African green monkey kidney (Vero) cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% (v/v) heat-inactivated foetal bovine serum (FBS), 1% non-essential amino acids (NEAA), 100 U/mL penicillin, 100 μg/mL streptomycin and 1% GlutaMAX at 37°C in a 5% CO₂ atmosphere (Zhang et al., 2019). The T. gondii RH stain tachyzoites used in our study were maintained in Vero layers in DMEM contained with 1% FBS, as described previously (Si et al., 2018). All the infection experiments with T. gondii were performed under biosafety level 2 (BSL-2) conditions.

Myrislignan (batch numbers DST180502-043, Desite Biotechnology Co., Ltd., China) was dissolved in dimethyl sulfoxide (DMSO, Sigma, USA) at a concentration of 4 mg/mL, then diluted in DMEM containing 1% FBS to different concentrations. All drugs were stored at 4°C.

T. gondii were isolated from infected Vero cells according to previously described methods. After treatment with different concentrations of myrislignan (32, 50 or 70 μg/mL) in DMEM or without any drug (as parasite control) for 24 h at 37°C, all the samples were washed with cold phosphate-buffered saline (PBS) and immediately stored at -80°C until they were used for RNA isolation. RNA-Seq analysis was based on three biological replicates per experimental group. Total RNA was extracted from T. gondii using TRIzol Reagent (Invitrogen, USA) and the concentrations were detected by an Agilent 2100 Bioanalyzer (Agilent RNA 6000 Nano Kit, Agilent Technologies, USA). Sequencing libraries were generated using an Illumina TruSeqTM RNA Sample Preparation Kit (Illumina, San Diego, CA, USA) and sequenced with the HiSeq 2000 System (TruSeq SBS KIT-HS V3, Illumina). RNA isolation, library construction, RNA sequencing, and read alignment were performed by BGI (Shenzhen, China) (He et al., 2019).

The level of gene expression was calculated in units of fragments per kilobase of transcript sequence per million base pairs sequenced (FPKM) of each gene. Differential expression analysis was performed using the DESeq R package. The P-values were adjusted as Q-values using the Benjamini-Hochberg and Storey-Tibshirani correction for multiple testing. As the |log2 (fold change)| ≥ 1 and Q-values ≤ 0.001, the transcripts were considered differentially expressed. DEGs were subjected to Gene Ontology (GO) (www.geneontology.org/) enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) (www.genome.jp/kegg/) analyses, which were performed as described previously (He et al., 2019).

Total RNA from treated and untreated T. gondii tachyzoites was extracted as described above, then used to synthesized cDNA. TB Green Premix Ex Taq II (Tli RNaseH Plus) (TaKaRa, Japan) was used to perform quantitative real-time polymerase chain reaction (qRT-PCR) reactions using a QuantStudio 6 Flex Real-Time PCR System (Life Technologies). The qRT-PCR primers used in this study are described in Supplementary Dataset S1 . α-tubulin was used as an internal standard reference gene. Each sample were carried out in biological triplicates.

3.74 mg/mL myrislignan in DMSO were spotted in 3D SPRi chips using a BioDot 1520 Array Printer to control the consistency of sample size. No myrislignan in DMSO were spotted in chips as the negative control spot. Freshly released T. gondii tachyzoites (1×10⁹) were lysed, and the protein concentration was detected with a Thermo Fisher BCA Protein Assay Kit (Number: 23227). The final concentration of the T. gondii sample was 200 µg/mL. Protein lysate was flowed through the chip surface to bind the compound on the chip surface, and PBST was also used as the negative control for the measurement of specific signals in oval regions of interest. After in situ enzymatic hydrolysis, the kinetic affinity between T. gondii peptides and myrislignan was calculated, and the protein or peptides captured on the chip surface were identified by HPLC-MS/MS (Nano Acquity UPLC System, Waters Corp., USA; AB SCIEX TOF/TOF Mass Spectrometry System, AB Sciex Pte. Ltd, USA).

Tachyzoites in Vero cells were treated with myrislignan (32 or 70 μg/mL) in DMEM or with no drug (as control) for 8 h, 16 h or 24 h, then fresh tachyzoites (approximately 1×10⁶/group) were extracted and incubated with 10 µM H2DCFDA (DCFH-DA, 2’,7’-Dichlorodihydrofluorescein diacetate) probe in DMEM for 20 min at 37°C. All the samples were washed with DMEM twice, and seeded into each well of a 24-well cell culture plate, then the luminescence was detected using a multilabel reader (EnSpire, PerkinElmer, USA) (Chen et al., 2018).

