2.1 Cell culture and T. gondii propagation
Human foreskin fibroblasts (HFF), African green monkey kidney cells transformed by SV40 (COS-7), human embryonic kidney 293T (HEK 293T) cells, and human keratinocytes (HaCaT) cells were bought from American Type Culture Collection (ATCC, USA), and cultured in Dulbecco’s Modified Eagles Medium (DMEM, Gibco/Invitrogen, USA) containing 10% (v/v) FBS (Gibco/Invitrogen, USA) and 1% gentamicin (10mg/ml, Invitrogen, USA) at 5% CO2 and 37°C. For fluorescence resonance energy transfer (FRET) experiments, COS-7 cells were grown on coverslips in 12-well plates (Corning, USA). For pSTAT3 detection, HaCaT or COS-7 cells were seeded on 6-well plates and grown to 100% confluence, and then cultured in serum-free medium overnight before treatment with IL-20 (R&D Systems), recombinant GST-ROP18 , or T. gondii infection.
The tachyzoites of CEP, CEP-TgROP18I, and CEP-TgROP18II were a generous gift from Jon Boyle at the University of Pittsburgh. The tachyzoites of T. gondii RH strain, rop16 knockout strain (RH-∆rop16), rop18 knockout strain (RH-∆rop18), and rop16 + rop18 double knockout strain (RH-∆rop16∆rop18) were propagated in HFFs with DMEM supplemented with 1% FBS, as well as the CEP and its mutant strains. Before experiments, the tachyzoites cultured in HFFs were purified by passing through a 3 µm filter after the host cells were ruptured or syringed broken, and then counted.
2.2 Mammalian eukaryotic expression
The cDNA of IL20RB on pDONR was generously gifted by Prof. Wen-Bin Ma at the School of Life Science, Sun Yat-Sen University. The cDNA of IL20RB was amplified using PCR with Pfu DNA polymerase (TransGen Biotech, China), to add the HA tag on its C-terminus and incorporate SalI and SacII restriction sites on the 5’- and 3’- ends, respectively. To perform FRET and co-immunoprecipitation (Co-IP) assays, IL20RB-HA, and its extracellular and intracellular domain was then cloned into pEYFP-C1 to form pEYFPC1-IL20RB-HA, pEYFPC1-IL20RB-Extr-HA (Extr, extracellular domain of IL20RB), and pEYFPC1-IL20RB-Cyt-HA (Cyt, intracellular domain of IL20RB). The construction of pCFPN1-ROP18-3×FLAG has been described in detail elsewhere (16). All the primers used are shown in Supplementary Data (Table S1).
2.3 CRISPR-CAS9 mediated generation of RH-∆rop16∆rop18 double knockout strain
RH-Δrop16 was constructed in our previous research. To disrupt the rop18 gene in the RH-Δrop16 strain, a recombinant CRISPR plasmid, pSAG1::CAS9-U6::sgROP18, which contains an sgRNA specifically targeting the site very close to and downstream from the start codon of rop18 gene, was constructed, using a Q5® Site-Directed mutagenesis kit (NEB, USA) following the published protocol(36). A chloramphenicol-resistance gene (chloramphenicol acetyltransferase, CAT) was used as the screening marker. The homologous recombination plasmid (pBlue-5’-3’-ROP18-homo-CAT) was constructed as follows (the procedure is also shown in Figure S1A); A 991 bp fragment and a 965 bp fragment homologous to the upstream and downstream of the gRNA targeted site of the rop18 gene, were respectively subcloned to the 5`- and 3`- end of the CAT gene cassette on pBlue-script SK II (-) plasmid (16). Subsequently, these two recombinant plasmids were co-transfected into RH-Δrop16 tachyzoites by electroporation. The transfected tachyzoites were used to infect HFF cells, and cultured in chloramphenicol free DEMEM complete medium for 48 h. To screen the recombinants, the tachyzoites were cultured in the DEMEM complete medium containing 20 mmol/L of chloramphenicol for 4 passages. Finally, the double-knockout RH-Δrop16Δrop18 tachyzoites were screened through monoclonal screening, and identified by PCR. All the PCR primers used for homologous template amplification, plasmid construction, and recombinant identification are shown in Table S1.
2.4 Detection of STAT3 activation in different T. gondii tachyzoites infected or IL-20 treated HaCaT cells
2.4.1 Cell preparation
- HaCaT cell infection with RH-WT, RH-Δrop16, RH-Δrop18, and RH-Δrop16Δrop18 strains: HaCaT cells were cultured in 6-well plates to 100% confluence. After the cells were washed 3 times with PBS and switched to serum-free medium and starved overnight, RH-WT, RH-Δrop16, RH-Δrop18, and RH-Δrop16Δrop18 strains were used to infect the cells by one well for each, at multiplicity of infection (MOI) 10 for 30min; another well was not infected for control.
