S was the most abundant and accounted for 89.07% of all the phosphorylated amino acids, followed by phospho-T (9.64%) and phospho-Y (1.30%). This figure appears in color at www.ajtmh.org.

Figure 3.

Gene ontology (GO) enrichment analysis of the 892 identified phosphoproteins. GO analysis of the 892 identified phosphoproteins revealed that the highest proportion were categorized as “binding” in the molecular function, “cell part” and “cell” in the cellular component, and “cellular process” in the biological process. This figure appears in color at www.ajtmh.org.

Figure 4.

Verification of the phosphorylation level change of the host cell phosphoproteins induced by Toxoplasma gondii infection. Human foreskin fibroblasts cell total proteins from the three groups of T. gondii infection for 0, 2, and 6 hours (indicated as uninfected, infected for 2 hours, infected for 6 hours) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and analyzed by Western blotting with antibodies against beta-actin, Phospho-Bad-S118, Phospho-MARCKS-S170, Phospho-Vimentin-S56, and Phospho-Vimentin-S42. Beta-actin was detected as loading control. From the Western blotting result, the trend change of the phosphorylation level of different host cell phosphoprotein phosphorylation sites induced by T. gondii infection at different time points was shown.