Toxoplasma gondii dense granule protein 15 induces apoptosis in choriocarcinoma JEG-3 cells through endoplasmic reticulum stress

Background Toxoplasma gondii, a single-celled parasite commonly found in mammals, has been shown to induce trophoblast cell apoptosis and subsequently cause fetal damage and abortion. Although dense granule protein 15 (GRA15) has been identified as a key component in innate immunity to T. gondii infection and its pathogenesis, its role in host cell apoptosis remains unclarified. Methods Type II GRA15 (GRA15II) cDNA was inserted into a plasmid encoding enhanced green fluorescent protein (pEGFP). Choriocarcinoma JEG-3 cells were transfected with either pEGFP or pEGFP-GRA15II and cultured for 24 h. Cell apoptosis and endoplasmic reticulum stress (ERS) responses were assessed. Inhibitors targeting inositol-requiring kinase 1α (IRE1α; 4μ8C, 100 nM) or c-Jun N-terminal kinase (JNK; SP6000125, 20 μM) were added 12 h after plasmid transfection, followed by testing the effect of GRA15II on ERS. Results When compared to pEGFP, pEGFP-GRA15II transfection facilitated cell apoptosis (P < 0.05), increased mRNA expression of caspase-3, caspase-4, 78-kDa glucose-regulated protein (GRP78), C/EBP homologous protein (CHOP) and X-box binding protein-1 (XBP1) (all P < 0.05), and promoted protein expression of cleaved caspase-3, cleaved poly(ADP-ribose) polymerase, Bax, CHOP, GRP78, phospho-JNK, and phospho-IRE1α (all P < 0.05). The 4μ8C and SP6000125 decreased apoptosis and protein expression of XBP1s, CHOP, TNF receptor-associated factor 2 (TRAF2), phosphorylated apoptosis signal-regulating kinase 1 (ASK1), cleaved caspase-3, phospho-JNK, and Bax (all P < 0.05) in pEGFP-GRA15II transfected cells. Conclusions Toxoplasma GRA15II induced ERS and subsequently caused apoptosis of choriocarcinoma JEG-3 cells.


Background
Toxoplasma gondii is an obligate intracellular singlecelled parasite that can invade all warm-blooded animals worldwide [1]. The strains of T. gondii circulating in Europe and North America can be grouped into three distinct genotypes, strains of Type I, Type II and Type III, according to the population structure [2][3][4][5]. During invasion, proteins from parasite organelles such as rhoptry proteins (ROPs) and dense granule proteins (GRAs) are released into host cells and are able to cause significant host damage [6,7]. The genotype/strain polymorphism of ROP16 and GRA15 have both been widely observed in the literature [8]. It has been reported that ROP16 from type I RH stain (ROP16 I ), but not from type II ME49 stain (ROP16 II ), could directly phosphorylate the signal transducer and activator of transcription STAT3 and STAT6, and subsequently polarize macrophages to an M2 phenotype. In addition, GRA15 from type II ME49 strain (GRA15 II ), but not from type I RH strain (GRA15 I ), could phosphorylate nuclear factor-kappa B (NF-κB), and subsequently drive macrophages to an M1 phenotype [9]. We have previously shown that both ROP16 I and GRA15 II were present in the majority of T. gondii Chinese 1 strains found in China [10][11][12][13][14].
Toxoplasma gondii can hijack host cell apoptotic machinery and promote either an anti-or pro-apoptotic program depending on the parasite virulence and load, as well as the host cell type [15]. In the literature, increased apoptosis following Toxoplasma infection has been observed in spleen cells [16], neuronal cells [17] and choriocarcinoma cells [18]. Previously, we found that endoplasmic reticulum stress (ERS) is involved in T. gondii-induced apoptosis [19,20], and that ROPs could trigger ERS-mediated apoptosis [21,22]. However, the effect of GRAs (e.g. GRA15) on host cell apoptosis remains unclear.
Importantly, maternal Toxoplasma infection may give rise to congenital transmission of the parasite to the fetus through the placenta [23][24][25][26] and/or via interfering with the immune tolerance on maternal-fetal interface. Our previous studies indicated that infection with TgCwh3 (a virulent strain of Chinese 1) induced apoptosis of trophoblast cells, and subsequently caused adverse pregnancy outcomes in mice [27]. Angeloni et al. [18] observed that ME49 (type II)-infected BeWo cells become more susceptible to apoptosis than RH (type I)-infected BeWo cells. In view of the M1 bias induced by GRA15 II , we postulated that a GRA15 II -induced NF-κB-dependent proinflammatory cytokine profile is more likely to cause cell apoptosis when compared to a ROP16 I -induced STAT3/STAT6dependent proinflammatory cytokines [18,28]. Here, we