- HaCaT cell treatment with IL20, or infection with RH-WT and RH-Δrop18 strains: HaCaT cells were cultured in 6-well plates to 100% confluence. After the cells were starved overnight, two wells of cells were treated with 400 ng of IL-20 for 30 min and 24 h, respectively; two wells were infected with RH-WT at MOI 10 for 30min and 24h, respectively, and two wells were infected with RH -Δrop18 under the same conditions; another well was not infected for control.
- HaCaT cell treatment with recombinant GST-ROP18 and GST at different amount: The construction of the prokaryotic expression vector and expression of GST-ROP18 and GST have been described in detail previously(16). The Escherichia coli expressed GST-ROP18 and GST proteins were purified using the GST fusion protein purification kit (GenScript Inc., China) following the manufacturer’s instructions. The concentration of the purified protein was detected with NanoDrop 2000 (Thermo Scientific, USA). HaCaT cells were cultured in 6-well plates to 100% confluence. The cells were starved overnight, and treated with 0.3, 0.6, 1.2, and 2.4mg of GST and GST-ROP18, separately, one well for each treatment. Another well was treated with 10 ul of elution buffer (20mM glutathione, 50mM Tris-HCl, pH 8.0) for control.
- HaCaT cell treatment with IL-20, GST-ROP18 and GST, and detection of the co-localization of ROP18 and IL20RB on HaCaT cell membrane
COS-7, HaCaT and HFF cells were grown in 6-well plates to 100% confluence. The serum starved cells were treated with IL-20 (200ng), recombinant GST (2 μg), or recombinant GST-ROP18 (2 μg) for 30min, and elution buffer (50mM Tris-HCl 8.0 with 20mM glutathione, pH 8.0) as a negative control, one well for each treatment.
- HaCaT cell infection with type III CEP, CEP-TgROP18I, and CEP-TgROP18II strains
HaCaT cells were grown in 6-well plates to 100% confluence, cells were starved overnight and infected with CEP, CEP-TgROP18I, and CEP-TgROP18II at MOI 10 for 30min, one well for each infection. Another well was uninfected for control.
2.4.2 Detection of STAT3 activation
The culture medium was aspirated, the cells were then washed with PBS three times, and the unrecruited parasites were washed off. The cells were harvested and lysed. Fifty miligrams of total proteins were subjected to SDS-PAGE and Western blot to detect activation of STAT3 (phosphorylation at Y705 or S727). b-actin was detected as the loading control for the whole cell lysate. Densitometric quantitation of each band in A was applied using Image J. The optical density of Stat3-pY705 and Stat3-pS727 was normalized to the density of the total STAT3, and divided by the uninfected group whose normalized value was set as 1.
2.4.3 Statistical analysis
All experiments were performed at least in triplicate. Data are presented as means ± SD of three independent experiments. Statistical significance was determined using the Kruskal–Wallis H-test with Bonferroni correction or independent t-test. Values of *p < 0.05, **p < 0.01 and ***p < 0.001 were defined as statistically significant.
2.5 Western blot analysis and co-immunoprecipitation
COS-7 cells transfected with recombinant plasmid or HaCaT cells infected with parasites were lysed in lysis buffer (Beyotime Biotechnology) containing protease inhibitor cocktail (Thermo Fisher Scientific) or/and phosphatase inhibitors cocktail (TransGen Biotech, China). The cell lysates were centrifuged at 14,000×g for 5 minutes at 4 °C. The supernatants (50mg of total proteins) were separated by SDS-PAGE and then transferred to PVDF membranes using Trans-Blot SD Semi-Dry Electrophoretic Transfer (Bio-Rad, USA). Then, the PVDF membranes were blocked in PBS or TBS containing 5% Bovine Serum Albumin (BSA, Sigma-Aldrich, German) and 0.05% Tween-20. After blocking, the PVDF membrane was incubated with primary antibodies overnight at 4 °C. Then, membranes were probed with secondary antibodies conjugated with horseradish peroxidase (HRP) for 2 h. The interest proteins were visualized by luminescence generated using ClarityTM Western ECL Substrate (Bio-Rad, USA) and imaged with ChemiDoc Touch Imaging System (BIO-RAD, USA).
For co-immunoprecipitation (Co-IP), the supernatants of cell lysates were incubated with 4μg of anti-HA tag antibody at 4°C for 1 h. Then, Protein A - Agarose (Santa Cruz, USA) was added to the mix and incubated for 12 h with gentle rotation at 4 °C. The agarose beads were collected by centrifugation for 5 min at 1,000×g at 4°C and washed four times with PBS. Then the immunoprecipitates were resuspended in SDS-PAGE sample buffer (Takara, Japan) and boiled for 10min followed by soaking immediately in ice water. The boiled samples were centrifuged at high speed, the supernatant was then loaded for SDS-PAGE, and then subjected to Western blot analysis with the antibodies described below.
