All animals and laboratory experiments were strictly performed in accordance with the recommendations of the Animal Ethics Committee, Nanjing Agricultural University, China. All experimental procedures used in this study were permitted by the science and Technology Agency of Jiangsu Province [ID: SYXK (SU) 2010-0005].

The native crossbred goats (3–6 months old) were housed indoor, provided with hay and corn (whole shelled) and water ad libitum. The anti-parasitic drug, levamisole (8 mg/kg body weight) was used with 2 weeks interval to keep goats free from naturally acquired helminth infection. The fecal samples were checked twice per week, using standard parasitological techniques and goats with no sign of helminths infection were used in subsequent experiments.

Sprague-Dawley (SD) rats with average body weight of 150 g, were bought at Experimental Animal Center of Jiangsu, PR China (Certified: SCXK 2008-0004). The rats were raised in microbe free condition.

The H. contortus strain was maintained by serial passage in helminth-free goats, at laboratory of immunology and molecular parasitology, Nanjing Agricultural University. The parasite eggs, larvae, and adults worms were collected and preserved according to the methods stated previously (20). The blood samples were taken from jugular vein of dewormed goats and PBMCs were collected by gradient centrifugation method (21). The PBMCs were cultured as the procedure stated previously (22).

The freshly adults H. contortus parasites were collected from abomasum of the infected goats and the extraction of RNA was carried out using Trizol method according to the previously described procedure (23), followed by cDNA synthesis as per the manufacturer’s guidelines of cDNA synthesis Kit (TaKaRa Biotech), and was preserved at −20°C for down-stream applications.

The open reading frame of HcSTP-1 was amplified by reverse-transcription-polymerase chain reaction (RT-PCR), from conserved domain sequences of H. contortus STP-1 gene (GenBank: GQ280010.1). For the subsequent cloning, underlined BamH I and Xho I restriction enzyme were inserted at the 5′-end of forward primer: (5′-GGATCCATGGACCCTACTCAAT-3′), and reverse primer: (5′-CTCGAGTTATTGACAAGGTGGAGC-3′). The amplified PCR fragment was electrophoresed and eluted with Gel purification Kit (Omega, USA) in accordance with kit protocol. The eluted product was then inserted into pMD19-T cloning vector (TaKaRa Biotechnology, China). The recombinant plasmid pMD19-T/HcSTP-1 was inserted into DH5α competent strain of Escherichia coli and cultured in Luria–Bertini medium with ampicillin (100 µg/ml). The positive clones followed by sequencing (Invitrogen Biotech, Shanghai, China) were validated by sequence analysis online using Blast program.¹ The obtained nucleotide sequence was translated into amino acids sequence and aligned and compared with different nematode species using GENETYX Version 7.0.9 software. In addition, a number of online approaches/programs were used to detect N-terminal signal peptides/http://www.cbs.dtu.dk/services/SignalP/, Transmembrane protein prediction/http://www.cbs.dtu.dk/services/TMHMM/, T cell motifs/DNAstar: EditSeq, Protean, B cell epitopes/http://tools.immuneepitope.org/tools/bcell/iedb_input, and GPI modification Site Prediction/http://mendel.imp.ac.at/sat/gpi/gpi_server.html.

The recognized recombinant pMD19-T/HcSTP-1 plasmid was digested with dual restriction enzymes BamH I and Xho I and ligated into prokaryotic expression vector pET32a (+) (Novagen, USA). Finally, the successful cloned STP-1 gene in a recombinant expression vector was sequenced to confirm its placement in the accurate reading frame. The recombinant plasmid pET32a (+)-HcSTP-1 was transferred into E. coli strain (BL21) and induced with 1 mM isopropyl-β-d-thiogalactopyranoside (IPTG; Sigma-Aldrich) after the OD₆₀₀ of the culture reached 0.6 at 37°C. The cell pellet after centrifugation was lysed using 10 µg/ml of lysozyme (Sigma-Aldrich) followed by sonication, and was resolved on 12% (w/v) sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The purification of recombinant protein was carried out according to the manufacturer’s instructions of Ni²⁺-nitrilotriacetic acid (Ni-NTA) column (GE Healthcare, USA). The histidine-tagged protein (empty pET32a) used as control protein in multiple assays in this study was purified and expressed similar to the procedure described for rHcSTP-1 protein and determined at 12% SDS-PAGE after Coomassie blue staining and quantified by Bradford method (24).

