Drugs and chemicals
The 36 compounds to be screened were small-molecule intermediate products of natural terpenoid chlorajaponilide C, the synthesis of which had been reported previously[15]. 100 nM stock solutions of each compound were prepared in dimethyl sulfoxide (DMSO, MP biomedicals, USA) and stored at -20°C. Pyrimethamine, azithromycin and staurosporine were purchased from MCE, USA.
Parasites strain
The type I virulent RH strain of T. gondii (generously donated by Guizhou Medical University, China) was used in this study. The RH strain was removed from liquid nitrogen and recovered in a water bath of 37°C for 30 min and then maintained in vitro by serial passage in human foreskin fibroblasts (HFF). When over 90% of the infected cells were lysed, the parasites were obtained together with the attached cells with a gentle scrape by a cell scraper, followed by centrifugation at 100×g for 10 min at 4°C before the supernatants were collected. Next, the mixture was passed through a 25-gauge syringe needle several times, centrifuged at 2,000×g for 10 min to remove the supernatants, and resuspended in either PBS (for infection) or RPMI 1640 medium (Gibco, USA) supplemented with 5% fetal bovine serum (Endo buffer) at 37°C in 5% CO2 (for treatment).
Cells
A murine macrophage-stable cell line (RAW 264.7) was cultured at 37°C in a 5% CO295% air mixture in RPMI 1640 medium containing 2.05 mM L-glutamine, Dulbecco’s modified Eagle’s medium (DMEM; Gibco, USA), respectively, supplemented with 10% fetal bovine serum (PAN, Germany) and 100 g/ml of antibiotics (penicillin and streptomycin; Ameresco, USA), with passage taking place every 3 to 4 days. When cells were over 80% confluent, 5 ml of trypsin with 0.25% EDTA and phenol red (Solarbio, China) was employed to digest the cells before they were washed off with fresh medium by pipetting for further seeding procedures.
Cytotoxicity assay
Cell Counting Kit-8 (DOJINDO, Japan) was used to evaluate the cytotoxic effects of compounds on RAW 264.7 macrophages. Macrophages (3×103 cells/well) were seeded in a 96-well cell culture plate and incubated at 37 °C in a 5% CO2 atmosphere for 2h. After that, the culture medium were removed and adhered cells were exposed to the compounds at 10μM to 1mM for 24h under the same incubation conditions. Each assay was performed in triplicate with independent experiment. Compounds with less cytotoxicity will be used in subsequent experiments.
In vitro assays for anti- Toxoplasma gondii activity
Tachyzoites were collected and loaded in triplicate in a 96-well cell culture plate at 5×105 cells/per well and challenged with compounds at 1μM and 10μM for 24 h. Parasite survival was performed after stimulation by a standard trypan blue dye exclusion test to determine the number of viable cells. Medium alone was used as control. All of the cultures were performed independent times, and the results are expressed as the percentage of inhibition (I%), which is equal to [(number of parasites in culture medium-number of parasites in medium with compounds)/number of parasites in culture medium] ×100%.
After that, several compounds with higher inhibition rate were selected. Parasites were collected and loaded as previously described but challenged with the selected compounds at different concentrations ranging from 0 to 200 M for 24 h. The results are expressed as the IC50 (50% inhibitory concentration) values.
In vivo assays for anti- Toxoplasma gondii activity
RAW 264.7 macrophages (2 × 105 cells/well) were seeded in a 24-well cell culture plate and incubated at 37 °C in a 5% CO2 atmosphere with sterile glass for 2h. Cells were infected with RH tachyzoites at a 10:1 parasite-to-cell ratio for 2 h, and after the replacement of the medium with new culture medium, azithromycin, pyrimethamine and compounds at IC50 were added for an extra 36h. Infected and stimulated cells were then observed with an optical microscope and Wright’s stain (NJJCBIO, China). The average number of tachyzoites in each cell was determined by randomly counting 200 infected cells in each of the duplicate coverslips.
Ultrastructural analysis by transmission electron microscopy
Approximately 4×107 tachyzoites were employed for co-culture with compounds, azithromycin, and pyrimethamine for 18h, and 4×107 tachyzoites without any treatment, followed fixed in 3% glutaraldehyde and immediately fixed again at 4°C with 1% osmium tetroxide solution. The samples were thoroughly rinsed with distilled water, and then dehydrated with an acetone gradient, and then transferred to SPI-Pon812 resin for polymerization and embedding. After sectioning, electron staining was performed sequentially with 2% uranyl acetate solution and Reynolds' lead citrate solution. Stained sections were imaged on a Hitachi HT7800 transmission electron microscope.
