Animals, parasites, reagents and ethics statement
Inbred mice used in this study were 8-week-old male BALB/c mice obtained from the Center of Laboratory Animals, Shanxi Medical University, Taiyuan. The mice were maintained in housing with a 12:12 h light-dark cycle with free access to food and water. Two New Zealand rabbits, approximately 60 days old, were kept in individual cages. All procedures were performed in accordance with the laws and conditions of the Guide for the Care and Use of Laboratory Animals and Institutional Animal Ethics Committee guidelines of Shanxi Medical University. T. gondii tachyzoites (RH strain) were kindly provided by the Peking University Health Science Center and were maintained via serial intraperitoneal passage in BALB/c mice [11].
The following antibodies used in this study: rabbit monoclonal anti-LC3B (No. 3868, Cell Signaling), rabbit monoclonal anti-Beclin-1 (No. 3495, Cell Signaling), rabbit monoclonal anti-p62 (No. 39749, Cell Signaling), rabbit monoclonal anti-Flag tag (No. 14793, Cell Signaling), rabbit monoclonal anti-phospho-Bcl-2 (Ser70) (No. 2827 Cell Signaling), rabbit polyclonal anti-JNK (No. 9252, Cell Signaling), rabbit monoclonal anti-phospho-SAPK/JNK (Thr183/Tyr185) (No. 4668, Cell r and rabbit monoclonal β-actin (No. 4970, Cell Signaling). Horseradish peroxidase (HRP)-linked anti-rabbit IgG secondary antibodies (No. 7074, Cell Signaling) were also used. Anti-FLAG M2 affinity gel (No. A2220) was obtained from Sigma-Aldrich).
Preparation of rabbit anti-T. gondii ROP17 serum
Purified T. gondii ROP17 protein (TgROP17) was prepared according to our previous study [12]. Antiserum against TgROP17 was produced in two male New Zealand rabbits (body weight: 2.0 ~2.5 kg). Before challenge, a 5 ml peripheral blood sample was collected from each rabbit ear vein for the preparation of a preimmune serum. The TgROP17 protein (500 μg) was hypodermically injected into the backs of the rabbits, and the same dose was used in four boosters administered every 10 days thereafter. Blood was taken from the marginal ear vein 10, 20, 30 and 40 days after the first challenge and on the 10th day after the final challenge. In addition, a blood sample was obtained from the jugular vein for establishing the generation of a specific antibody with enzyme-linked immunosorbent assay (ELISA) with TgROP17 used as a coating antigen. The antiserum phase was obtained by centrifugation separation at 2300 g for 10 minutes at 4 °C. After removing the precipitated residues, 0.02% NaN3 was added to the blood serum, which was stored at -80 °C.
Titre determination of rabbit anti-ROP17 serum
The titre of the ROP17 antiserum was detected by direct coating ELISA according to the published method [13]. First, a polystyrene microtitre plate (Corning, USA) was coated with 100 ng/100 µl of purified GST-ROP17, and the liquid was discarded after overnight incubation at 4 °C. By incubating the plate with PBST containing 1% bovine serum albumin (BSA) for 2 h at 37 °C, the nonspecific binding sites were blocked. The anti-ROP17 polyclonal antiserum was diluted to different concentrations (1: 1,000; 1: 2,000; 1:4,000; 1:8,000; 1:16,000; 1:32,000; 1:64,000; 1:128,000 ;1:256,000; and 1:512,000) with 1% BSA and incubated for 2 h at 37 °C. After being washed with PBST, the plate was incubated for 1 h with goat anti-rabbit IgG-HRP (Sigma, USA) diluted to 1:2,000 with 1% BSA. Tetramethylbenzidine (TMB) chromogenic reagent (Boster, Wuhan, China) was added to the plate that had been washed with TBST. After 50 ml of 2 M H2SO4 was added to plates to stop the reaction, the absorbance was measured at 450 nm by an ELISA plate reader (Epoch Multi-Volume Spectrophotometer System, BioTek, USA).
Immunohistochemistry
At the indicated time after intraperitoneal injection of 103T. gondii tachyzoites, the mice were sacrificed by intraperitoneal injection of sodium pentobarbital (100 mg/kg). The small intestine was dissected, and the faeces inside was removed by washing with cold PBS. Then, the small intestine was stored overnight in 4% paraformaldehyde at 4 °C. Tissues that were 5 μm thick were dewaxed in xylene and rehydrated in a series of ethanol solutions. Endogenous peroxidase activity was blocked using 3% hydrogen peroxide. The sections were heated in a microwave in citrate buffer solution to retrieve antigens, and non-specific binding was prevented with goat serum blocking for 1 h. The sections were incubated overnight at 4 °C with ROP17 (dilution 1:200), LC3B, Beclin 1, p62, Bcl-2 and phospho-Bcl-2 (Ser70) antibody (dilution 1:100), followed by incubation with horseradish peroxidase-coupled goat anti-rabbit secondary antibody at 37 °C for 1 h and staining with 3,3′-diaminobenzidine. The nucleus was counterstained blue with haematoxylin. The samples incubated with rabbit serum instead of indicated primary antibodies were used as negative control. The experimental procedure was performed strictly following the manufacturer’s instructions. One hundred cells were captured randomly in a field of view under the microscope, and the percentage of the positive cells was calculated.
