Inhibitory Effects and Analysis of RNA Interference on Thioredoxin Glutathione Reductase Expression in Schistosoma japonicum Author(s): Yanhui Han, Zhiqiang Fu, Yang Hong, Min Zhang, Hongxiao Han, Ke Lu, Jianmei Yang, Xiangrui Li, and Jiaojiao Lin Source: Journal of Parasitology, 100(4):463-469. 2014. Published By: American Society of Parasitologists DOI: http://dx.doi.org/10.1645/13-397.1 URL: http://www.bioone.org/doi/full/10.1645/13-397.1
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J. Parasitol., 100(4), 2014, pp. 463–469 Ó American Society of Parasitologists 2014
INHIBITORY EFFECTS AND ANALYSIS OF RNA INTERFERENCE ON THIOREDOXIN GLUTATHIONE REDUCTASE EXPRESSION IN SCHISTOSOMA JAPONICUM Yanhui Han, Zhiqiang Fu, Yang Hong, Min Zhang, Hongxiao Han, Ke Lu, Jianmei Yang, Xiangrui Li†, and Jiaojiao Lin* College of Veterinary Medicine, Nanjing Agricultural University, Nanjing, Jiangsu, 210095, P.R. China. Correspondence should be sent to: lixiangrui@njau. edu.cn ABSTRACT.
Schistosomes infect around 280 million people worldwide. The worms survive in the veins of the final host, where thioredoxin glutathione reductase (TGR) activity helps the parasites to survive in the aerobic environment. In the present study, we synthesized 4 small interfering RNAs (siRNA S1, S2, S3, and S4) targeting the Schistosoma japonicum (Sj) TGR gene and used them to knockdown the TGR gene. The knockdown effects of the siRNAs on SjTGR, and the thioredoxin reductase (TrxR) activity of SjTGR, were evaluated in vitro. The results of transfection with the siRNAs via the soaking method in vitro were confirmed by flow cytometry. S2 siRNA at a final concentration of 200 nM partially inhibited the expression of SjTGR at both the transcript and protein levels in vitro. TrxR-activity was lower in worms in the S2 siRNA-treated group compared with the control groups. Further analysis revealed that purified recombinant SjTGR could remove oxygen free radicals but not H2O2 directly, which may explain the incomplete effects of RNA interference on SjTGR. The results of this study indicate that SjTGR may play an important role in the clearance of oxygen free radicals and protection of S. japonicum parasites against oxidative damage.
TGR is an oxidoreductase with a broad range of substrate specificities conferred by a fusion between glutaredoxin (Grx) and thioredoxin reductase (TrxR) domains (Sun et al., 2001). TGR homologs have recently been detected in Echinococcus granulosus (Agorio et al., 2003), Taenia crassiceps (Rendon et al., 2004), S. mansoni (Alger and Williams, 2002), and Fasciola hepatica (Guevara-Flores et al., 2011), in which TGR presented a single enzyme responsible for recycling both thioredoxin (Trx) and glutathione (GSH(). In addition, a lag time was observed in enzyme assays using moderate or high concentrations of the substrate, the disulfide form of glutathione, in F. hepatica. However, it is difficult to purify sufficient native TGR protein from schistosomes for immunological or biochemical studies. Recombinant S. mansoni TGR was therefore expressed in Escherichia coli, and the purified recombinant protein exhibited substantial TrxR, glutathione reductase (GR), and Grx activities (Kuntz et al., 2007). RNAi is one promising methodology by which small interfering (si)RNAs trigger the degradation of homologous mRNA transcripts, resulting in effective, sequence-specific gene knockdown (Tabara et al., 1998; Hunter 1999; Grishok and Mello, 2002; Dykxhoorn et al., 2003). siRNAs were first discovered in Caenorhabditis elegans, since which they have been demonstrated in many other organisms (Fire et al., 1998; Grishok and Mello, 2002). In addition, double-stranded RNAs (dsRNA) have been shown to knockdown homologous transcripts and exert a continuous suppressive effect on the parasites (Skelly et al., 2003). The cathepsin B gene in S. mansoni was also suppressed by dsRNA using 2 different