E．tenella was obtained from the Key Laboratory of Animal Parasitology of the Ministry of Agriculture, Shanghai Veterinary Research Institute, the Chinese Academy of Agricultural Sciences，Shanghai, China. The parasites were maintained and propagated by passage through coccidia-free, 2-week-old chickens, as previously described . Coccidia-free 14-day-old chickens were inoculated with 1 × 104 sporulated oocysts of E. tenella. Unsporulated oocysts (UO) were collected from infected chicken ceca at 7 days postinfection. Sporulated oocysts (SO) were derived from UO that had undergone sporulation in 2% potassium dichromate at a temperature 28–30 °C for 72–120 h, under forced aeration with a suitable pump. When more than 90% of the oocysts had sporulated, the oocysts were collected and purified. The sporozoites (Spz) were purified from cleaned SO with in vitro excystation . Second-generation merozoites (sMrz) were isolated from infected chicken ceca at 115 h postinoculation, as described previously . All parasites were collected and frozen in liquid nitrogen.
Chickens and rabbits were fed and used according to a protocol approved by the Animal Care and Use Committee of the Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences.
The chicken embryo fibroblast cell line, DF-1, a derivative of the East Lansing Line (ELL-0) (Jiang et al. 2012), was used for BiFC and in vitro infection experiments.
Molecular cloning and sequence analysis of E. tenella-specific protein
Total RNA was extracted from E. tenella sporozoites with TRIzol Reagent (Invitrogen, USA). GeneRacer™ primers (GR5P and GR5N) were provided for the random amplification of PCR ends (RACE) in the GeneRacer™ Kit (Invitrogen) and gene-specific primers (GS5P and GS5N) were designed based on the expressed sequence tag (EST) sequence (GenBank accession number: JZ905773) which is 790 bp in length and contains a poly(A) at the 3¢ end (Table S). The 5¢ end of this gene was determined according to the manufacturer’s protocol. The PCR-amplified fragment was then ligated into the pGEM-T Easy Vector (Promega, USA) and used to transform competent Escherichia coli TOP10 cells. After PCR identification, the plasmid DNA was sequenced. After the resulting sequence was assembled and aligned with the original EST sequence, the full-length cDNA sequence of the gene was determined and submitted to the National Center for Biotechnology Information (NCBI) GenBank (accession number: MN161778). The full-length EtEsp cDNA sequence was used in a BLAST search of GenBank (http://www.ncbi.nlm.nih.gov/BLAST/) and the E. tenella genome database (http://www.genedb.org/Homepage/Etenella). The deduced amino acid sequence was obtained with the ORF Finder tool at NCBI. The molecular mass and theoretical isoelectric point were calculated with ProtParam tools (http://web.expasy.org/protparam/). The signal peptide sequence was identified with the SignalP 4.1 server (http://www.cbs.dtu.dk/services/SignalP/), and transmembrane regions were predicted with the TMHMM server v. 2.0 (http://www.cbs.dtu.dk/services/TMHMM/). Protein motifs were scanned with Motif Scan (http://myhits.isb-sib.ch/cgi-bin/motif_scan).
Recombinant protein expression and polyclonal anti-rEtEsp serum
The EtEsp open reading frame (ORF) cDNA was amplified with PCR using primers EtEsp-UP and EtEsp-LP (Table S), which contained BamHI and XhoI restriction sites, respectively. The PCR fragment was then ligated into the prokaryotic expression vector pET28a(+) digested with the same restriction endonucleases, to construct the recombinant expression plasmid pET–EtEsp. The recombinant protein His–EtEsp (rEtEsp) was expressed in Escherichia coli BL21 cells at 37 °C with 1 mM isopropyl-thio-α-d-galactoside. The cell pellet was lysed with sonication and digested with 10 µg/mL lysozyme (Sigma-Aldrich, USA). The lysate was then analyzed with 12% SDS-PAGE to confirm that the recombinant protein was present as a soluble protein or inclusion bodies. rEtEsp was purified with His·Bind® Resin (Merck, USA) and its concentration measured with a BCA Protein Assay Kit (Beyotime, China).
Two 2-month-old male rabbits were inoculated with 200 μg of purified rEtEsp emulsified in Freund’s complete adjuvant (Sigma-Aldrich). After 14 days, a booster of 200 μg of purified rEtEsp in Freund’s incomplete adjuvant (Sigma-Aldrich) was administered, followed by a second and third booster on days 28 and 42. One week after the final booster, the rabbit serum was collected and stored at −20 °C until use.