Cell culture and treatment
HEK 293T cells were purchased from the Cell Culture Center of the Chinese Academy of Medical Sciences (Beijing, China) and cultured in high-glucose Dulbecco’s modified Eagle’s medium (DMEM; Gibco, USA). The medium was supplemented with 10% (v/v) foetal bovine serum (FBS; Gibco, USA). The cells were grown at 37 °C in an 5% incubator with CO2. The cells were passaged every 2~3 days when they reached 70~80% confluency.
HEK 293T cells cultured in a 6-well plate (2.5 × 105 cells/well, 1 mL medium/well) were transfected with p3×Flag-CMV-14 and p3×Flag-CMV-14-TgROP17 using Lipofectamine 3000 (Thermo Fisher Scientific, USA) according to the manufacturer’s instructions. Forty-eight hours later, the cells were treated with serum-free medium and cultured for another 4 h and 8 h to induce autophagy.
Monodansylcadaverine (MDC) staining
The transfected HEK 293T cells were treated by serum starvation for 0, 4 and 8 h, and then, the cells were incubated with MDC (50 µmol/L) at 37 °C for 30 minutes. Then, the cells were washed two times with pre-cooled PBS and immediately observed and imaged under a fluorescence microscope (EVOS M5000, Thermo Fisher Scientific). One hundred cells per sample were analysed, and the percentage of autophagic cells was calculated.
Transmission electron microscopy (TEM)
After serum starvation treatment, the HEK 293T cells were collected and washed with cold PBS, fixed overnight in 3% glutaraldehyde at 4 °C and subsequently post-fixed in 1% osmium tetroxide for 30 minutes. The cells were gradient dehydrated with ethanol solutions ranging from 50% to 100% in a 10% graded series and then embedded in Eponate 12 resin (Ted Pella, Redding, CA, USA). The blocks were cut into ultrathin sections and then doubly stained with uranyl acetate and lead citrate. Autophagosomes were observed under a transmission electron microscope (JEOL-100CX, Japan).
Examination of GFP-LC3B puncta
HEK 293T cells were co-transfected with p3×Flag-CMV-14 or p3×Flag-CMV-14-TgROP17 and pCMV-GFP-LC3 (GenePharma Co., Ltd, China). After 48 h, the cells were cultured in serum-free medium for 4 h and 8 h. GFP-LC3 puncta were quantified by observation with a fluorescence microscope (EVOS M5000, Thermo Fisher Scientific) and counting at least 300 cells. The results are plotted as the mean ± SD of three independent experiments.
Co-immunoprecipitation
Co-immunoprecipitation analysis was performed in accordance with the manufacturer’s instructions. Briefly, HEK 293T cells were transfected with p3×Flag-CMV-14 or p3×Flag-CMV-14-TgROP17 for 48 h, and then, the cells were harvested and lysed using sonication in cell lysis buffer (50 mM Tris-HCl, pH 7.5; 150 mM NaCl; 1% Nonidet P-40; 0.5% sodium deoxycholate; and a 1% protease inhibitor cocktail (Roche, Switzerland)). The cell lysates were centrifuged, and the supernatant was incubated overnight with FLAG M2 Affinity Gel with gentle agitation at 4 °C. The following day, the beads were washed six times with cell lysis buffer and heated in 2 × sample buffer. After brief centrifugation, the supernatant was used for Western blotting.
Western blotting
The protein from 1×106 tachyzoites of T. gondii was prepared using 1×SDS loading buffer heated for 10 minutes. Total protein from the HEK 293T cells was extracted using RIPA buffer with protease inhibitors and quantified using BCA protein assay reagent (Thermo Scientific, USA). Equal amounts of protein samples were separated by 12% SDS-PAGE and transferred to a 0.2 μm PVDF membrane (Millipore, USA). The membrane was blocked in TBST buffer with 5% non-fat milk for 1 h before overnight incubation at 4 °C with different primary antibodies. Primary antibodies were detected with their corresponding HRP-IgGs using an ECL blot detection system (Transgene, China) with a Bio-Rad Universal Hood II Gel Doc XR system. β-actin on the same membrane was used as an internal control.
Statistical analysis
Data are presented as means ± standard deviation (SD). Differences between two groups were analysed using Student’s t-test. Pearson coefficient was used to analyse correlations between the expression of ROP17 with the expression of autophagy markers LC3B, beclin 1 and p62, and with phosphorylation of Bcl-2 and Bcl-2. An r-value ≥ 0.5 was considered as strong correlation, and an r-value < 0.5 was considered as poor correlation [14]. A p-value < 0.05 was considered as statistically significant. All data were analysed using SPSS software version 19.0 (SPSS Inc., Chicago, IL, U.S.A.).