methods, soaking and electroporation, both of which produced partial gene knockdown (Krautz-Peterson et al., 2007). Thus, although not all organisms display RNAi machinery, it is applicable to the investigation of schistosome genes (Correnti et al., 2005; KrautzPeterson et al., 2007; Morales et al., 2008; Patocka and Ribeiro, 2013). Several developmental stages of S. mansoni have proven to be susceptible to gene suppression by RNAi including sporocysts, adult worms, and schistosomula (Boyle et al., 2003; Skelly et al., 2003; Osman et al., 2006). Moreover, siRNAs have been shown to knockdown schistosome genes by attacking their homologous target mRNAs through joining with the nuclease complex to produce long-term effects (Tavernarakis et al., 2000). Because of the potential wide utility of RNAi for
Schistosomiasis affects approximately 280 million people worldwide and is a significant cause of morbidity in many countries (Montressor et al., 2002). The schistosomes are exposed to oxidative stress in the veins of their final host because of the presence of reactive oxygen species (ROS). However, antioxidants produced by the worms might help to remove ROS and thus increase their survival in the bloodstream (Kazura et al., 1981; Butterworth, 1984; LoVerde, 1998). Schistosomes lack catalase (Mkoji et al., 1988), which is a major hydrogen peroxide (H2O2)-neutralizing enzyme in many organisms, but H2O2 may be controlled by glutathione peroxidases (GPx) despite their poor reactivity toward H2O2 (Williams et al., 1992; Maiorino et al., 1996). Given the absence of catalase, we sought to identify additional antioxidant enzymes that could neutralize H2O2. The identification and characterization of these enzymes could aid in the development of an anti-schistosome vaccine or chemotherapy targeting the parasite’s redox defenses (Sayed and Williams, 2004). Peroxiredoxin (Prx), also known as thioredoxin peroxidase (TPx), is an important antioxidant recently described in a wide variety of organisms (McGonigle et al., 1998; Rhee et al., 2001; Hofmann et al., 2002), and the unique enzyme thioredoxin glutathione reductase (TGR) has also been described (Alger et al., 2002; Kuntz et al., 2007). Williams and colleagues (Kuntz et al., 2007) used RNA interference (RNAi) in Schistosoma mansoni and screened inhibitory compounds, including potassium antimonyl tartrate (PAT) and oltipraz (OPZ), which are no longer used to treat schistosomiasis, to test the hypothesis that TGR could be an essential parasite protein and a potentially important drug target. They found that TGR was essential for parasite survival and that auranofin was an efficient inhibitor of pure TGR. Furthermore, PAT and OPZ also inhibited TGR activity, suggesting that TGR was a key target of those compounds. These results validated parasite TGR as a schistosome drug target, based on a combination of genetic and biochemical approaches. Received 12 September 2013; revised 26 February 2014; accepted 10 March 2014. * Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Key Laboratory of Animal Parasitology, Ministry of Agriculture of China, Shanghai, 200241, P.R. China. DOI: 10.1645/13-397.1 463
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TABLE I. Sequences of siRNAs and primers. Name S1 siRNA S2 siRNA S3 siRNA S4 siRNA Control siRNA
Sequence
Target regions of SjTGR
Sense 5 0 -GUGGUUUCGAUCAACAAAUTT-3 0 Antisense 5 0 -AUUUGUUGAUCGAAACCACTT-3 0 Sense 5 0 -CCGGUAGAUACUGAGAAUATT-3 0 Antisense 5 0 -UAUUCUCAGUAUCUACCGGTT-3 0 Sense 5 0 -GGGCUAUAAAGUUUCACUATT-3 0 Antisense 5 0 -UAGUGAAACUUUAUAGCCCTT-3 0 Sense 5 0 -GGAGUCAAGUUCGCAAAGUTT-3 0 Antisense 5 0 -ACUUUGCGAACUUGACUCCTT-3 0 Sense 5 0 -GCGACGAUCUGCCUAAGAUdTdT-3 0 Antisense 5 0 -GCGACGAUCUGCCUAAGAUdTdT-3 0
963–983 1,067–1,089 628–649 1,016–1,038 No
Note: We tested the 5 siRNAs for 3 replicates to knockdown the expression of SjTGR. S3 siRNA, and S4 siRNA were ineffective in the experiments.