Phosphatidylserine (PS) exposure and cell death.
Approximately 1×106 tachyzoites were treated with compounds, azithromycin, pyrimethamine and the positive control staurosporine for 18h, followed by collection and centrifugation at 1,300 ×g for 15 min at 4°C to remove the supernatants before the addition of annexin V-FITC dye (BD, USA) in the dark for 10 min. After the addition of PI dye for another 5 min followed by the addition of the ligation buffer, the mixture was subjected to flow cytometry (BD, FACS Celesta) or confocal microscopy (Zeiss, LSM880) for further detection, as required by the protocol.
Measurement of changes in mitochondrial membrane potential.
1×106 tachyzoites were collected and centrifuged at 1,300 ×g for 15 min at 4°C employed after coculturing with compounds, azithromycin, pyrimethamine and 5μM carbonyl cyanide m-chlorophenylhydrazone (CCCP) (a respiratory uncoupler) for 18h. Then, JC-1 dye were added in the dark and incubate at 37°C for 20 min. After incubation, centrifuge tachyzoites for 15 minutes at 1300× g at 4°C and carefully aspirate the supernatants. Adding staining buffer (1×) to suspend tachyzoites, the mixture was subjected to flow cytometry (BD, FACS Celesta) or confocal microscopy (Zeiss, LSM880) for further detection, as required by the protocol.
Monitoring of changes in intracellular Ca2+ levels.
Approximately 1×106 tachyzoites were cocultured with compounds, azithromycin, and pyrimethamine for 18h.Next, the tachyzoites were collected and centrifuged followed by incubated (20 min at 37°C) in a cocktail of 5mM Fluo-4 AM, 20% Pluronic F-127 and HBSS (Solarbio, China). Then, 5 times the volume of HBSS containing 1% fetal bovine serum were added into the mixture followed by incubation at 37°C for 40 min. After incubation, the tachyzoites were washed third and suspend in HEPES buffer saline. The mixture was subjected to flow cytometry (BD, FACS Celesta) or confocal microscopy (Zeiss, LSM880) for further detection, as required by the protocol.
Invasion Activity
The tachyzoites were collected in 96-well culture plates at a concentration of 1×107cells/well with the addiction of compounds, azithromycin, and pyrimethamine. After cocultured at 37 °C in a 5% CO2 atmosphere for 18h, they were collected and centrifugated at 1,300 ×g for 15 min at 4°C.
The macrophages (2×105 cells/well) were seeded in a 24-well cell culture plate and incubated at 37 °C in a 5% CO2 atmosphere with sterile glass for 2h. The cells were infected with the tachyzoites that collected before at a 10:1 parasite-to-cell ratio for 4 h. Infected and stimulated cells were then observed with an optical microscope and Wright’s stain. The average number of tachyzoites in each cell was determined by randomly counting 200 infected cells in each of the duplicate coverslips.
Macrophage phagocytosis
Macrophages (1x105 cells/well) were cultured for 24 h in the presence of the following compounds, azithromycin, and pyrimethamine. Cells were washed by PBS and incubated with 50μg/ml zymosan (Solarbio, China) in new medium for 3h incubation. Cells were then observed with an optical microscope and Wright’s stain. The average number of zymosan granules in each cell was determined by randomly counting 200 infected cells in each of the duplicate coverslips.
Lysosomal Activity.
Macrophages (1 x 105 cells/well) were plated and incubated with compounds, azithromycin, and pyrimethamine for 24 h. After the replacement of the medium, 20μL of neutral red solution (Beyotime, China) was added and incubated for 2h. After that, the supernatant was discarded, the cells were washed with PBS, and 200μl of Neutral Red Detection Lysis Solution (Beyotime, China) was added. After 10 min on a shaker (QILINBEIER, TS-1), the plate was read at 540 nm by using an ELISA plate reader (TECAN, Sunrise-basic).
Statistics.
IC50 was calculated according to a nonlinear regression using a log inhibitor-versus-response equation with 95% confidence intervals by GraphPad Prism (version 8.0) software. The data were analyzed by Student’s t test for comparison of two groups or by one-way analysis of variance (ANOVA) for comparison of three or more groups using GraphPad Prism (version 8.0) software. Significant differences were determined and designated with asterisks as follows: *P < 0.05; **P < 0.01; ***P < 0.001 and ****P < 0.0001.