Introduction

Dense granules (DGs) are characteristic spherical organelles present in the merozoites of Plasrnodium spp., Sarcocystis spp., and Toxoplasma gondii. (Daszak et al. 1993). DGs have mostly been studied in the genus Sarcocystis, from which they have been isolated by subcellular fractionation and characterized and in which they were subsequently shown to be released into the secondary parasitophorous vacuole after host cell invasion (Leriche and Dubremetz 1990). DGs surrounded by a unique membrane are specialized secretory organelles of Apicomplexa parasites. Their number varies from a few DGs (Eimeria) to approximately 20 (Toxoplasma, Neospora) (Mercier et al. 2005). After invasion of the host cell, the contents of DGs are secreted into the parasitophorous vacuole (PVs); the dense granule proteins are considered to remodel the PVs and have important functions in parasite–host interrelation (Cesbron-Delauw et al. 1996).

Among the GRA proteins, GRA7 is expressed by all infectious forms of Toxoplasma (Ferguson et al. 1999), and anti-GRA7 antibodies are generated during murine and human infection (Carey et al. 2000; Neudeck et al. 2002). GRA7 has been detected on the surface of infected host cells (Neudeck et al. 2002). Within the infected host cell, GRA7 localizes to the PV lumen, the PV membrane, and strands extending from the PV membrane into the host cytosol (Fischer et al. 1998; Coppens et al. 2006).

Until now, limited information is available concerning the characterization of GRA proteins in Eimeria (Bonnin et al. 1995), with no report of sequence or function for these proteins. Therefore, we want to know if GRA7 of T. gondii could be expressed in E. tenella. In this paper, we report the expression of the GRA7 in E. tenella and localization of its protein products to different compartments of different stages of the parasites.

Materials and methods

Parasite and cell culture

E. tenella (BJ strain) was maintained and propagated in coccidia-free, 2–5-week-old AA broilers (Yan et al. 2009; Yin et al. 2011). Oocysts were collected from feces of chickens 6–9 days post-infection and were isolated, purified, and sporulated as described previously (Long et al. 1976; Huang et al. 2011). Sporozoite excystation and purification were achieved using a previously described method. Freshly purified E. tenella sporozoites were resuspended in cytomix buffer supplemented with 2 mM ATP and 5 mM glutathione (van den Hoff et al. 1992; Kelleher and Tomley 1998).

Primary chicken kidney (PCK) cells were used for in vitro transient transfection experiments. PCKs were cultured in DMEM medium supplemented with fetal bovine serum (10 %, v/v) and 1,000 U penicillin/streptomycin in a humidified atmosphere of 5 % CO2 at 41 °C.

Plasmid constructs

The vector pTgtubYFP-YFP/sagCAT plasmid (Gubbels et al. 2003) carries the tandem yellow fluorescent protein (YFP) gene and possesses the upstream region of the T. gondii tubulin gene. The coding sequence of GRA7 protein was amplified by PCR from the cDNA of the T. gondii (RH strain) parasites with the primers 5′-AGATCTATGGCCCGACACGCAATTT-3′ and 5′-CCTAGGCTGGCGGGCATCC TCCCCA-3′. The resulting fragment, bearing the BglII and AvrII restriction sites, was cloned into the pEASY-Blunt Simple Cloning Vector (TransGen Biotech, Beijing, China) and then inserted to the N-terminal of the YFP gene of the ptubYFP-YFP/sagCAT plasmid. The resulting recombinant plasmid was named pTgtubGRA7-YFP/sagCAT (Fig. 1). The pEtmic1GRA7-YFP/act plasmid was constructed from the pEtmic1YFP-YFP/act plasmid (Zou et al., unpublished data). First, GRA7-YFP fusion gene was amplified by PCR from the plasmid pTgtubGRA7-YFP/sagCAT with the primers 5′-GATGGTACCATGGCCCGACACG CAATTT-3′ and 5′-GGGCGGCCGCCTAAAGCTTCTTGTACAGCT-3′. The product was cloned into KpnI and NotI-digested pEtmic1YFP-YFP/act to produce the final expression vector pEtmic1GRA7-YFP/act (Fig. 1).

Fig. 1
figure 1

Schematic diagram of the expression vectors. Plasmid-containing tubulin gene promoter (Tgtub) from T. gondii and microneme protein 1 gene promoter (Etmic1) from Eimeria tenella, respectively

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Transfection

For plasmid transfection, five million sporozoites or tachyzoites, 10 μl linearized plasmid DNA together with 5 μl SnaBI were subjected to Nucleofector transfection. After AMAXA (Switzerland) nucleofection (ProgramU-033) (Clark et al. 2008; Yin et al. 2011), the transfected tachyzoites were then inoculated onto Vero (African green monkey kidney) cells in a humidified atmosphere of 5 % CO2 at 37 °C, and the electroporated sporozoites were inoculated onto 25-cm2 flasks (Corning, Costar, USA) with PCKCs, which were cultured in DMEM medium supplemented with fetal bovine serum (10 %, v/v) and 1,000 U penicillin/streptomycin in a humidified atmosphere of 5 % CO2 at 41 °C.

For the in vivo experiment, 2 × 106 electroporated sporozoites were inoculated via the cloacal route (Clark et al. 2008). The caecal mucous membranes of infected chickens were scraped and viewed under the fluorescence microscope to detect various fluorescent endogenous stages of E. tenella at 120 h post-inoculation (p.i.). Oocysts in feces excreted between days 6 and 9 post-inoculation were collected and checked by a fluorescence microscopy (Shi et al. 2008; Yan et al. 2009; Yin et al. 2011).

