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

He, Cheng; Kong, Ling; Zhou, Lijuan; Xia, Jing; Wei, Haixia; Liu, Min; Peng, Hongjuan

Given the interaction between host cell vimentin and TgROP18, and with the aim of exploring the effect of TgROP18 on vimentin, the solubility, phosphorylation, and expression levels of vimentin were determined in Cos7 cells infected with RH Δrop18 and RH WT. Host cell vimentin solubility was significantly different between cells infected with RH WT and RH Δrop18 at 1, 6, 24, 36, and 48h, suggesting that the solubility of vimentin was partly affected by TgROP18, in addition to other unknown parasitic factors ( Figure 7A , B, and C). To further investigate the effects of TgROP18 on vimentin solubility, Cos7 cells were transfected with pcDNA3.1-ROP18-flag. Much higher vimentin solubility was observed in cells over-expressing TgROP18 than in controls ( Figure 8A and B). The phosphorylation level of vimentin in Cos7 cells infected with RH ∆rop18 was also evaluated using Phos-tag assays, and the results indicated that there was a trend towards up-regulation of vimentin with the extension of infection time ( Figure 7D ). Comparison of these results with the changes in vimentin phosphorylation levels in cells infected with RH WT for different periods of time (Figure 3 ), suggested that TgROP18 affects the phosphorylation of vimentin ( Figure 7D ). To further explore the effects of ROP18 on vimentin, kinase assays in vitro were performed to determine whether vimentin was phosphorylated by the TgROP18 kinase. The results suggested TgROP18 could phosphorylate host cell vimentin in vitro ( Figure 7E ). Intriguingly, the changes in vimentin solubility and phosphorylation levels in cells infected with RH WT (Figure 7) were much more complex than those in cells over-expressing ROP18 (Figure 8) , suggesting that the dynamic patterns of vimentin solubility and phosphorylation induced by T. gondii infection were partly regulated by TgROP18, and may contribute to the reorganization of host cell vimentin. Furthermore, compared with the protein levels of vimentin in Cos7 cells infected with RH WT (Figure 3D ), no significant changes were observed in cells infected with RH ∆rop18 (Figure 7F ), indicating that the expression levels of vimentin were not clearly affected byTgROP18. Additionally, over-expression of ROP18 in Cos7 cells did not have obvious effects on host cell vimentin expression ( Figure 8C ). gondii RH/GFP for 30min and host-cell invasion was evaluated by a two-color assay to distinguish intracellular from extracellular parasites. Data are expressed as means ± SD from three independent experiments, each performed in triplicate, and analyzed by t-test (**p<0.01). C. Representative fluorescence images of B. Cell nuclei were stained with DAPI and extracellular parasites were stained with mouse anti-SAG1 antibody (scale bars, 10μm).D. Effect of vimentin on the proliferation of T. gondii in host cells. T. gondii RH/GFP were used to infect HBMEC and HBMEC ΔVim, and invaded parasites were allowed to replicate for 22 h. The number of vacuoles containing one, two, four, or eight parasites was visualized under a fluorescence microscope (100×). Data are expressed as means ± SD combined from three independent experiments, each performed in triplicate and analyzed by two-way ANOVA. No significant difference was identified between the two comparison groups. E. Representative fluorescence images of D (scale bars, 20μm). F. Effect of vimentin on the egress of T. gondii. HBMEC and HBMEC ∆Vim were infected with RH/GFP at a MOI of 3. After infection, T. gondii we