Clonorchis sinensis metacercariae were collected from naturally infected freshwater fish (Pseudorasbora parva) in an endemic area in Korea. Each of 100 metacercariae was orally infect to Sprague–Dawley rats. Worms were collected from the bile ducts at 1-, 2-, 3- and 4-weeks post-infection. Worms were washed more than 10 times with phosphate buffered saline (PBS, 100 mM, pH 7.4) at 4 °C and were stored at -80 °C. Fresh intact worms were immediately used in ex vivo stimulation experiments (see later section). Animals were housed in accordance with guidelines from the Association for the Assessment and Accreditation of Laboratory Animal Care.
Cloning of C. sinensis omega-class GSTs
Expressed sequence tags (ESTs) were constructed through a screening of randomly selected clones of an adult C. sinensis cDNA library [25, 26]. The similarity patterns of the EST sequences were analyzed against the non-redundant database using BLASTX at the NCBI (http://www.ncbi.nlm.nih.gov). Clones showing high-level sequence identities with Schistosoma mansoni (AAO49385) and Fasciola hepatica (JX156880) GSTo were selected. The adult C. sinensis cDNA library was screened by PCR using vectors (T3 and T7 promoter primers) and gene-specific primers, which contained BamHI (forward) and XhoI (reverse) restriction sites: CsGSTo1-forward (5′-CCG GAT CCA TGC CAA CCT GTT CCA AGC ATT TGC-3′); CsGSTo1-revese (5′-GGC TCG AGT TAC ATG TCC CAG TCA GGA TGA CCA-3′); CsGSTo2-forward (5′-ATG GAT CCA TGT GCT ATC TGG GAG ACG CAG GGA-3′); and CsGSTo2-reverse (5′-GGC TCG AGC TAG GCA ATT TCA AGA TTT GGC TTT CCA GC-3′). T7 promoter and forward primers were used to amplify the 3′-region; the T3 promoter and reverse primers were used for amplification of the 5′-region. The PCR thermal cycler profile included 35 cycles at 94 °C (50 s), 58 °C (50 s) and 72 °C (90 s), followed by 10 min extension at 72 °C. Amplicons were purified using a QIAquick PCR purification kit (Qiagen, Valencia, CA, USA), digested with respective enzymes and cloned into the pET-28a(+) vector (Novagen, Madison, WI, USA). The plasmids were transformed into Escherichia coli DH5α. Nucleotide sequences were determined from both strands. Two full-length cDNAs were obtained by overlapping the 5′- and 3′-region sequences.
The coding profiles and the homology patterns were analyzed using the ORF Finder and BLAST programs. The functionally/structurally conserved domains were searched using ProfileScan (http://myhits.isb-sib.ch/cgi-bin/motif_scan). The secondary structure elements were predicted by the Jpred (www.compbio.dundee.ac.uk/jpred/). The Expasy-Sib Bioinformatics Resource Portal (http://web.expasy.org/compute_pi/) was used to predict the theoretical molecular mass (M
r) and isoelectric point (pI). Tertiary structures were simulated using Swiss-PdbViewer (ver4.1) based on the human omega-class GST1 and 2 (pdb 1EEM and 3QAG). The amino acid sequences were employed as queries during sequence analyses using Hidden Markov Models (InterProScan, http://www.ebi.ac.uk/InterProScan/). The retrieved amino acid sequences were aligned with ClustalX 2.1 and optimized with GeneDoc (ver2.7) . The phylogenetic tree was constructed using the neighbor-joining method and the molecular evolution genetics analysis (MEGA) ver5.1 software . Statistical significance of each branching node was examined by a bootstrap analysis of 1000 replicates using SEQBOOT in the PHYLIP package .
Expression of recombinant proteins
The full-length CsGSTo1 and 2 cloned into the pET-28a(+) vector were introduced into E. coli BL21 (DE3). Expression of the recombinant proteins was induced with 0.1 mM isopropyl-β-D-thiogalactopyranoside (IPTG) for 4 h at 37 °C. Bacterial cells were sonicated and rCsGSTos were purified by Ni-nitrilotriacetic acid (NTA) affinity column (Qiagen) using Tris-HCl (50 mM, pH 8.0) supplemented with NaCl (200 mM) and imidazole (250 mM). His-tag was removed by a Thrombin CleanCleave kit (Sigma-Aldrich, St. Louis, MO, USA). The purified proteins were dialyzed against PBS (100 mM, pH 7.4) for 4 h at 4 °C, concentrated by lyophilization and analyzed by 12 % SDS-PAGE under reducing conditions.
