Toxoplasma gondii infection induces cell apoptosis via multiple pathways revealed by transcriptome analysis

Toxoplasma gondii infection induces cell apoptosis via multiple pathways revealed by transcriptome analysis 转录组分析揭示弓形虫感染可通过多途径诱导细胞发生凋亡 1 2 first-author 1 1 1 1 1 1 corresp Shaohong LU, lsh@zjams.com.cn Shaohong LU, lsh@zjams.com.cn lsh@zjams.com.cn https://orcid.org/0000-0001-9855-7154 1 corresp Xunhui ZHUO, zhuoxh@zjams.com.cn Xunhui ZHUO, zhuoxh@zjams.com.cn zhuoxh@zjams.com.cn https://orcid.org/0000-0001-5805-0711 School of Basic Medical Sciences and Forensic Medicine, Hangzhou Medical College, Hangzhou 310053, China School of Basic Medical Sciences and Forensic Medicine, Hangzhou Medical College, Hangzhou 310053, China Shandong Center for Disease Control and Prevention, Jinan 250021, China Shandong Center for Disease Control and Prevention, Jinan 250021, China Toxoplasma gondii is a worldwide parasite that can infect almost all kinds of mammals and cause fatal toxoplasmosis in immunocompromised patients. Apoptosis is one of the principal strategies of host cells to clear pathogens and maintain organismal homeostasis, but the mechanism of cell apoptosis induced by T. gondii remains obscure. To explore the apoptosis influenced by T. gondii , Vero cells infected or uninfected with the parasite were subjected to apoptosis detection and subsequent dual RNA sequencing (RNA-seq). Using high-throughput Illumina sequencing and bioinformatics analysis, we found that pro-apoptosis genes such as DNA damage-inducible transcript 3 ( DDIT3 ), growth arrest and DNA damage-inducible α ( GADD45A ), caspase-3 ( CASP3 ), and high-temperature requirement protease A2 ( HtrA2 ) were upregulated, and anti-apoptosis genes such as poly(adenosine diphosphate (ADP)-ribose) polymerase family member 3 ( PARP3 ), B-cell lymphoma 2 ( Bcl-2 ), and baculoviral inhibitor of apoptosis protein (IAP) repeat containing 5 ( BIRC5 ) were downregulated. Besides, tumor necrosis factor (TNF) receptor-associated factor 1 ( TRAF1 ), TRAF2 , TNF receptor superfamily member 10b ( TNFRSF10b ), disabled homolog 2 (DAB2)‍-interacting protein ( DAB2IP ), and inositol 1,4,5-trisphosphate receptor type 3 ( ITPR3 ) were enriched in the upstream of TNF, TNF-related apoptosis-inducing ligand (TRAIL), and endoplasmic reticulum (ER) stress pathways, and TRAIL-receptor 2 (TRAIL-R2) was regarded as an important membrane receptor influenced by T. gondii that had not been previously considered. In conclusion, the T. gondii RH strain could promote and mediate apoptosis through multiple pathways mentioned above in Vero cells. Our findings improve the understanding of the T. gondii infection process through providing new insights into the related cellular apoptosis mechanisms. 目的： 2 研究并阐明弓形虫感染宿主细胞后影响细胞凋亡及相关信号通路。 创新点： 2 通过转录组研究发现弓形虫感染促进了细胞凋亡，并发现了包括 DDIT3 、 GADD45A 、 CASP3 及 HtrA2 在内的多个关键上调基因。 方法： 2 首先通过流式细胞仪对感染及未感染弓形虫的细胞进行凋亡检测，提取RNA后进行转录组测序，通过转录组基因上调、下调分析，GO和KEGG通路、关键基因富集、基因网络关系图、RT-PCR验证等手段进行分析。 结论： 2 流式细胞仪检测发现弓形虫感染后能显著诱导细胞发生凋亡，进一步的转录组分析发现，与对照组相比，感染组有1579个基因表达有显著变化，其中有918个基因上调，661个基因下调；在富集的54条通路中，凋亡通路有16个关键基因上调，11个基因下调，其中caspase-3是我们发现的关键网络基因。细胞凋亡与细胞周期密切、与免疫及多种疾病有关，进一步深入研究和探索弓形虫与宿主细胞之间的凋亡机制可为寻找理想药物靶点提供理论基础。 Toxoplasma gondii Transcriptome analysis Apoptosis Infection 弓形虫 转录组 凋亡 感染 1 Introduction 1 Toxoplasma gondii is a worldwide protozoan that infects over 30% of the human population ( Parlog et al., 2015 Parlog et al., 2015 ). This parasite can also affect animals like cats, pigs, and goats, leading to considerable medical concerns in addition to veterinary and economic losses ( Zhuo et al., 2017 Zhuo et al., 2017 ). The three major strain types, namely, types I, II, and III of T. gondii differ regarding their virulence, propagation, and pathogenicity. The type I strain of high virulence, such as RH strain