Host Cell Vimentin Restrains Toxoplasma gondii Invasion and Phosphorylation of Vimentin is Partially Regulated by Interaction with Tg ROP18

Abstract

The obligate intracellular parasite, Toxoplasma gondii, manipulates the cytoskeleton of its host cells to facilitate infection. A significant rearrangement of host cell vimentin around Toxoplasma parasitophorous vacuoles is observed during the course of infection. ROP18 (TgROP18) is a serine-threonine kinase secreted by T. gondii rhoptry and a major virulence factor; however, the mechanisms by which this kinase modulates host factors remain poorly understood. Different and dynamic patterns of vimentin solubility, phosphorylation, and expression levels were observed in host cells infected with T. gondii strain RH and RH Δrop18 strains, suggesting that TgROP18 contributes to the regulation of these dynamic patterns. Additionally, host cell vimentin was demonstrated to interact with and be phosphorylated by TgROP18. A significant increase in T. gondii infection rate was observed in vimentin knockout human brain microvessel endothelial cells (HBMEC), while vimentin knockout or knock down in host cells had no impact on parasite proliferation and egress. These results indicate that host cell vimentin can inhibit T. gondii invasion. Interestingly, western blotting of different mouse tissues indicated that the lowest vimentin expression level was present in the brain, which may explain the mechanism underlying the nervous system tropism of T. gondii, and the phenomenon of huge cyst burdens developing in the mouse brain during chronic infection.

Keywords: Phosphorylation; Reorganization; TgROP18; Toxoplasma gondii; Tropism.; Vimentin.

Figure 1

CRISPR/Cas9-mediated gene disruption and tagging of the rop18 locus. A. Schematic of the CRISPR/CAS9 strategy used to inactivate rop18 by insertion of pyrimethamine-resistant DHFR (DHFR*). B. Verification PCR demonstrating homologous integration and gene disruption in a representative clone, compared with RH parental line tachyzoites. C. Schematic illustration of the CRISPR/CAS9 strategy used to tag endogenous ROP18 at the C-terminus with eGFP-FLAG. D. Verification PCR showing correct integration of eGFP-FLAG, SAG1 3′-UTR, and DHFR*. E. IF revealing that eGFP was successfully fused to the C-terminus of endogenous ROP18. F. Demonstration that FLAG tagged ROP18 was expressed by WB with rabbit anti-DDDDK antibody.

Figure 2

Vimentin rearrangement in Cos7 cells infected with T. gondii for different periods of time. Cos7 cells were infected with T. gondii RH strain tachyzoites for different periods of time (as indicated), and then fixed with paraformaldehyde and permeabilized with Triton X-100. Red, host cell vimentin labeled using mAb anti-vimentin; green, T. gondii tachyzoites labeled with rabbit anti-ROP2.Cell nuclei were stained with DAPI. The rearrangement of vimentin around T. gondii PVs was observed at different time points after infection (arrowheads).

Figure 3

Dynamics of vimentin characteristics during infection with T. gondii RH. A. Changes in vimentin solubility in Cos7 cells infected with T. gondii for different periods of time. B. Proportion of soluble and insoluble vimentin in control and T. gondii infected cells detected by WB. The percentage of soluble vimentin was determined by analysis of grey scale intensity using Image J software (n=3, *p<0.05 by t-test). The solubility of vimentin was significantly decreased during the first 6h post T. gondii infection. C. Cos7 cells were infected with T. gondii at an MOI of 3 or uninfected (Ctrl). Phosphorylation of host cell vimentin in the different groups was analyzed by a Phos-tag assay (top panel). As vimentin expression level changed with time post T. gondii infection, the amount of cell lysate protein loaded for SDS-PAGE was adjusted to maintain a consistent level of total vimentin protein. Equal loading of vimentin was confirmed by WB of the gel (bottom panel). Bands corresponding to phosphorylated and non-phosphorylated vimentin are indicated by black and white arrowheads, respectively. Similar results were obtained in another two separate experiments. Host cell vimentin phosphorylation peaked at 6h post-infection. D. Expression levels of vimentin in Cos7 cells infected with T. gondii (as described above) were detected by WB. Dynamic expression levels were observed after infection with T. gondii for different periods of time. These results suggest that the solubility, phosphorylation, and expression levels of vimentin were regulated by T. gondii infection, and that the higher phosphorylation level of vimentin may correlate with a decrease in its solubility. Statistical differences were evaluated by t-test, means ± SD combined from three independent experiments.

