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 cells (HEK 293T), and human keratinocytes cells (HaCaT) 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 are generous gift from Jon Boyle at 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 mutants 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 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 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, 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(15). 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 a sgRNA specifically targeting the site very close to and downstream the start codon of rop18 gene was constructed, using Q5® Site-Directed mutagenesis kit (NEB,USA) following the published protocol(35). A chloramphenicol-resistance gene, the chloramphenicol acetyltransferase (CAT) gene was used as the screening marker. The homologous recombination plasmid (pBlue-5’-3’-ROP18-homo-CAT) was constructed as follows (the procedure is shown in Figure S1A). A 991bp fragment and a 965bp fragment homologous to the upstream and downstream of the gRNA targeted site of rop18 gene, were respectively subcloned to the 5` and 3` end of the CAT gene cassette on pBlue-script SK II (-) plasmid (15). 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 20mmol/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 that used for homologous template amplification, plasmids construction, and recombinants identification were 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
A. HaCaT cells infection with RH-WT, RH-Δrop16, RH-Δrop18, and RH-Δrop16Δrop18 strains: HaCaT cells were cultured in 6-well plates to 100% confluence. 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.
B. HaCaT cells 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 30min and 24h, respectively; two wells were infected with RH-WT at MOI 10 for 30min and 24h, respectively, and two wells were infected with RH -Δrop18 at the same conditions; another well was not infected for control.
C. HaCaT cells treatment with recombinantGST-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(15). The E. 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 0.3mg, 0.6mg, 1.2mg, and 2.4mg of GST and GST-ROP18, respectively, 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.
D. HaCaT cells treatment with IL-20, GST-ROP18 and GST
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 (2μg), or recombinant GST-ROP18 (2μg) 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.
E. HaCaT cells infection with type III CEP, CEP-TgROP18I, and CEP-TgROP18II strains
HaCaT cells were grown in 6-well plates to 100% confluence, and infected with CEP, CEP-TgROP18I, and CEP-TgROP18II at MOI 10 for 30min, respectively, 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 for three times, and the unrecruited parasites were also washed off. The cells were harvested and lysed. Fifty mg of total proteins were subjected to SDS-PAGE and western blot to detect activation of STAT3 (phosphorylation at Y705 or S727). -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 were 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. After that, membranes were probed with secondary antibodies conjugated with horseradish peroxidase (HRP) for 2h. 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 12h with gentle rotation at 4°C. The agarose beads were collected by centrifugation for 5min at 1,000g at 4°C and washed four times with PBS. Then the immunoprecipitats were resuspended in SDS-PAGE sample buffer (Takara, Japan) and boiled for 10min followed by soaking immediately into 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 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 bought from ABcam Pharmatech, Inc, United Kingdom. Mouse mAb anti-DDDDk (1:2000), and rabbit polyclonal Ab anti-TNF (1:1000) were bought from ABclonal Biotechnology, USA. Mouse mAb anti-stat3 (1:1000), and rabbit mAb anti-b-actin (1:1000) were bought from Cell Signaling Technology, USA.
The secondary antibodies for western-blot: Goat anti-mouse IgG-HRP (1:2000) and goat anti-rabbit IgG-HRP (1:2000) were bought from Santa Cruz Biotechnology, USA. Goat anti-rat IgG-HRP (1:2000) was bought from Bioss Inc (China).
The antibody for IP or Co-IP: Rabbit anti-HA tag (4μg) was bought from Cell Signaling Technology, USA.
2.7 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 were 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 for three times. The difference between groups was analyzed with t-test using SPSS software (**p < 0.01).
2.8 Reverse Transcription PCR (RT-PCR) and Quantitative Reverse Transcription PCR (qRT-PCR)
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 cell were analyzed by qRT-PCR. 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). Relative transcription level of each gene was normalized to the mRNA of an internal control gene GAPDH. The transcription level of the target genes were compared by the change-in- cycling-threshold (∆∆CT) method.