Polyclonal antibodies against recombinant protein were generated from SD rats by subcutaneous injection of 300 µg of rHcSTP-1 protein mixed equally with Freund’s complete. After 2 weeks of first immunization, rats were injected three times at 1 week interval with same protein and Freund’s incomplete mixture. The sera were collected after 10 days of last injection and preserved at −80°C for later use. The sera against H. contortus parasites were collected from naturally infected goats (25), and sera collected from normal goats or rats were used as negative control.

The SDS-PAGE products of recombinant HcSTP-1 and soluble/ES products from adult parasites, were shifted nitrocellulose membrane (Millipore, USA) for western blot analysis. After blocked with 5% (w/v) skimmed milk powder in TBST (TBS with 0.5% Tween-20) at 37°C for 2 h, the strips were incubated with anti-H. contortus sera from goats or rat anti-sera against rHcSTP-1 (1:300 dilutions) as first antibody for treatment groups and normal goat serum or normal rat serum for control groups at 4°C overnight. Then, the membranes were incubated with the secondary antibody, HRP-conjugated goat anti-rat IgG (Santa Cruz, USA) in TBST (1:3,000 dilutions) at 37°C for 2 h. At end, the immunoreactions were visualized within 3–5 min, as per defined protocol of DAB Horseradish Peroxidase Color Development Kit (Beyotime Biotechnology).

The mature parasites were suspended in TISSUE-TEK^® O.C.T. compound (SAKURA, Torrance, CA, USA) and snap-frozen in liquid nitrogen. By using cryotome (CM1950, Wetzlar, Germany), worms were cut into sections with 10-µm thickness and fixed on poly-l-lysine hydrobromide glass slides. Non-specific bindings were confiscated by treating the slides with 10% normal goat serum, followed by incubation with rat-anti-STP-1 antiserum (1:300 dilutions) or normal rat serum as first antibody and Cy3-labeled Goat Anti-Rat IgG (1:3,000 dilutions) as second antibody (Beyotime, Shanghai, China) at 37°C for 2 h in each step. For DNA staining, the sections were marked with 1.5 µM 2-(4-amidinophenyl)-6-indolecarbamidine dihydrochloride (DAPI: Sigma, MO, USA) for 5 min. Finally, Anti-Fade Fluoromount Medium (Beyotime, Shanghai, China) was used and sections were examined under confocal microscope.

Freshly collected PBMCs, after washed with phosphate buffer saline (PBS: Ca²⁺/Mg²⁺-free, pH 7.4), were maintained at density of 1 × 10⁵ cells/ml in cell culture medium (RPMI 1640), containing 10% fetal bovine serum (FBS), 100 U/ml penicillin and 100 mg/ml streptomycin (Gibco, Life Technology), and incubated with rHcSTP-1 or control protein pET32a or control buffer (PBS) at constant temperature (37°C) and humidity (5% CO₂) for 4 h. Protein binding at surface of goat PBMCs were confirmed by immunofluorescence assay (IFA). Briefly, PBMCs after being washed and stabled with 4% paraformaldehyde on poly-l-lysine-treated slides were then permeabilized using 1% TritonX-100 in PBS at normal temperature. The PBMCs were subjected to primary antibodies rat anti-rHcSTP-1-IgG (1:300 dilutions) or normal rat serum followed by Cy3-coupled goat anti-rat IgG (Beyotime, China) (1:3,000 dilutions) as second antibody for 1 h at 37°C temperature. At last, the slides were stained in darkness with DAPI (Sigma, USA) for 5 min, and subjected to Anti-Fade Fluoromount solution (Beyotime, China) before laser scanning confocal microscopic examination (LSM710, Zeiss, Jena, Germany) at 100× magnifications.