and GT1 strain, could cause acute and fatal infections in immunocompromised patients; the type II of low virulence consistently results in chronic infection; type III is avirulent ( Fox and Bzik, 2015 Fox and Bzik, 2015 ). T. gondii is an obligatory parasite with complex organelles specialized for the invasion of and movement within host cells, and it resides in parasitophorous vacuole membrane (PVM) to escape host immune response. Polymorphic secreted effectors released from this parasite can manipulate gene expression and host cell disorder signaling pathways to establish life-long infection ( Venugopal and Marion, 2018 Venugopal and Marion, 2018 ). Cell apoptosis is one of the important defense mechanisms against infection by intracellular pathogens. It is a programmed death response to remove damaged cells and keep host homeostasis closely associated with cell replication, proliferation, immunity, and cancer; however, excessive apoptosis may cause disease ( Lin et al., 2018 Lin et al., 2018 ; Mammari et al., 2019 Mammari et al., 2019 ; Rajedadram et al., 2021 Rajedadram et al., 2021 ). Previous studies have demonstrated that apoptotic genesis is dependent on the host cell type and T. gondii strain involved; multiple proteins and pathways have been found to participate in apoptosis and act as important targets modulated by T. gondii . For instance, rhoptry protein 18 (ROP18) of the RH strain has been reported to promote apoptosis in mouse neuroblastoma Neuro2a cells ( Wan et al., 2015 Wan et al., 2015 ), but inhibit apoptosis in human SF268 neural cells and 293T cells ( Zhou et al., 2019 Zhou et al., 2019 ). In mice splenocytes, the T. gondii RH strain inhibited apoptosis, while ME49 strain promoted it ( Quan et al., 2013 Quan et al., 2013 ). Some researchers have confirmed that high-virulence Toxoplasma can decrease the expression of anti-apoptotic protein B-cell lymphoma 2 (Bcl-2) and cell survival, and increase the expression of pro-apoptotic proteins caspase-3 (CASP3) and C/EBP-homologous protein (CHOP) in mouse cells ( Wang et al., 2014 Wang et al., 2014 ). However, the type Ⅰ strain can also inhibit host cell apoptosis by inducing the expression of anti-inflammatory cytokines and increasing extracellular regulated protein kinase (ERK) protein phosphorylation in human trophoblastic cells ( Angeloni et al., 2013 Angeloni et al., 2013 ). The apoptosis-associated signaling pathways are also modulated differently when infected with various strains. ROP16 from type I and type III is responsible for activating the signal transducer and activator of transcription (STAT) signaling pathway ( Rosowski and Saeij, 2012 Rosowski and Saeij, 2012 ), while dense granule protein 15 (GRA15) of type II mediates nuclear factor κB (NF-κB) activation ( Rosowski et al., 2011 Rosowski et al., 2011 ). Therefore, it seems difficult to conclude the clear mechanism of cell apoptosis induced by T. gondii . Previous studies have mainly focused on some particular or few molecular targets on apoptosis. However, the integral apoptosis-associated gene changes in host cells were neglected, and the effect induced by T. gondii recombinant protein alone in some experiments may be quite different from that induced by the whole parasite ( Wei et al., 2018 Wei et al., 2018 ). In addition, a previous study demonstrated that the transcripts of mouse peritoneal cells were similar after infection with type I and type II T. gondii strains, and both showed greater changes than those infected with type III strain ( Hill et al., 2012 Hill et al., 2012 ). To explore apoptotic processes influenced by T. gondii as well as the apoptosis-related gene expression and signaling pathways involved in the host–parasite interaction, apoptosis detection and RNA sequencing (RNA-seq) were performed on the infected Vero cells and uninfected cells. The gene expression profile and differentially expressed genes (DEGs) were investigated by cell transcriptome analysis. The detection results and RNA-seq data demonstrated the promotive effect of T. gondii on apoptosis in Vero cells, which uncovered the basis of apoptosis regulation and could provide novel information of the potential mechanism for future research ( Cong et al., 2018 Cong et al., 2018 ). 