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

Abstract

The invasion of tachyzoites into the host cell results in extensive host cell signaling activation/deactivation that is usually regulated by the phosphorylation/dephosphorylation. To elucidate how 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 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- 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 . Similar biological process profiles but different molecular function categories of host cells infected with for 2 and 6 hours, which suggested that the host cell processes were not affected significantly by 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.

Host Cell Vimentin Restrains Toxoplasma gondii Invasion and Phosphorylation of Vimentin is Partially Regulated by Interaction with TgROP18

“…Interestingly, host cell vimentin was found to interact with Tg ROP18 in our previous protein interaction screening based on BiFC, and the results of this study verified those findings further using FRET and Co-IP experiments (Figure 6 ). In our previous phosphoproteinomics assays of T. gondii infected HFF cells, host cell vimentin exhibited a complex phosphorylation pattern at different infection times 34 . In this study, a dynamic phosphorylation pattern of vimentin was identified in host cells infected with T. gondii RH and RH Δ rop18 strains using phos-Tag assays, and the results indicated that the phosphorylation pattern of vimentin was partly affected by T. gondii ROP18; moreover, vimentin phosphorylated by Tg ROP18 was also demonstrated by our kinase assay in vitro (Figure 7 ).…”

“…The assembly and solubility of vimentin are regulated by the phosphorylation/dephosphorylation of vimentin at Ser38 and Ser55 induced by Rab7a 33 . Moreover, phosphorylation level of vimentin Ser38 was significantly upregulated in host cell infected with T. gondii at both 2h and 6h comparing to host cell without T. gondii infection 34 . Consistent with the above reports, higher vimentin expression corresponding to much lower solubility in host cell with T. gondii infection for 2h and 6h, which also suggest that vimentin phosphorylation is associated with its solubility.…”

Global Lysine Crotonylation and 2-Hydroxyisobutyrylation in Phenotypically Different Toxoplasma gondii Parasites

“…Qualitative analysis indicated that 1061 and 984 proteins with 3735 and 3396 modified sites in RH and ME49 strain T. gondii , respectively, were crotonylated, and 1950 and 1720 proteins with 9502 and 8092 2-hydroxyisobutyrylation sites, respectively, in the two parasite strains were identified. In addition to the finding that modification of proteins was more prevalent in the RH strain than in the ME49 strain, lysine crotonylation and 2-hydroxyisobutyrylation were the most prominent PTMs in T. gondii except for phosphorylation (11, 12, 49–57) (phosphorylation can occur on diverse amino acids) (supplemental Table S6), suggesting critical roles of crotonylation and 2-hydroxyisobutyrylation in the regulation of various biological processes of the parasites.…”

iTRAQ-Based Phosphoproteomic Analysis of Toxoplasma gondii Tachyzoites Provides Insight Into the Role of Phosphorylation for its Invasion and Egress

“…The dried peptides were reconstituted in a solution containing 65% (v/v) acetonitrile (ACN) and 3.5% (v/v) trifluoroacetic acid (TFA) and then saturated with glutamic acid. Phosphopeptides were enriched using TiO 2 , as previously described ( He et al, 2017a ). Briefly, iTRAQ-labeled peptides were added into the freshly prepared TiO 2 beads (GL Sciences) at peptides-to-beads ratio of 1:4 (mass/mass) and then incubated for 20 min at 37°C with end-over-end rotation.…”