Plasmid construction and transfection
A plasmid encoding enhanced green fluorescent protein-C2 (pEGFP-C2) was purchased from BD Biosciences (Franklin Lakes, NJ, USA). The open reading frame encoding TgGRA15 II (omitted signal peptide of 1500 bp; http://toxodb.org) cDNA was reconstituted by RT-PCR using total RNA isolated from TgCtwh3 tachyzoites. The pEGFP-GRA15 II plasmid was constructed by inserting TgGRA15 II cDNA into the pEGFP plasmid as previously described [29]. JEG-3 cells were plated in 96-well plates (Corning, Corning, NY, USA) at a density of 10 4 cells/ ml, cultured for 24 h, then transfected with either pEGFP or pEGFP-GRA15 II using Lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions.

The presence of GRA15 II
The expression of green fluorescent protein (GFP) was recorded using fluorescent microscopy (Olympus BX60, Tokyo, Japan) 24 h after transfection. The presence of

Cell viability and apoptosis
Twenty-four hours following transfection, JEG-3 cell viability was measured using the Cell Titer 96 Aqueous One Solution Cell Proliferation assay kit (MTS, Promega, Madison, WI, USA) according to the manufacturer's instructions. A phycoerythrin-annexin V apoptosis detection kit (flow cytometry based assay, BD Biosciences, Franklin Lakes, NJ, USA) was used to determine cell apoptosis. Briefly, cells were washed twice using cold phosphate buffered saline (PBS) and suspended in binding buffer (100 μl). Phycoerythrin-annexin V (5 μl) and 7-aminoactinomycin D (7-AAD, 5 μl) were added to the suspension and incubated for 15 min at room temperature, followed by addition of binding buffer (400μl). A flow cytometry assay was conducted within 1 h using FACSVerse (BD Biosciences) with FCS Express 4.0. Annexin V + /7-AADrepresented early apoptotic cells and annexin V + /7-AAD + represented late apoptotic cells. Untransfected cells served as a negative control. Cells treated with staurosporine   Table 1 were synthesized by Shenggong Biotechnology (Shanghai, China). Gene expression levels were normalized to glyceraldehyde-3phosphate dehydrogenase (GAPDH) levels and data was analyzed using the 2 -ΔΔCt method.

Statistical analysis
A two-tailed independent Student's t-test (GraphPad Prism 5.0 software, GraphPad Prism, San Diego, CA, USA) was used to determine the differences between control and pEGFP-GRA15 II -transfected JEG-3 cells. Data are presented as the mean ± standard error (SE). All statistical tests were considered as significant at P < 0.05.

Results
The presence of GRA15 II To investigate whether the pEGFP-GRA15 II construct could be expressed in JEG-3 cells, the expression of GRA15 II protein was determined in both pEGFP-GRA15 II -and pEGFP-transfected JEG-3 cells. GFP fluorescence was detected in both pEGFP and pEGFP-GRA15 II -transfected JEG-3 cells at 24 h (Fig. 1a). pEGFP-transfected cells had an increased fluorescence signal when compared to pEGFP-GRA15 II -transfected cells. The GFP protein (28 kDa) and the GFP-GRA15 II fusion protein (85 kDa) were expressed in JEG-3 cells, 24 h after transfection (Fig. 1b).