Specific antibodies and immunoblotting
Specific-pathogen free 6-week-old female BALB/c mice were immunized with rCsGSTos (100 μg each) emulsified with 2 % ammonium hydroxide gel adjuvant (InvivoGen, San Diego, CA, USA). Two weeks later, proteins (100 μg) mixed with emulsifier were boosted three times at one-week intervals. One-week later, the sera were collected and IgG fractions were purified using Protein G affinity column.
Proteins were separated by 12 % reducing SDS-PAGE and/or isoelectrically focused using IPG strips (pH 3–10; GE Healthcare, Piscataway, NJ, USA) for 30 kVh followed by 12 % SDS-PAGE (2-dimensional electrophoresis; 2-DE). The proteins transferred onto nitrocellulose membranes (Santa Cruz Biotechnology, Santa Cruz, CA, USA) were blocked in Tris-buffered saline (100 mM, pH 8.8) containing 0.01 % Tween 20 and 3 % skim milk for 1 h. The membrane was incubated with anti-rCsGSTo1 or 2 antibody (1:1,000 dilution) overnight at 4 °C and subsequently with horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (1:4,000 dilution; Cappel, West Chester, PA, USA) for 2 h. Signals were detected using West-Q Pico enhanced chemiluminescence (ECL) kit (GenDEPOT, Dallas, TX, USA). All images were obtained after 2 min exposure for quantitative analysis.
Binding characteristics of native CsGSTos against S-hexylglutathione (SHG) and GSH
We determined the binding specificity of CsGSTos toward SHG and GSH. Adult C. sinensis (4-week-old) were homogenized with a Teflon-pestle homogenizer in PBS (100 mM, pH 7.4) containing a protease inhibitor cocktail (Roche, Basel, Switzerland). The supernatant was obtained by centrifugation at 20,000 g for 30 min at 4 °C. Proteins (200 μg protein per column) were loaded onto a SHG-agarose column (Sigma-Aldrich) or a glutathione-Sepharose 4B column (GE Healthcare). The columns were washed with 20 bed volumes of Tris-HCl buffer (50 mM, pH 7.8) containing 200 mM NaCl. Bound proteins were eluted using Tris-HCl buffer (50 mM, pH 7.8) with 0, 2 and 4 mM step-wise gradient fashions of SHG or GSH. Purified proteins resolved by 12 % SDS-PAGE/2-DE were stained with Coomassie brilliant G-250 (CBB) or further processed by immunoblotting using anti-rCsGSTo1 and 2 antibodies.
GST activity was spectrophotometrically determined employing a panel of substrates (Sigma-Aldrich); 1-chloro-2,4-dinitrobenzene (CDNB; pH 6.5, 340 nm), 1,2-dichloro-4-nitrobenzene (DCNB; pH 7.5, 345 nm), 4-nitrobenzyl chloride (4-NBC; pH 6.5, 310 nm), 4-nitrophenyl acetate (4-NPA; pH 7.0, 400 nm), 4-hydroxy nonenal (pH 7.5, 340 nm), cumene hydroperoxide (CHP; pH 6.5, 340 nm) and ethacrynic acid (pH 7.5, 340 nm). The reactions were recorded for 5 min at 25 °C in 100 mM potassium phosphate buffer (pH 7.2, 200 μl) containing each 4 mM substrate and 4 mM GSH. The formation of ascorbate by the glutathione-dependent DHAR was detected in potassium phosphate buffer (50 mM, pH 7.2) supplemented with 1 mM GSH and 0.25 mM dehydroascorbate (DHA) at 265 nm. Thioltransferase activity was assayed using hydroxylethyl disulfide (HEDS, 2 mM) in potassium phosphate buffer (50 mM, pH 7.2) containing 0.2 mM NADPH, 0.5 mM GSH and 0.5 units of glutathione reductase for 2 min at 340 nm. One unit of enzyme activity was defined as the amount of enzyme that catalyzed the formation of one micromole of product per min in the presence of respective substrates. Vmax and appKm were determined by one site saturation assays in ranges of 0.01–5 mM DHA with 5 mM GSH (saturating concentration). We also used variable concentrations of GSH ranging from 0.01–5.0 mM with 5 mM DHA (saturating concentration). Enzyme activity was monitored by changes of absorbance and was converted to specific activity using a molar extinction coefficient (Δε = 5.3). Non-enzymatic reaction was concomitantly monitored and subtracted from the entire reaction rate. All enzyme assays were independently performed in triplicate at 25 °C. Data were analyzed by best fit algorithm in SigmaPlot10.0.1 (Systat, San Diego, CA, USA).