2 Materials and methods 1 2.1 Preparation of T. gondii tachyzoites and Vero cells 2 Vero cells were cultured in Dulbecco's modified Eagle medium (DMEM; Gibco, Thermo Fisher Scientific Inc., Waltham, MA, USA) nutrient solution supplemented with 5% (volume fraction) fetal bovine serum (FBS; Gibco), penicillin (100 U/mL, Life Technologies, Gaithersburg, MD, USA), and streptomycin (100 μg/mL, Life Technologies) at 37 ℃ in an incubator under 5% CO 2 . Tachyzoites of the RH strain of T. gondii were maintained in Vero cells. 2.2 In vitro infection 2 After culture in the Vero cells for 3–4 generations, the freshly egressed tachyzoites were passed through 25-gauge (25G) needles and then purified by a 5-‍μm filter. The tachyzoites were counted and then subjected to Vero cells at a multiplicity of infection (MOI) value of 3:1. The infected and uninfected cells were cultured under the same conditions as described above ( Cui and Shen, 2020 Cui and Shen, 2020 ). 2.3 Detection of apoptotic phenotype 2 The apoptosis of Vero cells induced by T. gondii infection was detected by a Becton-Dickinson FACSCalibur flow cytometer (Becton Dickinson, San Jose, CA, USA). Vero cells (0.5×10 6‍ –‍1.0×10 6 ) were seeded into a six-well cell plate and infected with T. gondii tachyzoites (MOI=3:1) for 12 or 24 h. The Vero cells were then stained with fluorescence isothiocyanate (FITC)‍-conjugated Annexin V and propidium iodide (PI) using the instructions of the apoptotic detection kit (Beyotime, China). Briefly, the Vero cells were washed twice with phosphate-buffered saline (PBS), and added with 195 μL Annexin V-FITC-binding buffer, 5 μL Annexin V-FITC reagent, and 10 μL PI dye. The samples were then evaluated with the flow cytometer, and data processing was carried out using FlowJo-V10 software (Becton Dickinson, USA). 2.4 RNA extraction and qualification 2 After being infected with T. gondii under the same conditions as described previously, the total RNA was extracted using TRIzol ® Reagent (Invitrogen, Carlsbad, CA, USA) according to manufacturer's protocol. Two replication experiments were performed, in which Vero cells were infected or uninfected with T. gondii for 24 h. The RNA quality and concentration were checked by a Nanodrop 2000 device (Thermo Scientific Inc., Wilmington, DE, USA), and RNA integrity was determined by an Aglient 2100 BioAnalyzer (Agilent Technologies, Santa Clara, CA, USA). The samples that met the quality control requirements (standard RNA 28S/18S ratio of ≥1.5 and integrity number of ≥8.0) were selected for RNA-seq. 2.5 Transcriptome sequencing and alignment reading 2 The messenger RNA (mRNA) was enriched by binding to magnetic beads with oligonucleotide attachment, and then fragmented for complementary DNA (cDNA) synthesis. The cDNA was prepared for polymerase chain reaction (PCR) amplification and construction of sequence libraries, followed by steps of purification, repairing blunt ends, and adding adaptor to 3'-ends. As soon as the library quality assessment was up to the required standard, the sequencing was performed on an Illumina Novaseq TM 6000 platform (LC Sciences, Houston, Texas, USA), with read length of 150 bp paired-end. After removing the adaptors by Cutadapt (Version 1.9; https://cutadapt.readthedocs.io/en/stable https://cutadapt.readthedocs.io/en/stable ) and verifying the quality of sequence through the FastQC program (Version 0.11.9; https://www.bioinformatics. https://www.bioinformatics. ‍babraham.