The PBMCs were stimulated with Concanavalin A (ConA/10 μg/ml), and treated with rHcSTP-1 (10, 20, and 40 µg/ml) or pET32a protein (10 µg/ml) and control buffer (PBS) in 24-well plate containing RPMI 1640 medium at 5% CO₂ and 37°C. The PBMCs were collected after 48 h of culture, by centrifugation and PrimeScript™ RT reagent kit (TaKaRa, CA, USA) was utilized to extract RNA from cells sediment as per the manufacturer’s guidelines. The cDNA-based quantifications of cytokine transcriptions were evaluated as previously stated protocol of RT-PCR. The reaction conditions and set of primers used for cytokines [interleukin-2 (IL-2), IL-10, TGF-β1, IL-6, IFN-γ, and IL-17] and endogenous reference gene (β-actin), as well as their amplification efficiencies are mentioned in Tables S1–S3 in Supplementary Material. The data were analyzed based on Raw cycle thresholds (Ct), obtained from the ABI Prism 7500 software (Applied Biosystems, USA) by comparative Ct (2^−ΔΔ Ct) method (26).

After separation of PBMCs by standard Ficoll-hypaque (GE Healthcare, USA) gradient centrifugation and two times washing with the PBS (Ca²⁺/Mg²⁺-free, pH 7.4), were poured into flat-bottom six well culture plates (Corning Inc., USA) containing RPMI 1640 medium (Invitrogen, USA) supplemented with 10% FBS and penicillin + streptomycin (Invitrogen, USA). The monocytes stick to the bottom of the plate, were collected (27) and adjusted at density of 1 × 10⁶ cells/ml, whereas, Non-sticky cells were discarded by multiple washing steps. The purified monocytes were incubated in 24-well culture plates with different concentrations of rHcSTP-1 (treatment group) or pET32a protein and PBS (control group) at 37°C for 24 h. Subsequently, the monocytes were marked with monoclonal antibodies MHC-I (MCA2189A647, AbDserotec, Bio-Rad, USA) and MHC-II (MCA2225PE, AbDserotec), followed by flow cytometric analysis at FACS Calibur cytometer (BD Biosciences, San Jose, CA, USA) (Figure 7A).

The 100 µl of freshly isolated PBMCs at density of 1 × 10⁶ cells/ml was poured into 96-well culture plate and stimulated with ConA (10 µg/ml). The PBMCs were incubated with serial concentrations of rHcSTP-1 protein (10, 20, and 40 µg/ml) in treatment group and pET32a empty protein or control vehicle (PBS) were added into control groups at 5% humidity and 37°C temperature for 72 h. Detection of PBMC proliferation was carried out according to the previously mentioned procedure of cell proliferation assay kit (28). 10 µl of CCK-8 solution (Beyotime China) was mixed in every well of plate 6 h prior to harvest and absorbance were checked by using spectrophotometer (Bio-Rad, USA) at wavelength 450 nm (OD₄₅₀). Result presented here are from three independent experiments.

The migration activity of cultured PBMCs was measured with 8.0-µm pores Millicell^® insert (Merck-Millipore, USA) as stated previously. Briefly, in treatment group 200 µl of freshly separated cells were poured into the upper chamber with various concentrations of rHcSTP-1 (10, 20, and 40 µg/ml) and pET32a control protein or equal volume of control buffer (PBS) were added in control groups. Subsequently, 1,300 µl cell culture medium was filled in the lower chamber for 2 h incubation at 37°C and 5% CO₂. After that, columns were removed, and PBMC passed through polycarbonate layer were figure out by a Neubauer counting chamber. Data were resulted as percentage of seeded PBMCs. Three independent experiments were conducted.

The goat PBMCs were separated and washed with Ca²⁺/Mg²⁺-free PBS (pH 7.4). 100 µl of cells containing DMEM medium was poured in 96-well plate with varying concentrations of rHcSTP-1 (10, 20, and 40 µg/ml), similarly the control groups were incubated with recombinant protein of pET32a or PBS (control buffer). NO production by cells was determined by Griess assay (29), by using Total Nitric Oxide Assay Kit (Beyotime, China) according to the manufacturer’s instructions. The values in the reaction mixture were measured at microplate spectrophotometer (Bio-Rad Laboratories, USA) at 540 nm (OD₅₄₀) wavelength. Data presented here are results of triplicate experiments.