We determined the inhibition mode of CsGSTos employing SHG and the anthelminthic drug praziquantel (PZQ; Shinpoong, Seoul, Korea). rCsGSTos (each 100 ng) were preincubated with Dulbecco’s PBS supplemented with 10–1,000 μM PZQ or 10–500 nM SHG for 2 min, after which the reaction was initiated by adding 1 mM GSH and 1 mM DHA. The increase in absorbance of the resulting GSH conjugate was recorded spectrophotometrically at 265 nm. A set of reactions under identical conditions was done for each inhibitor concentration and for controls. To examine the inhibition mode of the specific active sites, rCsGSTos were preincubated with saturating concentrations of GSH (5 mM) for 2 min before conjugating reaction with varying concentrations of DHA (0.01–5 mM). To determine the inhibition mode of inhibitors against the ligand-binding sites, the initial velocity of the enzyme reactions was observed in the presence of the respective inhibitors. rCsGSTos were incubated with saturating concentration of DHA (5 mM) for 2 min prior to the conjugating reaction with variable concentrations of GSH (0.01–5 mM). All measurements were independently done in triplicate. Data were analyzed by Lineweaver-Burk plots.
In order to observe tissue distribution pattern of CsGSTos, immunohistochemical staining was done on adult worm sections. Clonorchis sinensis adult worms were fixed in 4 % neutral paraformaldehyde, embedded and cut into 4 μm-thick pieces. Sections were treated with 3 % H2O2 for 10 min, subsequently with Tris buffered saline (100 mM, pH 8.0) containing 3 % BSA and 0.1 % Tween 20 (TBS/T-BSA) for 1 h. The slides were incubated overnight at 4 °C with anti-rCsGSTo1 or 2 antibody (1:400 dilution in TBS/T-BSA) and further incubated with HRP-conjugated goat anti-mouse IgG (1:1,000 dilution; Cappel). Color reactions were developed using HighDef blue chromogen (Enzo Life Sciences, Farmingdale, NY, USA) with PBS (100 mM, pH 7.4) supplemented with 0.05 % 3,3'-diaminobenzidine blue and 0.015 % H2O2 for 5 min. The images were photographed under a TissueFAXS plus (TissueGnostics, Vienna, Austria).
In vitro induction of CsGSTos under oxidative stresses
To assess biological reactivity of CsGSTos under oxidative stressful conditions, we observed induction profile of CsGSTos upon treatment with oxidizing chemicals. Fresh intact worms were stabilized for 1 h at 37 °C in 5 % CO2 atmosphere in serum- and phenol red-free RPMI medium. Worms (10 worms per group per 1 ml of medium) were transferred into fresh medium containing different doses of 5-hydroxy-1,4-naphthoquinone (Juglone; Sigma-Aldrich) (25–100 μM) or CHP (1–4 mM) and incubated for 1 h at 37 °C. The worms were harvested and fractionated into individual compartments, such as seminal receptacle, vitelline follicle-enriched parenchyma and eggs under a dissecting microscope. The conditioned medium containing excretory-secretory products (ESP) was also harvested and sperm was separately collected under a dissecting microscope. Proteins of the respective compartments were extracted in PBS (100 mM, pH 7.4) containing protease inhibitor cocktail (one tablet/25 ml PBS; Complete; Roche) and centrifugation at 12,000 g for 30 min at 4 °C. The proteins prepared from individual compartments of the worms incubated without oxidative treatment were used as controls. Cs tubulin (CsTub; DF143021), whose constant expression was verified by RT-PCR in association with the respective stimuli , was used as an internal control. The induction profiles of CsGSTo were examined by immunoblotting probed each with anti-rCsGSTo antibody and by quantitative real-time RT-PCR (qRT-PCR). At least three independent experiments were done with freshly prepared worms.