Discussion
Toxoplasma gondii infection can cause abortion, preterm delivery, stillbirth and fetal abnormalities in pregnant animals and humans [30,31] through three possible mechanisms. First, Toxoplasma gondii can be directly transferred to the fetus through the placenta and cause a congenital infection [25]; secondly, maternal physiological and immunological disorders caused by Toxoplasma gondii infection can adversely affect fetal development [26]; and thirdly, Toxoplasma gondii can induce apoptosis in placental cells [27]. It has been found that the majority of host cells act as bystanders during an acute infection, and apoptosis of these host cells may result from the secretion of certain soluble factors by parasite-infected cells [32,33]. The composition of the T. gondii excreted-secreted antigens (ESAs) is surprisingly complex and only a few microneme proteins, ROPs and GRAs, have been identified [34,35]. ROP16-and ROP18-induced host cell apoptosis has been previously reported [21,22]. In this study, GRA15 II transfection significantly increased apoptosis and decreased cell viability in choriocarcinoma JEG-3 cells as early as 24 hours post-transfection. These results suggest that GRA15 II , which exists in Toxoplasma gondii strains such as ME49 (type II Toxoplasma gondii) and ToxoDB#9 (major Toxoplasma gondii strain in China), is a virulence antigen. The GRA15 II -induced apoptosis was accompanied with ERS in choriocarcinoma JEG-3 cells. The mRNA transcription and protein expression level of GRP78, a key ERS sensor protein, were significantly increased by GRA15 II transfection at 24 hours. The finding indicates that GRA15 II -induced apoptosis at least partially resulted from ERS. In other studies, PERK, activating transcription factor (ATF) 6, and IRE1α have been proposed as three major proteins downstream of GRP78 signaling during ERS [36,37]. In the current study, the expression level of phospho-IRE1α, but not PERK or ATF6, was increased by GRA15 II transfection. In line with this, the GRA15 II -related ERS/apoptosis was mainly induced by the GRP78-IRE1α signaling pathway. It has been reported that the activation (phosphorylation) of IRE1α could lead to apoptosis by either mediating the splicing of XBP1 to XBP1s, and subsequently increasing the expression of CHOP [38], or recruiting TRAF2, activating ASK1, and stimulating JNK [39]. In the current work, we found the expression of proteins in both of these pathways was significantly increased by GRA15 II transfection. Thus, the result clearly demonstrates that both the IRE1α-XBP1-CHOP and IRElα-TRAF2-ASK1-JNK pathways contributed in GRA15 IIinduced apoptosis as proposed schematically in Fig. 7. We further illustrated this hypothesis by treating the GRA15 II -transfected choriocarcinoma JEG-3 cells with 4μ8C (IRElα inhibitor) and SP6000125 (JNK inhibitor); both 4μ8C and SP6000125 suppressed the expression levels of apoptosis-associated proteins in GRA15 II -transfected choriocarcinoma JEG-3 cells, and subsequently decreased apoptosis and increased cell viability. Fig. 7 Schematic diagram of the signaling pathways involved in GRA15 II -induced apoptosis in choriocarcinoma JEG-3 cells. Abbreviations: GRA15, dense granule protein 15; ERS, endoplasmic reticulum stress; IRE1α, inositol requiring kinase 1; XBP1, X-box binding protein-1; CHOP, C/EBP homologous protein; TRAF2, TNF receptor-associated factor 2; ASK1, apoptosis signal-regulating kinase 1; JNK, c-Jun N-terminal kinase During pregnancy, fetal development is directly related to the proliferation, differentiation, and apoptosis of trophoblast cells [23,24]. Increased trophoblast cell apoptosis could be damaging to fetal health and even cause adverse pregnancy outcomes [40,41]. The above finding contributes novel knowledge to our current understanding in regards to Toxoplasma gondii-induced apoptosis, and may help to illustrate the underlying mechanism of Toxoplasma gondii-induced pregnancy failure. The objective of the current study was to determine whether ERS was involved in GRA15 II -induced apoptosis in choriocarcinoma JEG-3 cells. It was not investigated whether ERS was the only (or the major) factor that caused apoptosis in GRA15 II -transfected choriocarcinoma JEG-3 cells. It is possible that other pathways (e.g. mitochondrial pathway, death receptor pathway) may also contribute to GRA15 II -induced cell apoptosis, and this will need to be investigated in future studies. Additionally, the host target protein of GRA15 II remains unknown. There may be some differences in "normal" in vivo cells when compared to the choriocarcinoma JEG-3 cells that were used in the current study.

Conclusions
Toxoplasma-derived GRA15 II increased the expression of ERS-and apoptosis-associated proteins in choriocarcinoma JEG-3 cells. GRA15 II -induced ERS and apoptosis were alleviated by treatment with 4μ8C and SP6000125. GRA15 II induces apoptosis at least partially through endoplasmic reticulum stress.