Figure 4

Function analysis of host cell vimentin on the infection of T. gondii. A. Verification of vimentin knockout in HBMEC by WB. B. Effect of host cell vimentin on the invasion of T. gondii into host cells. HBMEC and HBMEC ΔVim cells were infected with T. 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 were cultured in cells for 30h and then induced to egress using A23187; DMSO was used as a control. Data are expressed as means ± SD combined from three independent experiments, each performed in triplicate, and were analyzed by t-test. G. Representative fluorescence images of F (scale bars, 10μm). White arrows indicate the non-released PVs, and brown arrows indicate the egressed tachyzoites from one PV.

Figure 5

Differences in vimentin mRNA and protein expression in various mouse tissues. A. Transcriptional levels of vimentin were detected by qRT-PCR. Data are expressed as means ± SD combined from three independent experiments and were analyzed by t-test. **p≤0.01, ***p≤0.001. B. WB was performed to detect the expression of vimentin in different mouse organs. Beta-actin was used as a loading control. The lowest transcription and expression levels were observed in mouse brain, relative to the other organs.

Figure 6

Identification of the interaction between host cell vimentin andTgROP18. A. FRET was used to investigate the interaction between vimentin and ROP18. Image acquisition and parameter calculation were performed using the sensitized emission (SE) method. B. The calculated efficiency of FRET was approximately 55% (t-test, **p<0.01). C. Interaction of vimentin with ROP18 was further demonstrated by immunoprecipitation assay. HFF cells were infected with T. gondii RH-ROP18-eGFP-FLAG at a MOI of 5 for 36h and then lysed for immunoprecipitation with mAbs anti-vimentin and anti-FLAG. Starting fractions (Input) and immunoprecipitates (IP) were analyzed by WB using Rbs anti-vimentin and anti-DDDDK.

Figure 7

Dynamic patterns of vimentin solubility and phosphorylation were partially affected by TgROP18. A, B. Cos7 cells were infected with T. gondii RH Δrop18. The solubility of vimentin was then detected and analyzed by WB. C. Comparison analysis of vimentin solubility in Cos7 cells infected with T. gondii RH and RH Δrop18. (t test, *p<0.05) D. Analysis of vimentin phosphorylation in Cos7 cells infected with RH Δrop18. E. TgROP18 phosphorylates host cell vimentin. Recombinant GST-ROP18 and vimentin proteins expressed and purified from E. coli were co-incubated in the presence of unlabeled ATP in vitro. Total vimentin and phosphorylated vimentin were detected with rabbit anti-vimentin and anti-phospho Ser/Thr antibodies, respectively. The phosphorylated vimentin band was only visible in the presence of TgROP18. F. Analysis of vimentin expression levels in host cells infected with RH Δrop18.

Figure 8

Effect of overexpression of TgROP18 on Cos7 cell vimentin solubility. A. Verification of over-expression of ROP18 in Cos7 cells and determination of the percentage of soluble vimentin, relative to total vimentin, based on WB signals. B. Analysis of the percentage of soluble vimentin, relative to total vimentin, using Image J software. Data are expressed as means ± SD combined from three independent experiments and were analyzed by t-test. ***p≤0.001. Much higher vimentin solubility was observed in cells overexpressingTgROP18. C. Detection of vimentin expression in Cos7 cells overexpressing TgROP18 (transfected with pcDNA3.1-ROP18) and controls (untransfected cells (ctrl) and cells transfected with pcDNA3.1). The results indicate that the expression of vimentin was not affected by TgROP18 overexpression.