schistosome gene analysis, we studied the function of the SjTGR gene using RNAi to knockdown its expression using 2 siRNAs and examined the DNA-protection and peroxidase activities of recombinant SjTGR. MATERIALS AND METHODS
transfection efficiency in parasites in vitro. Parasites were collected 3 hr post-treatment in the dark, washed with fresh RPMI 1640, cut into small slivers, and then digested for 30 min with trypsinogen (0.25%) at 37 C. After centrifugation for 5 min, the precipitates were collected and suspended in RPMI 1640 (Bayne et al., 1994). The parasite cells were analyzed by flow cytometry (FCM) to determine the ratio of fluorescent cells. Untreated parasites were used as controls.
Parasites and animals
qRT-PCR analysis
Schistosoma japonicum (Chinese strain) life-cycle was maintained in Oncomelania hupensis snails and New Zealand rabbits at the Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences. Cercariae were collected by exposing infected snails to light, and the numbers and viability of cercariae were determined under a light microscope prior to use for challenge. Male BALB/c mice (6-wk-old) and rabbits (2.0–2.5 kg body weight) were purchased from the Shanghai Experimental Animal Center, Chinese Academy of Sciences (China). Animal care and all procedures involving animals were conducted according to the principles of Shanghai Veterinary Research Institute for the Care and Use of Laboratory Animals.
At 2 days post-siRNA treatment, total parasite RNA was extracted using Trizol reagent (Invitrogen, Carlsbad, California) and cDNA was synthesized using a PrimeScript RT Reagent Kit With gDNA Eraser (Takara, Shiga, Japan). qRT-PCR was performed in a reaction mixture of 20 ll containing 0.8 ll primers (10 lM), 1 ll cDNA, 8.2 ll EASY Dilution Buffer (Takara), and 10 ll 23SYBR Green PCR Premix Taq (Takara) in a Mastercycler ep Realplex (Eppendorf, Hamburg, Germany). The thermal cycling profiles were as follows: 95 C for 2 min followed by 40 cycles of amplification (95 C for 15 sec, 60 C for 15 sec, 72 C for 20 sec). Primers for SjTGR were as follows: forward primer: 5 0 -CTACTGGCGAGCGTCCAAAATACC-30 , reverse primer: 5 0 -AACATCACCGCCCAAACTGACAAG-3 0 . The amplification fragment was 170 bp long. Shistosoma japonicum a-tubulin (Sjatubulin) was used as an internal control for normalization. Primers for the Sja-tubulin gene were: sense: 5 0 -CTGATTTTCCATT-CGTTTG-30 ; antisense: 5 0 -GTTGTCTACCATGTTGGCA-3 0 , which amplified a product of 213 bp.
Preparation of siRNA Two specific siRNAs (S1 and S2) targeting different regions of the S. japonicum TGR gene (GenBank EU938325) were designed (Table I), and these and a non-schistosome control siRNA (NC-L) were chemically synthesized by GenePharma Company (Shanghai, China). Preparation of recombinant SjTGR Purified recombinant SjTGR was prepared as described by Han et al. (2012). Recombinant SjTGR (rSjTGR) was eluted in 60 mM imidazole and dialyzed against phosphate-buffered saline (PBS). The concentration was determined using a Bradford assay kit (Sangon, Shanghai, China). rSjTGR was stored at 80 C until use. Treatment of parasites with siRNAs by soaking in vitro Rabbits were infected with approximately 6,000 cercariae and the parasites were artificially perfused at 11 days post-infection. Isolated parasites were cultured in RPMI 1640 medium with 10% rabbit serum (Gibco, Grand Island, New York), 0.1% lactoalbumin hydrolysate (Sigma, St. Louis, Missouri), 0.2 U/ml insulin (Sigma), 1 lM hydrocortisone (Sigma), 0.5 lM hypoxanthine (Sigma), 1 lM 5-hydroxytryptamine (Sigma), and antibiotics (100 U/ml penicillin and 100 lg/ml streptomycin) at 37 C in an atmosphere of 5% CO2, 95% air in 6-well plates (150 6 10 parasites per well). The parasites were treated with siRNAs by soaking for 2 or 3 days at a final concentration of 200 nM (Cheng et al., 2009). The knockdown effect was observed using quantitative real-time polymerase chain reaction (qRT-PCR) and western blotting.