‍ac.‍uk/projects/fastqc), the clean reads were mapped to the genome of Chlorocebus sabaeus (International Nucleotide Sequence Database Collaboration (INSDC): JACDXN000000000.1) by using HISAT2 (Version 2.0.4; https://daehwankimlab. https://daehwankimlab. ‍ github.‍io/hisat2), followed by assembling the reads and calculating reads per kilobase per million mapped reads (RPKMs) via StringTie (Version 2.1.4; https://ccb. https://ccb. ‍jhu.‍edu/software/stringtie). The gene expression value that meets fragments per kilobase per million (FPKMs) of ≥10 was utilized for further analysis. 2.6 Gene expression level and differential expression analyses 2 The edgeR Bioconductor package (Version 3.14.0; https://bioconductor. https://bioconductor. ‍org/packages/release/bioc/html/edg‍eR.html) was used to identify DEGs and visualize the gene expression profiles. The threshold of significant DEGs was adjusted as P < 0.01 and |log 2 (fold change)|‍ > 1 ( Zhou et al., 2016 Zhou et al., 2016 ). The Gene Ontology (GO) Version 2019.5 and Kyoto Encyclopedia of Genes and Genomes (KEGG) Version 2019.5 databases were used to identify the biological functions of DEGs and the overrepresented biological processes of infected cells. The Search Tool for Retrieval of Interacting Genes/Proteins (STRING; https://www. https://www. ‍string-db.‍org) database was employed to explore the information on protein–protein interaction (PPI) of apoptosis-associated DEGs, and the required confidence score of PPI network was chosen as ≥0.4 ( Szklarczyk et al., 2015 Szklarczyk et al., 2015 ). Hub gene screening was performed using cytoHubba, a plugin of Cytoscape software (Version 3.7.1; https://cytoscape. https://cytoscape. ‍org) ( Bader and Hogue, 2003 Bader and Hogue, 2003 ). All of the DEGs enriched in the apoptosis pathway were counted and ranked by the maximal clique centrality (MCC), a topological analysis method, to improve the precision of predicting essential proteins from the yeast PPI network ( Chin et al., 2014 Chin et al., 2014 ). 2.7 Gene expression validation by qRT-PCR 2 To verify the authenticity of gene expression trend induced by T. gondii , the extracted RNA templates were reversely transcribed to cDNA through reverse transcriptase (TOYOBO, Japan) and quantitative real-time PCR (qRT-PCR). The 20-‍μL reaction mixture contained SYBR Green Ⅰ Master Mix (TOYOBO) with special primers ( Table 1 Table 1 ) was performed on a CFX-Manager real-time system (Bio-Rad, USA). The <math id="M1"><msup><mrow><mn mathvariant="normal">2</mn></mrow><mrow><mo>-</mo><mi mathvariant="normal">Δ</mi><mi mathvariant="normal">Δ</mi><msub><mrow><mi mathvariant="normal">C</mi></mrow><mrow><mi mathvariant="normal">T</mi></mrow></msub></mrow></msup></math> http://html.publish.founderss.cn/rc-pub/api/common/picture?pictureId=38096954&type= http://html.publish.founderss.cn/rc-pub/api/common/picture?pictureId=38096951&type= 7.61999989 3.21733332 method was used to calculate the relative gene expression as previously reported ( Livak and Schmittgen, 2001 Livak and Schmittgen, 2001 ). Three independent replicate experiments were carried out. qRT-PCR: quantitative real-time polymerase chain reaction; F: forward; R: reverse. Gene Primers Product length (bp) TRAF1 F: 5'-CTCTTCTCCCCAGCCTTCTAC-3' R: 5'-CAGCAACGCATCATACTCCC-3' 141 DDIT F: 5'-ACCTCCTGGAAATGAAGAGGAAG-3' R: 5'-TGTGACCTCTGCTCGTTCTG-3' 103 GADD45A F: 5'-AGCAGAAGACCGAAAGGATGG-3' R: 5'-GCACAACACCACGTTATCGG-3' 143 CASP3 F: 5'-TGGAAGCAAATCAGTGGACTCT-3' R: 5'-ACCGAGATGCCATTCCAGTG-3' 128 ATF3 F: 5'-AAGAGGCGGCGAGAAAGAAAT-3' R: 5'-AGCTTCTCTGACTCTTTCTGCA-3' 95 AIFM1 F: 5'-GCCACAGAGCAGTCAGGAAC-3' R: 5'-TGACACCTTTGCCGTAGTCC-3' 136 LAMN F: 5'-GGCTGCGTAACAAGTCCAAT-3' R: 