2.6 Fluorescence Resonance Energy Transfer (FRET) experiment
COS-7 cells were seeded on coverslips in a 12-well plate, and grown to 80% confluence. The experimental group was co-transfected with pCFPN1-ROP18-3×FLAG and pEYFPC1-IL20RB-HA for 48h. The mono-fluorescence groups were transfected with pCFPN1-ROP18-3×FLAG or pEYFPC1-IL20RB-HA separately for adjustment of the fluorescence base line of donor (eCFP) and acceptor (eYFP). The negative control group was co-transfected with pECFPN1 and pEYFPC1, and the positive control group was transfected with pEYFP-ECFP. The co-localization of ECFP-ROP18 with EYFP-IL20RB was observed under a FluoView FV1000 confocal microscope (Olympus, Japan), and the FRET efficiency and intermolecular distance were calculated by sensitized emission (SE) program. The detection was repeated in different cells three times. The difference between groups was analyzed with the t-test using SPSS software (**p < 0.01).
2.7 Reverse Transcription PCR (RT-PCR) and Quantitative Reverse Transcription PCR (qRT-PCR)
The cells were starved overnight before infection and detection. The transcription level of IL20RB in COS-7, HEK293T, HFF, and HaCaT cells was evaluated by RT-PCR. The transcription levels of IL20RB, IL22RA1, and TNF-α in infected cells were analyzed by qRT-PCR. The normal HaCaT cells, and the cells infected with RH-WT or RH-∆rop18 at MOI 10 for 10, 20 and 30 min, were harvested separately. Transcription level of iNOS, IL20, IL22, and IL10 was detected by qRT-PCR. All the PCR primers are shown in Table S1. The translation level of IL10 was detected by Western blot.
Total RNA isolation from infected or uninfected cells was performed using Trizol® Reagent (AmbionTM, USA), and genomic DNAs were immediately removed by TURBO DNAase (Invitrogen, USA). cDNAs were generated with the RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, USA). PCR was conducted using Pfu DNA polymerase (TransGen Biotech, China), and qRT-PCR was performed using SuperReal PreMix Plus (Tiangen Biotech, China). All of the procedures were performed following the manufacturer’s instructions. Gene-specific primers are shown in Table S1. Each experiment was performed in triplicate and the reactions were conducted in a BioRad Applied Biosystems 7500 qPCR System (BioRad laboratories, Inc, USA). The relative transcription level of each gene was normalized to the mRNA of an internal control gene, GAPDH. The transcription levels of the target genes were compared by the change-in- cycling-threshold (∆∆CT) method.
2.8 Detection of the co-localization of ROP18 and IL20RB on HaCaT cell membrane
HaCaT cells were grown on coverslips in 12-well plates to 100% confluence. The overnight serum starved cells were treated with recombinant GST (1μg), or recombinant GST- ROP18 (1μg) for 30min, and then fixed in 300μl 4% paraformaldehyde. The cells were then permeabilized with 0.5% Triton X-100/PBS for 10 min , then washed with PBS three times, 5 min for each. The cells were blocked with 500 μl 10% BSA for 1 h at room temperature, followed by incubation with Rabbit anti-IL20RB and Mouse anti-GST monoclonal antibody in 10% BSA overnight at 4 °C. After washing with PBS three times, 5 min for each, the cells were incubated with the secondary antibodies Alex Fluor 488 Goat Anti-Rabbit IgG and Alex Fluor 594 Goat Anti-Mouse IgG in 10% BSA for 1 h at room temperature in the dark. The cells were washed with PBS for three times, 5 min for each. The coverslips were mounted with DAPI mounting oil and observed under a fluorescence microscope (Nikon eclipse Ni) with a 100× objective lens (NA 1.40).
2.9 Antibodies:
The primary antibodies for Western blot: Mouse poly-clonal Ab anti-TgROP18 (1:1000) were raised in our laboratory; mouse mAb anti-TgSAG1 (1:1000), rabbit anti-HA tag (1:5000), rabbit mAb anti-stat3 (phosphor Y705, 1:200,000), rabbit mAb anti-stat3 (phosphor S727, 1:5000), and rat mAb anti-IL20RB (1:1000) were purchased from ABcam Pharmatech, Inc, United Kingdom. Mouse mAb anti-GST (1:2000), mouse mAb anti-DDDDk (1:2000), and rabbit polyclonal Ab anti-TNFa (1:1000) were purchased from ABclonal Biotechnology, USA. Mouse mAb anti-stat3 (1:1000), and rabbit mAb anti-b-actin (1:1000) were purchased from Cell Signaling Technology, USA. Rabbit anti IL-10 monoclonal antibody (1:1000) was purchased from Affbiotech Biosciences, UK.
The secondary antibodies for Western blot: Goat anti-mouse IgG-HRP (1:2000) and goat anti-rabbit IgG-HRP (1:2000) were purchased from Santa Cruz Biotechnology, USA. Goat anti-rat IgG-HRP (1:2000) was purchased from Bioss Inc (China). Alex Fluor 488 Goat anti-Rabbit IgG (1:2000) was purchased from Invitrogen, and Alex Fluor 594 Goat anti-Mouse IgG (1:2000) was purchased from Thermo Fisher Scientific.
The antibody for IP or Co-IP: Rabbit anti-HA tag (4μg) was purchased from Cell Signaling Technology, USA.