Western blotting At 3 days post-treatment with siRNA, soluble proteins from differentlytreated parasites were extracted using ProteoJET Mammalian Cell Lysis Reagent (Fermentas, Ontario, Canada), supplemented with a cocktail of protease inhibitors and measured using a Bradford assay kit (Sangon). The proteins were then fractionated by 10% sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) and electrotransferred to a 0.45-lm pore size nitrocellulose membrane (Whatman, Lower Saxony, Germany) at 260 mA for 2 hr at 4 C. Non-specific protein–protein interactions were blocked with 5% nonfat dry milk in PBS (pH 7.4) containing 0.1% Tweent 20 (Sigma) (PBST). The membrane was incubated for 1 hr at room temperature with mouse anti-SjTGR serum or anti-a-tubulin (Beyotime, Shanghai, China) at 1:100 dilutions. The membrane was then washed 3 times for 10 min each with PBST and incubated with horseradish peroxidase-conjugated goat anti-mouse IgG (HþL) (1:1,000) (Takara) for 1.5 hr. The result was finally detected using an HRP-DAB visualization Kit (Tiangen, Shanghai, China) according to the manufacturer’s instructions. The results of western blots were converted into a histogram by measuring the optical density of the bands using Image J2x software (National Institutes of Health [NIH], Bethesda, Maryland). Determination of TrxR activity
Evaluation of siRNA transfection efficiency in vitro A fluorescein-labeled siRNA (final concentration 200 nM) was chemically synthesized by GenePharma and used to examine the
In vitro, soluble proteins were extracted from untreated, control, and siRNA-treated worms. Protein concentrations were determined using a Bradford assay kit (Sangon) according to the manufacturer’s instructions.
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FIGURE 1. Efficiency of siRNA transfection analyzed by FCM. Fluorescein-labeled siRNA was marked by FITC. Gray region indicates background cells. Black region (J region) indicates fluorescent cells. (A) represents cells of treated parasites, (B) represents cells of untreated parasites. All data are expressed as the mean 6 SE of triplicate experiments.
The enzymatic assay was performed at 25 C in 0.1 M potassium phosphate (pH 7.5) containing 10 mM ethylenediaminetetra-acetic acid. 5, 5 0 dithiobis (2-nitrobenzoic acid) (DTNB) (Sigma) was used as a substrate to determine TrxR activity. The enzymatic assay contained 100 lM bnicotinamide adenine dinucleotide phosphate (NADPH) (Sigma) and 3 mM DTNB, and the initial increase in A412 was recorded during the first 2 min by the addition of DTNB. One unit of TrxR was defined as that required for the NADPH-dependent production of 2 lM of 2-nitro-5thiobenzoic acid per min using e412 nm ¼ 13.6 mM1cm1. DNA-protection activity of recombinant SjTGR The antioxidant activity of SjTGR was investigated using a DNAcleavage (nicking) assay to assess protection from DNA damage caused by a metal-catalyzed oxidation (MCO) system, as previously described (Kim et al., 1988; Li et al., 2004). Briefly, 100 ll of reaction mixture containing 33 lM FeCl3, 3.3 mM dithiothreitol (DTT), and a final concentration of purified SjTGR fusion protein ranging from 100–600 lg/ml were incubated for 2 hr at 37 C. pUC19 super-coiled plasmid DNA (300 ng) was then added into each reaction mixture and incubated at 37 C for 2.5 hr. Finally, the mixtures were examined by subjecting 20-ll samples to agarose gel electrophoresis with ethidium bromide to assess DNA protection. Bovine serum albumin (BSA) was assayed as a control. Peroxidase activity of recombinant SjTGR The ability of recombinant SjTGR to reduce H2O2 was determined using the method described by Lim and colleagues (Lim et al., 1993). Briefly, a reaction mixture (100 ll) containing different concentrations (0– 50 lg/ml) of rSjTGR and 50 mM reaction buffer (Tris-HCl, pH 8.0) was incubated for 30 min at 37 C. H2O2 was added to a final concentration of 30 lM and incubated for an additional 30 min. Subsequently, 0.9 ml (8% v/v) of trichloroacetic acid was added to terminate the reaction, and protein was then removed by centrifugation. Finally, 0.2 ml ferrous ammonium sulfate (10 mM) and 0.1 ml potassium thiocyanate (2.5 M) were added into the mixture and the absorbance was measured at 480 nm. Reduced H2O2 was determined using a known amount of H2O2 as a standard. Statistical analysis The data were analyzed to compare the differences among groups using Student’s t-tests.