5'-TGCAGCCCAGATCGTTACC-3' 143 BIRC5 F: 5'-CTCAAGGACCACCGCATCTC-3' R: 5'-GCCAAGTCTGGCTCGTTCT-3' 125 IL12A F: 5'-CACGCTTCAGAAGGCCAGACA-3' R: 5'-AATTCCAATGGTAAACAGGCCTCT-3' 120 IL1A F 5'-GACCTTCAAGCAGAGCATGG-3' R: 5'-GACCTGGGCTTGATGATTTCTTC-3' 144 IL6 F: 5'-ACATGTGTGAAAGCAGCAAAGAG-3' R: 5'-TCACCAGGCAAGTCTCCTCAT-3' 114 CCL20 F: 5'-ACATCAATGCTGTCATCTTTCACA-3' R: 5'-AATCAATTTCACCCAGGTCTGC-3' 80 CXCL8 F: 5'-AGCTCTGTGTGAAGGTGCAGTT-3' R: 5'-CAGTGTGGTCCACTCTCAATCA-3' 132 RPS18 F: 5'-AAATAGCCTTTGCCATCACTGC-3' R: 5'-TGATCACACGTTCCACCTCATC-3' 132 GAPDH F: 5'-AAGGTCGGAGTCAACGGATTT-3' R: 5'-ATGGGTGGAATCATACTGGAACA-3' 147 2.8 Statistical analysis 2 The GO and KEGG pathway enrichment analyses were performed by Fisher's exact test, and the P -value was corrected by the Q -value. The gene expression differences between the infected and uninfected groups were evaluated by independent-samples t -test. A P -value of < 0.05 was considered as statistically significant. 3 Results 1 3.1 Effect of T. gondii on Vero cell apoptosis 2 We observed that the early apoptotic cells infected with T. gondii tachyzoites increased significantly compared with control cells. As revealed by flow cytometry, the rates of early and late apoptotic cells of the control group were about 2.99% and 2.40%, respectively, while in those infected with T. gondii , the early and late apoptotic cells reached 72.30% and 7.53% for 12 h and 64.80% and 11.59% for 24 h, respectively ( Fig. 1 Fig. 1 ). These results showed that T. gondii could induce apoptosis in the early stage in the infected group compared with the negative control group. 3.2 General features of the transcriptome data 2 After removing the adaptor and low-quality reads, 160 million clean reads were acquired, which accounted for nearly 80% of the total reads. Each sample had at least 97% clean reads that reached the Phred Quality score of 30 (Q30), and the GC content was about 48%, representing a high-level quality of the sequenced bases. In the infection groups, an average of 57% clean reads could be uniquely mapped to reference genomes, and the sites of approximately 85% of mapped sequences were within exons ( Table 2 Table 2 ). * Data are expresses as number (percentage). A read: an inferred sequence of base pairs corresponding to all or part of a single DNA fragment. Q30: Phred Quality score of 30 (rate of incorrect base sequencing is less than 0.001); Ctrl: control; T.g: Toxoplasma gondii . 2 Sample 2 Valid data 2 Valid ratio(%) 2 Q30(%) 2 GC content(%) 3 Number of reads * 2 Exon (%) Read Base (Gb) Mapped Unique mapped Multi-mapped Ctrl1 41 047 806 6.16 80.63 97.88 48.50 39 064 606 (95.17%) 29 344 754 (71.49%) 9 719 852 (23.68%) 85.31 Ctrl2 42 838 750 6.43 78.45 98.02 48.00 41 062 033 (95.85%) 30 843 666 (72.00%) 10 218 367 (23.85%) 85.77 T.g1 39 040 728 5.86 81.19 97.94 49.00 29 830 596 (76.41%) 22 292 307 (57.10%) 7 538 289 (19.31%) 84.88 T.g2 37 745 558 5.66 80.72 97.97 49.50 28 665 634 (75.94%) 21 512 741 (56.99%) 7 152 893 (18.95%) 85.29 3.3 Effect of T. gondii infection on gene expression profiles in Vero cells 2 In order to identify the Vero cell's DEGs infected by T. gondii , genes with the corrected FPKM value greater than 10 was regarded as expressed genes. Among the expressed genes, those with the log 2 (fold-change) of gene expression value of > 1 or < -1 and adjusted P -value of < 0.01 were regarded as DEGs. Under these requirements, the screening resulted in 1579 DEGs, of which 918 genes were upregulated and 661 genes were downregulated ( Fig. 2 Fig. 2 ). Among the DEGs, 27 genes related to apoptosis were significantly expressed, including 16 upregulated and 11 downregulated genes. 