Fluorescence observation

The transfected T. gondii and E. tenella were investigated using a fluorescence microscope (Olympus, IX71, Tokyo, Japan) with 488-nm excitation and 508-nm emission filters, under which, the expressed fluorescence appeared green (Hao et al. 2007; Yin et al. 2011).

Results and discussion

Expression of GRA7 in transfected T. gondii tachyzoites

To confirm the expression and localization of the GRA7-EYFP fusion protein in T. gondii tachyzoites, T. gondii tachyzoites were transfected with the pTgtubGRA7-YFP/sagCAT construct. Fluorescence was detected in the transfected parasites throughout the observation period (12–120 h after electroporation) (Fig. 2). At 12 h p.i., only a few tachyzoites displayed weak fluorescence. By 24 h, each tachyzoite expressing YFP multiplied to two or four tachyzoites, all of which remained fluorescent (Fig. 2a). Between 48 and 72 h, greater fluorescence was displayed by the tachyzoites expressing YFP (Fig. 2b, c). Fluorescence was expressed mainly into PVs of the parasites (Fig. 2); this is consistent with the previous report (Ferguson et al. 1999; Carey et al. 2000; Neudeck et al. 2002). When the tachyzoites were transfected with the ptubYFP-YFP/sagCAT plasmid, the fluorescence was uniform, expressed mainly into the cytoplasm of the parasites (Zou et al. 2009).

Fig. 2
figure 2

Expression of GRA7-YFP protein in T. gondii transfected with the plasmid pTgtubGRA7-YFP/sagCAT. Transfected tachyzoites were inoculated onto PCK cells and observed by fluorescence microscopy during different times. The images show that the fluorescent protein was targeted into the PV by GRA7. Multiplication of tachyzoites in the vacuoles could be observed post-inoculation. a Four tachyzoites at 24 h post-inoculation (p.i.), b 16 tachyzoites 48 h p.i., c 32 tachyzoites 64 h p.i. Bar = 20 μm

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Transient expression of GRA7 in E. tenella

The tubulin gene promoter of T. gondii is a strong promoter in T. gondii, and it also worked in E. tenella (Zou et al. 2009), but the transfection efficiency was so low that it was difficult to get the transgenic parasites, especially the oocysts. Then, we used the MIC1 promoter of E. tenella to drvie the expression of the foreign gene. At 16 h p.i., sporozoites expressing YFP were observed in parasites transfected with pEtmic1GRA7-YFP/act. Fluorescence was expressed mainly into PVs of the parasites (Fig. 3a), immature first-generation schizonts expressing YFP were observed at 42–60 h p.i. (Fig. 3b), and thin membranous extensions of PV into the host cytosol (MEHC) can also be observed (data not shown). Same structures, called duct-like structures, were observed in stages of sporozoites and immature schizonts by Zgrzebski et al. (1993), who described that the duct-like structures always extend from the posterior pole of the sporozoite and the PV, marking the pathway of the parasite after host cell invasion. In a previous study by Shi et al. (2009), they also observed MEHC, and they postulated that MEHC provides a trafficking pathway between the parasite and the host cell via which macromolecules can passively diffuse into or out of the parasite (Shi et al. 2009). The immature second-generation schizonts still expressed the YFP into the PVs (Fig. 3c), but the mature schizonts expressed YFP into the parasite to form small particles (Fig. 3d). In the stage of the sporulated oocysts, YFP was targeted into the cavity of the sporocysts (Fig. 3e). As for the extracellular sporozoites, YFP gathered to form small particles in the parasite (Fig. 3f). When the sporozoites were transfected with the pEtmic1YFP-YFP/act plasmid, which has no GRA7 gene sequence, the fluorescence was uniform in the transfected parasites, suggesting a cytoplasmic localization of the reporter protein (data not shown).

Fig. 3
figure 3

Expression of GRA7-YFP protein in E. tenella transfected with the plasmid pEtmic1GRA7-YFP/act. Transfected sporozoites were inoculated with host cells (PCK cells) and observed by fluorescence microscopy during different times. a PV harboring sporozoite. b Immature first-generation schizonts in PVs. c Immature second-generation schizonts in PVs. d A second-generation schizont with releasing merozoites. e Sporulated oocysts with the fluorescent protein into the cavity of the sporocysts. f Released sporozoites from the oocysts. Bar = 20 μm

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In a previous study, fluorescent protein can be targeted into the PVs of E. tenella by signal peptide of GRA8 derived from T. gondii, but no fluorescent zoites and oocysts were observed, indicating that YFP is only targeted to the PVs in that system (Shi et al. 2009).

In our study, we utilized the GRA7 coding sequence, including the signal sequence to target the fluorescent protein, and we got the transgenic oocysts. In the intracellular stage, GRA7-YFP fusion protein was targeted into the PVs of the sporozoites and the immature schizonts; during the extracellular stage, YFP gathered to form small particles in the released merozoites and sporozoites; the small particles in the parasites may be the secretory organelles of the E. tenella, but the detailed pathway needs to be determined in future study. Interestingly, the fusion protein could be secreted into the cavity of the sporocysts in sporulated oocysts. The ability to secret foreign proteins by GRA7 is of great importance for developing transgenic eimerian parasites into a eukaryotic vaccine vector, as researches indicated that secreted antigens were superior in inducing protective immunity (Hess et al. 1996; Shen et al. 1998). The experiments described in this paper suggest that a system using E. tenella to study the localization of T. gondii antigen is appropriate; in addition, our results suggested that GRA proteins have a conserved function across species of Apicomplexa in targeting proteins to the PV.