RESULTS Efficiency of siRNAs transfection The efficiency of siRNA transfection was determined by soaking treated schistosomes with fluorescent siRNAs and analyzing the results by FCM. The proportion of fluorescent cells in the treated group was 6.25% 6 1.28 (Fig. 1A) compared with 0.64% 6 0.16 in the untreated group (Fig. 1B). Effect of RNA interference on SjTGR in vitro at the transcript level The knockdown efficiencies of the 4 SjTGR-specific and control siRNAs on SjTGR transcripts in vitro were analyzed by qRTPCR (Fig. 2). SjTGR mRNA was reduced by 9.66% by the S1 siRNA and 46.01% by the S2 siRNA compared with untreated parasites, while levels remained almost unchanged by treatment with the S3 siRNA, S4 siRNA, and the control siRNA. These results indicate that the S2 siRNA sequence affected the targetgene expression in vitro. Effect of RNAi on SjTGR in vitro at the protein level The effect of siRNA knockdown on SjTGR at the protein level in vitro was examined by western blotting. SjTGR protein expression was inhibited in parasites at 3 days post-S2 siRNA treatment (Fig. 3A). Western blot results were normalized and converted into a histogram using Image J2x software (NIH) (Fig. 3B). The results of western blotting were similar to those seen at the mRNA level. TrxR activity in the S2 siRNA group was 1.16 U/mg, which was significantly lower (P , 0.01) than in the untreated, control siRNA, and S1 siRNA groups (1.51 U/mg, 1.55 U/mg, and 1.51 U/mg, respectively).
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FIGURE 2. Effects of RNAi on SjTGR transcription in vitro analyzed by qRT-PCR. The value in each column is the ratio of the optical density of SjTGR to that of Sja-tubulin. All data are expressed as the mean 6 SE of triplicate experiments.
Super-coiled DNA-protection activity and peroxidase activity of SjTGR The activity of recombinant TGR was detected in the same way as for native TrxR (Han et al., 2012). Recombinant SjTGR was defrosted from 80 C and analyzed by SDS-PAGE to check that it was not degraded. A DNA-nicking assay was used to determine the DNAprotection activity of SjTGR using pUC19 super-coiled plasmid DNA and the MCO system. Oxygen free radicals produced by the MCO system caused nicking of pUC19 super-coiled plasmid DNA. pUC19 super-coiled plasmid DNA was measured by gelmobility shift after treatment. Separately incubated MCO components did not damage pUC19 super-coiled plasmid DNA. DNA nicking was prevented by the addition of purified recombinant SjTGR, but not in the absence of rSjTGR or by addition of the control protein, BSA, under the same conditions. Furthermore, the protective effect of rSjTGR was concentration dependent, and decreasing levels of rSjTGR in the reaction reduced plasmid protection from oxidative cleavage. The lowest concentration of purified rSjTGR that conferred protection was 100 lg/ml, and almost 100% of DNA-nicking was prevented by rSjTGR at a concentration of 600 lg/ml after 2.5 hr incubation at 37 C (Fig. 4). To determine the antioxidant properties of purified rSjTGR, peroxidase activity was measured using H2O2 as the substrate. The reduction of peroxide was monitored based on the remaining H2O2 in the ferrithiocyanate system. rSjTGR failed to remove H2O2 even with increasing concentrations (Fig. 5), indicating that rSjTGR did not possess the peroxidase activity required to remove H2O2 directly. DISCUSSION Schistosomes can survive in the bloodstream of their final host for 5–30 yr. Several survival mechanisms have been proposed,
FIGURE 3. Effect of siRNA knockdown on SjTGR protein levels in vitro by western blotting analysis. Worms were collected at 3 days posttreatment. (A) The soluble proteins were extracted from the treated worms. Sja-tubulin was used as a loading control. (B) The results of western blots were converted into a histogram by measuring the optical densities of the bands. All data are expressed as the mean 6 SE of triplicate experiments. *Statistically significant compared with untreated group (P , 0.05), **(P , 0.01).