Analysis of EtEsp transcript levels with real-time quantitative PCR (qPCR)
The expression profiles of EtEsp mRNA were examined in four developmental stages of E. tenella (UO, SO, Spz, and sMrz) with qPCR. cDNA samples were synthesized from DNaseI-treated total RNAs of the E. tenella developmental stages using SuperScript™ II Reverse Transcriptase (Invitrogen) and random pd(N)6 primer. The housekeeping gene 18S rRNA was used as the internal control. The primers used to amplify the EtEsp cDNA (EtEsp-SP and EtEsp-AP) and the 18S rRNA gene (18S-SP and 18S-AP) were designed with Primer3 v. 0.4.0 (http://bioinfo.ut.ee/primer3-0.4.0/) (Table S). qPCR was performed with the StepOnePlus™ Real-Time PCR System using the SYBR® Premix Ex Taq™ II kit (Takara, Japan). All experiments were performed twice, with separate biological replicates. In each experiment, the reactions were performed in triplicate. A dilution series of cDNA templates of the sporozoites was used to establish standard curves, and all standard curves had correlation coefficients of R2 > 0.99. The comparative 2−ΔΔCt method was used to analyze the relative levels of gene expression.
SDS-PAGE and western blotting
Protein samples were prepared from the four E. tenella developmental stages (UO, SO, Spz, and sMrz), and from DF-1 cells transfected with the recombinant plasmids, for western blotting. The protein concentrations were determined with a BCA Protein Assay Kit (Beyotime,China). The purified rEtEsp and protein lysates were separated with SDS-PAGE and transferred electrophoretically to polyvinylidene difluoride membranes. Rabbit antiserum (1:100) against sporozoite proteins, previously produced in our laboratory , a rabbit anti-rEtEsp antibody (1:100), a mouse monoclonal anti-α-tubulin antibody (1:1000) (Sigma-Aldrich), and a monoclonal anti-His antibody (1:1000) were used as the primary antibodies to detect rEtEsp or native EtEsp. Naïve rabbit serum (1:100) was used as the negative control. IRDye-800CW-labelled goat anti-rabbit IgG antibody (1:25,000) and IRDye-680RD-labeled donkey anti-mouse IgG antibody (1:25,000; LI-Cor, Lincoln, NE, USA) were used as the secondary antibodies. The IRDyes were detected with the Odyssey Infrared Imaging System (LI-Cor).
The ORF fragments of EtEsp and the EtAMA1 ectodomain, with no stop codon, were amplified from the first-strand cDNA with two pairs of primers (BfEtEsp-UP/BfEtEsp-LP and BfEtAMA1-UP/BfEtAMA1-LP, respectively), which contained EcoRI and BglII restriction sites (EtEsp) or EcoRI and XhoII restriction sites (EtAMA1). The fragments were then digested with the appropriate restriction enzymes and ligated into the pBiFC-VN155 and pBiFC-VC155 vectors digested with the same enzymes, respectively, to construct the recombinant plasmids pBiFC-VN155–EtEsp and pBiFC-VC155–EtAMA1, respectively. Before the BiFC assay, the uptake of the expression vectors by the cells was confirmed. DF-1 cells were transfected with the recombinant plasmid pBiFC-VN155–EtEsp or pBiFC-VC155–EtAMA1 using Lipofectamine™ 2000 Transfection Reagent (Invitrogen, USA), according to the manufacturer’s instructions. At 48 h after transfection, the cells were harvested and the proteins were extracted with RIPA Lysis Buffer (Beyotime). Western blots were probed with rabbit anti-rEtEsp antibody and rabbit anti-rEtAMA1 antibody, which were previously produced in our laboratory . After confirmation that the cells had expressed the two constructs, DF-1 cells were cotransfected with pBiFC-VN155–EtEsp and pBiFC-VC155–EtAMA1. DF-1 cells were also cotransfected with pBiFC-bJunVN55 (I152L) and pBiFC-bFosVC155 or pBiFC-bJunVN55 (I152L) or pBiFC-bFos(deltaZIP)VC155 vector as the positive or negative control, respectively. The DF-1 cells were observed with ﬂuorescence microscopy 24 h after transfection with the different constructs.