Efficiency of rHcSTP-1 to induce apoptosis of PBMC was assessed by flow cytometric analysis (BD Biosciences, USA). The PBMCs (1 × 10⁷ cells/ml) treated with different rHcSTP-1 protein concentrations (5, 10, 20, and 40 µg/ml) and empty pET32a protein, were stained with Annexin V-FITC kit (Miltenyi Biotec, Germany) according to the manufacturer’s protocols. The apoptosis rate was calculated by the percentage of early (Annexin V⁺ PI−) and late (Annexin V⁺ PI⁺) PBMC apoptosis. Cells without any treatment were set as control. Data were analyzed using Flow Jo 7.6 software (Tree Star, USA).

Data analysis regarding all experiments was performed by using the statistical package, GraphPad Premier 6.0 (GraphPad Prism, San Diego, CA, USA). Data are presented as mean ± SEM. The differences between groups were compared by one-way ANOVA, followed by a Tukey test and were considered statistically significant at p < 0.05.

The RT-PCR generated fragment of STP-1 gene with molecular size of 951 bp, was obtained from H. contortus cDNA and successfully cloned into an expression vector system, confirmed by enzymatic digestion with BamH I and Xho I (Figure 1). After that, cloned HcSTP-1 sequence was translated into 316 amino acids with predicted 35 kDa molecular mass and calculated pI of 6.328. The nucleotide and amino acid sequence identity HcSTP-1 using BLASTx and BLASTp revealed highly sequence resemblance with STPs from other parasite species, existing at NCBI database (see text footnote 1). In this study, characterization of HcSTP-1 cDNA showed 98% significant sequence identity to H. contortus (ADJ96628.1), 80% with C. briggsae (XM_002631635.1) (data not shown). Multiple sequence alignment showed that HcSTP-1 polypeptide shares a significant similarity of 91% with T. vitrinus (GenBank: CAM84506.1), 62% with T. canis (GenBank: KHN86897.1), 62% with Ascaris suum (GenBank: CAJ98743.1), and 58% with Caenorhabditis elegans (GenBank: NP_505086.2). The BLAST analysis predicted number of conserved metal ion-binding sites and histidine residue predicted to be involved in proton donation. The amino acids expressing STP protein motif LRGNHE was found during sequence analysis. The aligned STPs sequences were highly similar in central regions, particularly in term of characteristics sequence motifs, such as GDXHG, GDYVDRG, GNHE, HGG, RG, and H (30), whereas there was a higher variation occurred regarding to N- and C-terminal regions among all align sequences (Figure 1). Further sequence analysis indicated lack of signal peptide, transmembrane domain, and GPI anchor site, whereas T and B cell motifs were predicted in deduced protein structure (Figures S1–S4 in Supplementary Material).

The recombinant product of HcSTP-1 gene inserted into E. coli was expressed after IPTG induction and detected after staining with Coomassie brilliant blue R-250 on SDS-PAGE. The rHcSTP-1 protein was expressed with molecular weight of about 53 kDa (Figure 2, Lane P) along with 18 kDa calculated mass of vector protein (pET32a). The target protein was confirmed at its original size of 35 kDa after reduction from vector protein. Polyclonal antibodies against rHcSTP-1 were identified by immuno-blot analysis, which revealed that rHcSTP-1 could be recognized by sera from goats infected with H. contortus and native STP-1 protein from H. contortus could be recognized by antibodies generated against rHcSTP-1 protein (Figures 2C,D, Lane 1). However, no protein was detected with normal sera taken from un-immunized goats/rats (Figures 2C,D, Lane 2).

The partial body sections of male and female adult worms are shown in Figure 3. Immunohistochemical analysis was performed to detect distribution of native STP-1 protein within the worms. The red lining at outer and inner layer of membranes and in gut region strongly indicated the presence of this protein within worms sections. However, no fluorescence was detected in the control section (Figure 3).

The interaction of HcSTP-1 with PBMCs, which are key elements for immune responses, was investigated by using IFA. As shown in Figure 4, the Cy3-labeled target protein and the DAPI labeled nuclei displayed red and blue fluorescence, respectively. The red visualization on the surface of cells in treatment group (rHcSTP-1) indicated the binding of the protein whereas, no red labeling was detected in the control groups (pET32a or PBS), which clearly specified that rHcSTP-1 could bind on the surface of goat PBMCs and exerts its immune effects in a multiple manners (Figure 4).