including the production of protective antioxidant proteins to neutralize oxidative damage resulting from the host’s immune response and from self-generated oxygen radicals (LoVerde, 1998). These antioxidant proteins have been an important focus of research with regard to schistosomiasis. Three types of Prxs, classified as novel antioxidant proteins, have recently been cloned and sequenced from S. mansoni and S. japonicum. These proteins work predominantly to remove H2O2 in schistosomes (Kwatia et al., 2000; Williams et al., 2001; Sayed and Williams, 2004; Kumagai et al., 2006). Another antioxidant protein, TGR, has also been cloned and characterized from S. japonicum, and the protective efficacy of SjTGR against schistosome infection in mice has been examined (Han et al., 2012; Song et al., 2012). Immunization with recombinant SjTGR induced a 33.5–36.51% decrease in worm burden. TGR is thus an important vaccine candidate for the treatment of schistosomiasis, and we speculated that it might therefore also represent an important protein in terms of parasite survival.
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FIGURE 4. Protection of super-coiled DNA cleavage by rSjTGR in an MCO system. Lanes (1–6) pUC19 plasmid þ FeCl3 þ DTT þ recombinant SjTGR (600, 500, 400, 300, 200, 100 lg/ml, respectively); (7) pUC19 plasmid þ FeCl3; (8) pUC19 plasmid þ DTT; (9) pUC19 plasmid þ FeCl3 þ DTT; (10) pUC19 plasmid þ FeCl3 þ DTT þ 600 lg/ml BSA; (11) pUC19 plasmid. NF, nicked form of pUC19 plasmid; SF, super-coiled form of pUC19 plasmid.
In the present study, we studied the biological function of SjTGR using 4 siRNAs (S1, S2, S3, and S4) based on regions of the SjTGR transcript. The results showed that S2 siRNA could partially knockdown SjTGR expression in schistosomes in vitro at both the transcript and protein levels. The different knockdown efficacies of these 4 siRNAs may correlate with their distinct locations in the coding sequence of the SjTGR mRNA. We used a simple soaking method to transfect the siRNAs into the parasites. This is an effective and convenient method that causes minimal physical damage to the schistosomes (Kuntz et al., 2007; Ndegwa et al., 2007; Cheng et al., 2009). Electroporation and Lipofectaminet have also been used to transfect siRNAs; the former has proven to be an effective delivery method but the latter was shown to be toxic to the cultured parasites, leading to parasite death (Correnti et al., 2005; Krautz-Peterson et al., 2007). Both soaking and electroporation were found to be effective methods for knockdown of schistosome genes with siRNA (KrautzPeterson et al., 2007). We compared the efficacies of these two methods for gene knockdown in S. japonicum and found that electroporation was associated with a higher mortality, especially in larval schistosomes. We previously determined that 200 nM was the optimal concentration of siRNA for inducing gene suppression (Cheng et al., 2009). We therefore used the soaking method and a concentration of 200 nM was chosen in the present study. RNAi has previously been used to identify gene function in other parasites. In the first study of RNAi in vitro in an animal parasite, Samarasinghe et al. (2011) found that genes expressed at sites accessible to the environment were likely to be susceptible to RNAi in the sheep gastrointestinal nematode Haemonchus contortus. In addition, knockdown of the H. contortus H11 gene prior to infection reduced the worm burden in sheep by 40% and egg output by 57%. RNAi has been reported in the trematodes F. hepatica, in which successful knockdown of the cysteine proteases cathepsin B and L in the infective stage resulted in marked reductions in target transcript levels and in the expression of the encoded proteins in the gut (McGonigle et al., 2008). In the first report of targeted manipulation of endogenous gene expression, researchers reported that dsRNA treatment of S. mansoni sporocysts resulted in disrupted expression of the genes encoding the GAPDH and the glucose transporter protein 1 (SGTP1) in the human blood fluke stage (Boyle et al., 2003). The transforming growth factor-b (TGF-b) type II receptor (SmTbR II) was suppressed by RNAi in adult worms. That study was the first to used RNAse III to digest a long dsRNA encoding a portion of SmTbR II into smaller fragment siRNAs (Osman et al., 2006). Kuntz et al. (2007) found that TGR was essential for survival of S. mansoni. Using RNAi and selecting a long double-stranded
FIGURE 5. Peroxidase activity of rSjTGR protein. Peroxidase activity was assayed using purified SjTGR fusion protein and H2O2 (30 lM) in a concentration-dependent manner. Remaining H2O2 was measured spectrophotometrically and calculated as the percentage of H2O2 removed by rSjTGR. Values are the mean 6 SE from 3 similar experiments. Error bars represent SE (n ¼ 3).