3.4 GO analysis 2 GO is an international standard to classify genes into various biological functions. After GO analysis for DEGs, 1579 genes obtained functional annotations and were significantly enriched in biological process, cellular component, and molecular function. The top three GO terms, including microtubule binding, nucleus, and negative transcription regulation, were significantly different between T. gondii -infected and mock-infected control groups (Figs. 3 3 and 4 4 ). 3.5 KEGG analysis 2 The KEGG analysis represents pathways with known functions ( Lu et al., 2019 Lu et al., 2019 ). The results of KEGG analysis showed that 54 pathways were enriched, with the pathway in cancer being the most significant one. Others within the top 10 significant pathways were involved in virus infection, axon guidance, cancer, and cell senescence ( Fig. 5 Fig. 5 ). 3.6 Gene expression changes in the apoptosis signaling pathway 2 Among DEGs in the apoptosis signaling pathway, most of the upregulated genes belonged to pro-apoptosis genes, such as DNA damage-inducible transcript 3 ( DDIT3 ), growth arrest and DNA damage-inducible α ( GADD45A ), CASP3 , and high-temperature requirement protease A2 ( HtrA2 ), and most of the downregulated genes were anti-apoptosis, such as poly(adenosine diphosphate (ADP)‍-ribose) polymerase family member 3 ( PARP3 ), Bcl-2 , and baculoviral inhibitor of apoptosis protein (IAP) repeat-containing 5 ( BIRC5 ) ( Fig. 6 Fig. 6 ). The apoptosis signaling pathway map including DEGs was shown in Fig. 7 Fig. 7 . The PPI network of apoptosis-associated genes and the hub gene showed that CASP3 was the top hub gene with the most interactions ( Fig. 8 Fig. 8 ). The result of RNA-seq was confirmed by qRT-PCR. Fifteen genes were randomly selected from the apoptosis pathway and immune-related pathways. The gene expression was normalized to RPS18 as a reference gene, and the data were represented as mean value±standard error of mean (SEM). Linear correlation analysis with r =0.976 meant that the qRT-PCR results were nearly consistent with RNA-seq data ( Fig. 9 Fig. 9 ). 4 Discussion 1 Toxoplasmosis has been considered as a serious public health issue, and studies on the interaction between T. gondii and host cells can provide theoretical suggestions for treatment and prevention. T. gondii has been reported to change the gene expression and transcription profiles of host cells; however, few studies have focused on the expression of apoptosis-related genes and pathways. Therefore, based on the observed phenomenon of apoptotic genesis, Vero cells were sequenced and the comparative analysis of gene expression was undertaken comparing infected and uninfected groups, in order to identify the significant genes and molecular apoptosis pathways for an in-depth knowledge about host–parasite interactions. Overall, 1579 DEGs enriched to 54 distinct pathways were characterized and identified. Unsurprisingly, apoptosis pathways were significantly regulated among these pathways. The classic apoptosis signaling pathways include the membrane receptor pathway and the mitochondria pathway ( Lima and Lodoen, 2019 Lima and Lodoen, 2019 ), and overwhelming endoplasmic reticulum (ER) stress signal also promotes apoptosis ( Ariyasu et al., 2017 Ariyasu et al., 2017 ). In the apoptosis signaling pathway, 27 genes were significantly expressed, including 16 upregulated and 11 downregulated genes. Most upregulated genes were pro-apoptosis, such as DDIT3 , GADD45A , CASP3 , and HtrA2 , and most downregulated genes were anti-apoptosis, such as Bcl-2 , PARP3 , and BIRC5 . Among the upregulated genes, tumor necrosis factor (TNF) receptor-associated factor 1 ( TRAF1 ) was the most significant one, and TRAF2 was also upregulated. TRAFs are intracellular adaptors that bind to TNF receptor-associated death domain (TRADD) and act in several pathways involved in apoptosis regulation, cell death, immunity, inflammation, cancer, etc. ( Sajjad