The mRNA levels of cytokines produced by cultured goat immune cells (PBMCs) in response to STP-1 protein were tested at RT-PCR machine. As depicted in results of Figure 5, transcriptions level of IL-2 [ANOVA, F_(5,12) = 198.9, p = 0.0001] and transforming growth factor-β1 (TGF-β1) [ANOVA, F_(5,12) = 108.0, p = 0.0001] were increased significantly at dose-dependent manner to that of pET32a protein and PBS control group. However, the IL-10 transcription was decreased [ANOVA, F_(5,12) = 19.04, p = 0.0001] dose dependently to that of control groups. Here, we also noted that interferon gamma (IFN-γ) at protein concentration of 10 µg/ml showed no difference (p = 0.337); however, at 20 µg/ml (p = 0.002) and 40 µg/ml [ANOVA, F_(5,12) = 105.6, p = 0.0001] the transcription level was augmented significantly. After incubation with rHcSTP-1 protein, the expressions of IL-17 at 10 µg/ml reached at its significant level [ANOVA, F_(5,12) = 11.76, p = 0.0003], while at 20 and 40 µg/ml remained non-significant. However, IL-6 was not significantly different [ANOVA, F_(5,12) = 0.604, p = 0.699] when compared with control groups (Figure 5).

Antiproliferative effects of rHcSTP-1, compared to the negative control and control protein treated groups were evaluated by using cell counting kit (CCK8). The results demonstrated that, cells stimulated with ConA and incubated with rHcSTP-1 protein downregulated [ANOVA, F_(5,12) = 67.38, p = 0.0001] the multiplication efficiency dose dependently (Figure 6). However, no significant change was observed between control groups [ANOVA, F_(5,12) = 67.38, p = 0.557].

In response to the rHcSTP-1, the expression of MHC-I and -II surface markers on monocytes were evaluated, and results showed that as compared to expression in control groups, rHcSTP-1 significantly decreased MHC-I (Figures 7B,D) molecule expression [F_(5,12) = 780.3] as well as MHC-II [F_(5,12) = 157.2] on the surface of the goat monocytes in a dose-dependent manner p < 0.0001 (Figures 7C,E).

To investigate impact of the rHcSTP-1 on immune cell migration, the percentage of migrated cells through Millipore polycarbonate membrane into the lower chamber was calculated. Results showed that 16.67 ± 0.8819% cells with control and 16.33 ± 1.764% cells with control protein group (pET32a) were migrated into lower chamber (p = 0.087). At 10 µg/ml (27.00 ± 1.528%), 20 µg/ml (32.67 ± 2.603%), and 40 µg/ml (35.00 ± 2.309%) rHcSTP-1 protein concentrations, significant amount of cells were passed through membrane (Figure 8). However, no difference was found between control and 5 µg/ml (20.00 ± 1.155%) protein concentration (p = 0.084).

Nitric oxide plays a crucial role in the host defense mechanism by preventing growth or killing the parasite directly during infection. By using total nitric oxide kit, we found that no significant production was occurred [ANOVA, F_(5,12) = 25.92, p = 0.504] at 5 and 10 µg/ml compared with the groups treated as control. Here, we also observed that at 20 and 40 µg/ml, a significant level of NO was produced [ANOVA, F_(5,12) = 25.92, p = 0.0001]. However, production of NO between control (PBS) and pET32a group was also non-significant [ANOVA, F_(5,12) = 25.92, p = 0.473] (Figure 9).

It has been suggested that STPs obviously involved in various cellular processes including cell viability and cell death (10). The apoptotic activity of rHcSTP-1 on PBMCs was evaluated by using the externalization of membrane phospholipid phosphatidylserine (PS) as a marker of cell apoptosis. Flow cytometry assay revealed that cells incubated with different protein concentrations (5, 10, 20, and 40 µg/ml) showed significantly increased frequency of apoptotic percentage in both early [ANOVA, F_(5,12) = 69.08, p = 0.0001] and late stage apoptosis [ANOVA, F_(5,12) = 68.30, p = 0.0001] compared with the control groups. Meanwhile, no significant change was observed [ANOVA, F_(5,12) = 200.8, p = 0.0986] between control and pET32a group (Figure 10).