To confirm the interaction between EtAMA1 and EtEsp317 in vitro, a GST pull-down assay was performed with the Pierce™ GST Protein Interaction Pull-Down Kit (Thermo Scientific, USA), according to the manufacturer’s instructions. The recombinant plasmid pGEX-6P–EtAMA1 was previously constructed in our laboratory . The expression of the recombinant protein GST–EtAMA1 was induced and the protein purified with GST resin for use as the bait protein. The ORF of EtEsp was inserted into the pET-28a vector to express the recombinant protein His–EtEsp (rEtEsp) as the prey protein. GST–EtAMA1 was incubated with equilibrated glutathione-agarose to immobilize the bait protein. rEtEsp was then added to the glutathione-agarose and incubated with the bait protein. The bait and prey proteins were eluted from the glutathione-agarose. Escherichia coli BL21 cells were transformed with recombinant plasmid pET–EtMIC2, constructed previously in our laboratory , to express the recombinant protein His–EtMIC2 as the negative control. All the proteins were then resolved with 12% SDS-PAGE and detected with western blotting using the appropriate antibodies, as described above.
Assay of EtEsp secretion
Freshly excysted sporozoites (4 × 106) were incubated in 100 μL of complete medium (CM; Dulbecco’s modified Eagle’s medium [DMEM] supplemented with 10% fetal bovine serum [FBS], 100 U/mL penicillin/streptomycin, 2 mM l-glutamine) for 2 h at 41 °C under 5% CO2 for the secretion experiments. They were then incubated with 5, 10, or 20 μM staurosporine (Sigma; dissolved in dimethylsulfoxide [DMSO]) or an appropriate volume of carrier DMSO, as described previously . The secretion of EtMIC2 was used as the control. The sporozoites were then pelleted by centrifugation for 10 min at 6000 × g. The supernatants were recovered and analyzed with western blotting using a rabbit anti-rEtEsp antibody and rabbit anti-rEtMIC2 antibody generated previously in our laboratory .
Immunofluorescence localization analysis
Purified differentially developed parasites (Spz, sporocysts [Spo], and sMrz) were transferred to glass slides and air-dried, as previously described [10, 29]. Purified freshly sporozoites were infected DF-1 cells after incubation in CM for 2 h at 41°C. At different time points after infection, the DF-1 cells were collected, washed, transferred to glass slides, and air-dried. The slides were then fixed in 2% paraformaldehyde in phosphate-buffered saline (PBS) and placed in 1% Triton X-100 in PBS for 15 min to increase their permeability. The slides were blocked with PBS containing 2% (w/v) bovine serum albumin for overnight at 4 °C. A rabbit anti-rEtEsp antibody (1:100) was added and the cells were incubated for 1 h at 37 °C. A 1:500 dilution of fluorescein isothiocyanate (FITC)-conjugated goat anti-rabbit IgG antibody (Sigma-Aldrich) was then added and the cells incubated for 1 h at 37 °C. The cell nuclei were stained by incubation in 10 μg/mL 4¢,6-diamidino-2-phenylindole (Beyotime) at room temperature for 10 min. After each step, the slides were washed three times for 10 min each with PBS containing 0.05% Tween 20. The slides were finally mounted with 50 μL of Fluoromount Aqueous Mounting Medium (Sigma-Aldrich) before observation with a fluorescence microscope (Olympus, Tokyo, Japan).
Invasion inhibition assay in vitro
The invasion inhibition assay was based on previous reports the invasion of DF-1 cells by E. tenella sporozoites. Antibodies were purified with Protein A+G Agarose (Beyotime). DF-1 cells (2 × 105 cells per well) were cultured in 24-well plates (Corning) in CM for 24 h at 37 °C under 5% CO2. The freshly purified sporozoites were counted and labeled with carboxyfluorescein diacetate succinimidyl ester (Beyotime). The labeled sporozoites were incubated at 37 °C with 50, 100, 200, 300 or 400 μg/mL purified IgG directed against rEtEsp for 2 h. The same quantity of IgG from naïve rabbit serum (Sigma-Aldrich) was used as the negative control, and an equivalent volume of PBS as the normal control. After they were washed twice with sterile PBS, DF-1 cells (105/well) were infected with the labeled sporozoites (105/well) in 24-well plates and cultured for 16 h at 41 °C under 5% CO2. The cells were then collected and analyzed with flow cytometry on a Cytomics™ FC 500 (Beckman Coulter, Indianapolis, IN, USA). The controls were uninfected DF-1 cells. The infected cells, uninfected cells, and free sporozoites were gated with the CXP software to count the infected (labeled sporozoites) and uninfected (fluorescence-free) cells. All assays were performed in triplicate. The percentages of infected cells in the presence or absence of an anti-rEtEsp polyclonal antibody were used to calculate the inhibition rates, as previously described .