et al., 2019 Sajjad et al., 2019 ; Kim and Park, 2020 Kim and Park, 2020 ). Furthermore, the genes expressing TRAF2-interacting proteins, including receptor-interacting serine/threonine kinase 1 ( RIPK1 ), disabled homolog 2 (DAB2)‍-interacting protein ( DAB2IP ), and endoplasmic reticulum to nucleus signaling 1 ( ERN1 ), were also upregulated. All the above-mentioned genes were enriched in the upstream of TNF and ER stress pathways, and their common downstream signal was the apoptosis signal-regulating kinase 1 (ASK1)‍-c-Jun N-terminal kinase (JNK) pathway. This implied that T. gondii could mediate apoptosis through the TNF and ER stress pathways, then via the ASK1-JNK pathway, and finally act on mitochondria ( Fig. 7 Fig. 7 ). Furthermore, the upregulation of TNF receptor superfamily member 10b ( TNFRSF10b ) revealed that another membrane receptor pathway was also affected. The TNFRSF10b expresses membrane receptor TNF-related apoptosis-inducing ligand-receptor (TRAIL-R), which interacts with extracellular protein TRAIL and recruits intracellular protein Fas-associated protein with death domain (FADD), and then it triggers the caspase cascade leading to apoptosis ( Yuan et al., 2018 Yuan et al., 2018 ). Thus far, few studies have focused on the TRAIL pathway in T. gondii infection; however, the important role of FADD and its close relationship with other proteins shown by the PPI network require further investigation. CASP3 is a hub gene in the apoptosis pathway, playing a pivotal role in apoptotic execution ( Lossi et al., 2018 Lossi et al., 2018 ). It could activate deoxyribonuclease and destruct lamin, resulting in DNA fragmentation, nuclear protein degradation, and cell proliferation inhibition ( Estaquier et al., 2012 Estaquier et al., 2012 ). It also hydrolyzes anti-apoptosis proteins such as IAP, PARP, and Bcl-2 to promote apoptosis ( Enari et al., 1998 Enari et al., 1998 ). Based on the sequencing data, CASP3 was upregulated, and the genes PARP2 , PARP3 , BIRC5 , Bcl-2 , lamin A ( LMNA ), tubulin α-‍1A ( TUBA1A ) and β‍-actin ( ACTB ), expressing proteins hydrolyzed by CASP3 , were downregulated, which indicated that the pro-apoptosis factors were increased and anti-apoptosis factors were decreased. Moreover, CASP3 could be activated by Ca 2+ mediated in the ER stress pathway. Inositol 1,‍4,‍5-trisphosphate receptors (ITPRs) are responsible for Ca 2+ channel formation ( Tantral et al., 2004 Tantral et al., 2004 ; Mangla et al., 2020 Mangla et al., 2020 ), and the upregulation of ITPR1 and ITPR3 indicated that T. gondii infection can also promote cellular apoptosis through this signaling pathway through the release of more Ca 2+ . The other pro-apoptosis genes, such as DDIT3 , GADD45A , Fos proto-oncogene/AP-1 transcription factor subunit ( FOS ), and HtrA2 , were also upregulated, which play roles either in cytochrome c (Cyt-C) release or IAP cleavage ( Gao et al., 2009 Gao et al., 2009 ; Estaquier et al., 2012 Estaquier et al., 2012 ; Hu et al., 2019 Hu et al., 2019 ). However, we also observed the upregulated expression of anti-apoptosis genes such as phosphoinositide-3-kinase regulatory subunit 3 ( PIK3R3 ) and NF-‍κB subunit 1 ( NFKB1 ), and the downregulated expression of pro-apoptosis genes such as apoptosis-inducing factor mitochondria-associated 1 ( AIFM1 ) and tumor protein p53 ( TP53 ), which seemed contradictory to the apoptotic process. A possible explanation may be that the cell receives the invasion signal from T. gondii and initiates apoptosis, while the upregulation of NFKB1 and PIK3R3 may target the activation of immune response and autophagy or other biological processes aiming to clear the parasite at the same time. Simultaneously, the parasite can also disturb gene expression for survival and proliferation