Fresh tachyzoites (1×10⁶ per group) from Vero cells were lysed after incubation with myrislignan (32 or 70 μg/mL) or without drug (control), then centrifuged at 12,000 g for 5 min at 4°C. The supernatant was added to a 96 well plates, and the absorbance of each sample was measured at 450 nm by a total SOD assay kit (WST-8, Beyotime, China) after incubation at 37°C for 30 min in dark (Chen et al., 2018).

Vero infected with T. gondii for 8 h and incubated with 32 or 70 μg/mL myrislignan for 16 h or 24 h, digested with TrypLE Express for 2 min, washed twice with PBS. Then, the cells were processed for TEM, as described previously (Si et al., 2018).

For each sample, tachyzoites in Vero cells were treated with myrislignan (32 or 70 μg/mL) for 16 h in DMEM or with no drug (as a control). After extraction, the fresh tachyzoites were suspended in the MDC solution (100 μM) at 37°C for 60 min, and then washed with PBS, resuspended in 500 μL PBS. The fluorescence in each group was visualized by laser scanning confocal microscopy (ZEISS LSM-800, Jena, German). The experiment was repeated three times (Zhang et al., 2021).

After myrislignan (16, 32, 50, 60 or 70 μg/mL) treatment for 16 h, T. gondii were lysed with RIPA lysis buffer, all protein samples were separated on 15% urea SDS-polyacrylamide gel electrophoresis and transferred onto 0.22 μm polyvinylidene fluoride (PVDF) membranes (Merck Millipore, US) (Besteiro et al., 2011; Kong-Hap et al., 2013). After blocking, membranes were incubated with the corresponding primary antibodies against TgATG8 (1: 250, presented by researcher Dr. Jia of Harbin Institute of Veterinary Medicine) at 4°C overnight. The membranes were washed with TBST buffer and incubated with horseradish peroxidase-conjugated secondary antibodies (1:2000, Cell Signaling Technology, USA), and chemiluminescent detection was completed with enhanced chemiluminescence Western blot agent (Millipore, Billerica, MA, USA). The protein signals were detected with Amersham Imager 600 system (GE, Boston, MA, USA) and were normalized to the corresponding internal control tubulin to eliminate the variance in total protein (Wang et al., 2010; Lee et al., 2013).

T. gondii tachyzoites in infected host cells and incubation with myrislignan (32, 50 or 70 μg/mL) for 24 h. Intracellular parasites were collected by passage of host cells, and approximately 1×10⁶ tachyzoites were centrifugation at 1,500 g, 15 min at 4°C and washed with PBS. Then, the samples were suspended in 100 μL of binding buffer with 5 μL of annexin V-PE and 5 μL of 7-AAD dye (Becton Dickinson Company, 559763) in the dark for 20 min at 37°C. Double mixtures were analysed by Guava easyCyte flow cytometer (Merck, USA) (Chen et al., 2018). The experiment was repeated three times.

Data comparisons between the control and myrislignan treatment groups in the ROS and SOD tests, flow cytometry assay was statistically analysed by one-way analysis of variance (ANOVA) using SPSS software (SPSS Inc., Chicago, IL, USA). Differences were considered statistically significant at a p value <0.01.

RNA-Seq was used to investigate the gene expression patterns of T. gondii treated with myrislignan. To verify the RNA-Seq results, 6 candidate genes were randomly selected and evaluated by qRT-PCR) in this study. The results indicated that the expression levels of DEGs obtained by qRT-PCR were nearly consistent with those obtained by RNA-Seq, demonstrating the validity of the transcriptomic RNA-Seq data ( Figure 1A ).

DEGs analysis was carried out by comparing the gene transcriptional levels in myrislignan-treated T. gondii and that in untreated control T. gondii, the DEGs in each treatment group in Table 1 . Venn diagram analysis revealed 63 genes that were differentially co-expressed in myrislignan treated groups (32, 50 or 70 μg/mL) ( Figure 1B ). GO enrichment analysis of DEGs revealed changes in biological processes, molecular functions and cellular components in T. gondii after treatment with myrislignan ( Figure 1C ), and the most common GO terms in these categories were “catalytic activity”, such as “oxidoreductase activity”, “oxidation−reduction process” and “electron transfer activity”. KEGG pathway analysis showed that the DEGs were mainly associated with “oxidative phosphorylation”, as shown in Figure 1D .