(ds) RNA in vitro, 92% of parasites died within 4 days after knockdown of TGR expression. Cercariae were mechanically transformed to schistosomula by vortexing, and the schistosomula were then cultured with 54 lg/ml of each dsRNA after separating the bodies from tails. After 3 days of TGR dsRNA treatment, TGR protein activity (using DTNB as substrate) was reduced by 63.5%, and a marked decrease in TGR mRNA was detected by RT-PCR. In our study, we collected 11-day-old schistosomula from infected rabbits and cultured them with different siRNAs. Early stages of schistosomula are commonly used for siRNA analysis, but the parasites are difficult to collect before 10 days post-infection, and schistosomula around 11 days are therefore usually used for siRNA studies in our lab. After 3 days, TGR activity was reduced by 25.16% in vitro. A partial decrease in the expression of TGR was also seen. siRNAs have recently been applied instead of dsRNAs to avoid inducing the stress (toxic) response while retaining the ability to induce genespecific knockdown (Elbashir et al., 2001; Harborth et al., 2001). However, siRNAs can also induce toxicity, though this is generally slower and less severe than that associated with dsRNAs (Chopra et al., 2002). RNAi offers great potential for in vitro target validation and as a potentially novel therapeutic strategy based on the efficient knockdown of a target gene (Aigner, 2006). The effects of RNAi in this study were less than expected. TGR is an antioxidant protein in schistosomes, and we hypothesized that TGR could reduce H2O2, oxygen free radicals, or both. We therefore used a DNA-nicking assay to determine the antioxidant activity of SjTGR and showed that SjTGR could protect schistosome nucleic acids from oxidative damage by reducing oxygen free radicals. We simultaneously determined the peroxidase activity of SjTGR, and found no change in reduced H2O2 with increasing concentrations of rSjTGR, suggesting that SjTGR could scavenge oxygen free radicals to protect the schistosomes but could not directly reduce H2O2. H2O2 is reduced directly by GPx and Prxs, which are downstream enzymes of TGR and regulated by TGR. GPx combined with Prxs could thus reduce H2O2 to H2O in
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schistosomes in the absence of catalase (Mkoji et al., 1988; Simurda et al., 1988; Maiorino et al., 1996). However, GPx and Prxs are themselves maintained in the reduced, active state by GSH, Trx, or both. Electrons from NADPH are transferred to GSH and Trx by the unique parasite reductase, TGR (Sayed and Williams, 2004). Knockdown of SjTGR, therefore, did not completely block the actions of GPx and Prx, which could still eliminate ROS. This may help to explain why knockdown of TGR with S2 siRNA only reduced the TGR activity by 25.16% in vitro. In summary, the present investigation identified and characterized SjTGR, which may play an important role in the clearance of oxygen free radicals and protection of S. japonicum parasites against oxidative damage. RNAi targeting SjTGR partially knockdown its expression at both the transcript and protein levels. However, SjTGR could directly remove oxygen free radicals but not H2O2, which might help to explain the observed effects of RNAi. ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation of China (31172315), the Special Fund for Agro-Scientific Research in the Public Interest (200903036), the basic scientific research operation cost of state-level public welfare scientific research countryard (2013JB18), the Science and Technology Commission of Shanghai Municipality (12140902700), and the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD).