via the NF-‍κB and PI3K-Akt pathways ( Yang et al., 2017 Yang et al., 2017 ; Ihara et al., 2020 Ihara et al., 2020 ). Moreover, AIF and p53 could induce apoptosis in a caspase-independent manner ( Ranjan and Iwakuma, 2016 Ranjan and Iwakuma, 2016 ; Delavallée et al., 2020 Delavallée et al., 2020 ), and the downregulation of AIFM1 and TP53 may imply that the caspase-independent mechanism cannot play a major role in T. gondii -induced apoptosis. Herein, the apoptosis-related gene expression and pathways induced by T. gondii infection were analyzed systematically through transcriptomics. From the sequencing data, it was found that the T. gondii RH strain promoted apoptosis in Vero cells mainly by inducing the upregulation of pro-apoptosis genes and downregulation of anti-apoptosis genes, mediated by the TNF and ER stress pathways and through the ASK1-JNK pathway to disrupt mitochondrial homeostasis. Besides, T. gondii could act on ER to promote Ca 2+ release and disorganize the cell, which constitutes another method to induce apoptosis. It was also indicated that T. gondii could influence the less-studied TRAIL pathway, whose important role of apoptosis-regulation is worth further in-depth research. Overall, T. gondii infection is a signal for apoptosis to eliminate impaired cells and limit parasite dissemination, but the parasite can obstruct cell death to survive and propagate. Apoptotic genesis is not only associated with the strain type of the parasite but is also dependent on cell metabolism and sensitivity. 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J Zhejiang Univ-Sci B (Biomed & Biotechnol) , 18 ( 6 ): 453 - 461 . https://doi.org/10.1631/jzus.B1600316 https://doi.org/10.1631/jzus.B1600316 Evaluation of potential anti-toxoplasmosis efficiency of combined traditional herbs in a mouse model Kaige DU, Fei LU, and Xunhui ZHUO performed the experimental research and data analysis. Chengzuo XIE, Haojie DING, Yu SHEN, and Yafan GAO analyzed the data. Kaige DU and Xunhui ZHUO wrote and edited the manuscript. Shaohong LU contributed to the study design and editing of the manuscript. All authors have read and approved the final manuscript, and therefore, have full access to all the data in the study and take responsibility for the integrity and security of the data. This research was supported by the National Natural Science Foundation of China (Nos. 81802037 and 81871684), the Qingshan Lake United Fund of Zhejiang Province (No. LQY19H190002), the Zhejiang Provincial Natural Science Foundation of China (No. LY22H190003), the Zhejiang Provincial Program for the Cultivation of High-level Innovative Health Talents, and the Basic Scientific Research Funds of Department of Education of Zhejiang Province (Nos. KYZD202104 and KYYB202101), China. 10.1631/jzus.B2100877 1673-1581（2022）04-0315-13 This research was supported by the National Natural Science Foundation of China (Nos. 81802037 and 81871684), the Qingshan Lake United Fund of Zhejiang Province (No. LQY19H190002), the Zhejiang Provincial Natural Science Foundation of China (No. LY22H190003), the Zhejiang Provincial Program for the Cultivation of High-level Innovative Health Talents, and the Basic Scientific Research Funds of Department of Education of Zhejiang Province (Nos. KYZD202104 and KYYB202101), China. 2021-10-14 2022-01-16 2022-04-15 2022-12-27 2022-03-01 Copyright © Zhejiang University and Springer-Verlag GmbH Germany, part of Springer Nature 2022 2022 浙江大学出版社 Kaige DU, Fei LU, Chengzuo XIE, Haojie DING, Yu SHEN, Yafan GAO, Shaohong LU, and Xunhui ZHUO declare that they have no conflict of interest. This article does not contain any studies with human or animal subjects performed by any of the authors. Compliance with ethics guidelines Data availability statement 浙江大学学报（英文版）（B辑：生物医学和生物技术） 4 23