To more precisely identify a target protein, SPR analysis investigated the affinity and interactive effect between myrislignan and the T. gondii proteins. A total of 58 T. gondii proteins were captured by myrislignan ( Supplementary Dataset S2 ). The kinetic affinity between T. gondii peptides and myrislignan was calculated. Accordingly, 26 specific binding proteins indicating high affinity with binding scores of greater than 1,000, were selected for the following experiments. According to Gene Ontology (GO) database, the target proteins were further analysed by functional clustering and enrichment. The results of protein classification were shown in Figure 2A , the results of molecular function classification were shown in Figure 2B , the biological process classification results were shown in Figure 2C , and the cell component classification of the results were shown in Figure 2D .

In order to verify and identify new mechanisms of action of myrislignan, RNA-Seq data and SPR-MS data (26 proteins) were compared and combined. We found the DEGs and high affinity proteins were enriched in the oxidation-reduction process ( Figure 2E ). Therefore, myrislignan may play a key role of anti-T. gondii activity by affecting the oxidation-reduction process of T. gondii.

We investigated whether myrislignan stimulated the increase of ROS production in T. gondii tachyzoites. After myrislignan incubation, ROS activity was also significantly (p<0.01) upregulated ( Figure 3A ). SOD is an important antioxidant produced by parasitic protozoa. It can maintain the stability of the internal environment and prevent the clearance of host immune cells. Therefore, we evaluated the SOD activity of T. gondii RH tachyzoites and found that the content of SOD was increased after myrislignan incubation, but did not increase over time ( Figure 3B ).

Myrislignan treatment for 16 h caused many autophagic vacuoles to emerge in the cytoplasm, as indicated by the arrows; all of these effects are hallmarks of autophagy ( Figures 4C, E ). In addition, after 24 h of treatment with myrislignan, the cytoplasmic structure and parasitophorous vacuole (PV) membranes of tachyzoites had completely disappeared, and progressive degeneration of the parasites was observed, as shown in ( Figures 4D, F ). However, autophagic vacuoles were not frequently discovered in untreated T. gondii. The TEM results confirmed that the untreated parasites displayed a well-preserved intracellular space with typical apicomplexan structural features, including rhoptries (R), dense granules (DGs), a nucleus (N), and a mitochondrion (M) ( Figures 4A, B ). However, confirmation of these structures as autophagosomes will require the generation of specific markers.

To further confirm autophagy in T. gondii stimulated by myrislignan, MDC staining was exploited to detect numerous autophagic vacuoles in T. gondii after incubation with myrislignan. In MDC staining, myrislignan treatment resulted in obvious fluorescent spot-like structure of T. gondii, indicating a large number of autophagic vacuoles ( Figures 5B, C ), while there were no fluorescent dot-like structures in the untreated T. gondii ( Figure 5A ), the fluorescence intensity mean value of each group was shown in Figure 5D .

Furthermore, the expression of the typical autophagic marker TgATG8-PE was assessed in T. gondii in the absence or presence of myrislignan by western blotting analysis. As expected, the results showed that the autophagy marker TgATG8-PE was upregulated after myrislignan treatment in a dose-dependent manner ( Figure 5E ).

Furthermore, the cell death of inhibiting extracellular growing tachyzoites after treatment with different concentrations of myrislignan for 24 h was determined by flow cytometry ( Figure 6A ). The different patterns in the Annexin V-PE/7-AAD analysis were used to identify the different T. gondii populations where 7-AAD-negative and Annexin V-PE-negative cells were designated as viable tachyzoites. The proportion of viable tachyzoites varied from 88.53% ± 1.31% in control groups to 42.02% ± 1.29%, 26.83% ± 3.29%, 17.22 ± 1.37% in 32, 50 or 70 μg/mL myrislignan treatment groups, respectively. The results showed that myrislignan treatment had a concentration-dependent significant (p < 0.01) increase in cell death effect of T. gondii tachyzoites ( Figure 6B ).