Reverse-transcription PCR (RT-PCR) and qRT-PCR
Expressional changes of CsGSTos during worm’s maturation and in response to oxidative stress were determined. Total RNA was extracted from the experimental worms or different developmental stages of worms using a RNeasy Mini kit (Qiagen). RT-PCR was done using RT-PCR PreMix kit (iNtRON, Seongnam, Korea). CsGSTo transcripts in the RNA (1 μg) were amplified by PCR with the following primers: CsGSTo1-forward, 5′-GTT TCC ATT TGT GGA C-3′ and -reverse, 5′-TGG TAG CTG CAA TAC G-3′; CsGSTo2-forward, 5′-TCG TTT GAG CGA ATC G-3′ and -reverse, 5′-CAG CGA GAC TGA GTT G-3′. The thermal cycler profile included pre-heating for 30 min (50 °C) and 10 min (94 °C), 30 cycles of 50 s (94 °C), 50 s (58 °C) and 90 s (72 °C) with 10 min final extension (72 °C). CsTub gene was amplified by PCR using gene specific forward primer (5′-ATT CAG CTG TCC TGG GAA AC-3′) and reverse primer (5′-ACT GCA TTG ATA ACG AAG CG-3′). Thermal cycler profile included 25, 30 and 35 cycles at 94 °C (50 s), 58 °C (50 s) and 72 °C (90 s), followed by a 10 min final extension (72 °C). The PCR products were analyzed on 1 % agarose gel with ethidium bromide staining.
qRT-PCR was conducted using the Rotor-Gene SYBR Green PCR kit and the Rotor-Gene Q Real-time PCR (Qiagen). Total RNA (200 ng) treated with DNase was reverse-transcribed into cDNA using the SuperScript First-Strand Synthesis system (Thermo Fisher Scientific, Waltham, MA, USA). The cDNA was used to examine transcriptional activities of CsGSTo genes with specific primers. The qRT-PCR program included pre-denaturation for 5 min (94 °C), 40 cycles of amplification (94 °C for 15 s, 60 °C for 30 s and 72 °C for 30 s) and a melt cycle from 65 °C to 95 °C. Control reactions were done with RNAs that had not been reverse-transcribed. mRNA abundancy was evaluated in three independent samples for each group with three technical repeats. Data were normalized against those obtained with the CsTrop (ΔCT). Fold inductions (ΔΔCT) were calculated by comparison of the non-stimulated controls. Data were analyzed with the Rotor-Gene Q ScreenClust HRM software using the 2-ΔΔC
T method .
Disc diffusion, cell growth and survival assays under oxidative stress
We observed effects of CsGSTo during oxidative stress employing CsGSTo overexpressing E. coli. Escherichia coli BL21 cells transformed with CsGSTo1 or 2 expression plasmids and control cells transformed with mock vector were induced for expression of recombinant proteins by adding 0.1 mM IPTG for 4 h at 37 °C. The cells (5 × 108) were cultured on LB-kanamycin agar plates for 1 h at 37 °C. Discs (6-mm diameter) soaked with 10, 50, 100 and 200 mM CHP or Juglone were placed on the surface of the top agar. The cells were grown for 24 h at 37 °C and the inhibition zones were measured. For the cell growth assay, stationary-phase cultures of CsGSTo overexpressing bacterial cells and control cells were diluted to an optical density at 600 nm (OD600) of 0.004. The cells were grown in LB broth at 37 °C until exponential phase (OD600 = 0.13–0.14). Aliquots were treated with 0.5, 1, 2 and 4 mM CHP. Growth curves were obtained in Erlenmeyer flasks at 37 °C and 225 rpm. The cultures were diluted to an OD600 of 0.01 in LB broth and the OD600 was measured every 1 h for 25 h. CsGSTo overexpressing E. coli and control cells were subjected to survival assay against oxidative injury. Escherichia coli cells were grown (OD600 = 0.5), after which CHP or Juglone was added to exponential bacterial suspension (0.49 ml) to final concentrations of 1, 2 and 4 mM, and incubated from 20–60 min at 37 °C with shaking (225 rpm). The cells diluted in PBS (0.1 ml) were plated on LB agar plates and grown for 48 h at 37 °C. Cell viability was determined by counting colony-forming units per ml (CFU/ml) as percentage of surviving cells compared to untreated cells. The limit of detection was 100 CFU/ml. All assays were done independently in triplicate.
Data are expressed as mean ± standard deviation (SD) of 3–5 independent experiments. Statistical significance was evaluated by a one-way analysis of variance (ANOVA) using the Statistical Package for the Social Sciences (SPSS; ver20.0) software (SPSS, Chicago, IL, USA), or Student’s t-test followed by a Bonferroni correction, as appropriate. Differences in mean values were considered statistically significant at P < 0.05.