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J. Parasitol., 100(4), 2014, pp. 463–469 Ó American Society of Parasitologists 2014
INHIBITORY EFFECTS AND ANALYSIS OF RNA INTERFERENCE ON THIOREDOXIN GLUTATHIONE REDUCTASE EXPRESSION IN SCHISTOSOMA JAPONICUM Yanhui Han, Zhiqiang Fu, Yang Hong, Min Zhang, Hongxiao Han, Ke Lu, Jianmei Yang, Xiangrui Li†, and Jiaojiao Lin* College of Veterinary Medicine, Nanjing Agricultural University, Nanjing, Jiangsu, 210095, P.R. China. Correspondence should be sent to: lixiangrui@njau. edu.cn ABSTRACT.
Schistosomes infect around 280 million people worldwide. The worms survive in the veins of the final host, where thioredoxin glutathione reductase (TGR) activity helps the parasites to survive in the aerobic environment. In the present study, we synthesized 4 small interfering RNAs (siRNA S1, S2, S3, and S4) targeting the Schistosoma japonicum (Sj) TGR gene and used them to knockdown the TGR gene. The knockdown effects of the siRNAs on SjTGR, and the thioredoxin reductase (TrxR) activity of SjTGR, were evaluated in vitro. The results of transfection with the siRNAs via the soaking method in vitro were confirmed by flow cytometry. S2 siRNA at a final concentration of 200 nM partially inhibited the expression of SjTGR at both the transcript and protein levels in vitro. TrxR-activity was lower in worms in the S2 siRNA-treated group compared with the control groups. Further analysis revealed that purified recombinant SjTGR could remove oxygen free radicals but not H2O2 directly, which may explain the incomplete effects of RNA interference on SjTGR. The results of this study indicate that SjTGR may play an important role in the clearance of oxygen free radicals and protection of S. japonicum parasites against oxidative damage.
TGR is an oxidoreductase with a broad range of substrate specificities conferred by a fusion between glutaredoxin (Grx) and thioredoxin reductase (TrxR) domains (Sun et al., 2001). TGR homologs have recently been detected in Echinococcus granulosus (Agorio et al., 2003), Taenia crassiceps (Rendon et al., 2004), S. mansoni (Alger and Williams, 2002), and Fasciola hepatica (Guevara-Flores et al., 2011), in which TGR presented a single enzyme responsible for recycling both thioredoxin (Trx) and glutathione (GSH(). In addition, a lag time was observed in enzyme assays using moderate or high concentrations of the substrate, the disulfide form of glutathione, in F. hepatica. However, it is difficult to purify sufficient native TGR protein from schistosomes for immunological or biochemical studies. Recombinant S. mansoni TGR was therefore expressed in Escherichia coli, and the purified recombinant protein exhibited substantial TrxR, glutathione reductase (GR), and Grx activities (Kuntz et al., 2007). RNAi is one promising methodology by which small interfering (si)RNAs trigger the degradation of homologous mRNA transcripts, resulting in effective, sequence-specific gene knockdown (Tabara et al., 1998; Hunter 1999; Grishok and Mello, 2002; Dykxhoorn et al., 2003). siRNAs were first discovered in Caenorhabditis elegans, since which they have been demonstrated in many other organisms (Fire et al., 1998; Grishok and Mello, 2002). In addition, double-stranded RNAs (dsRNA) have been shown to knockdown homologous transcripts and exert a continuous suppressive effect on the parasites (Skelly et al., 2003). The cathepsin B gene in S. mansoni was also suppressed by dsRNA using 2 different methods, soaking and electroporation, both of which produced partial gene knockdown (Krautz-Peterson et al., 2007). Thus, although not all organisms display RNAi machinery, it is applicable to the investigation of schistosome genes (Correnti et al., 2005; KrautzPeterson et al., 2007; Morales et al., 2008; Patocka and Ribeiro, 2013). Several developmental stages of S. mansoni have proven to be susceptible to gene suppression by RNAi including sporocysts, adult worms, and schistosomula (Boyle et al., 2003; Skelly et al., 2003; Osman et al., 2006). Moreover, siRNAs have been shown to knockdown schistosome genes by attacking their homologous target mRNAs through joining with the nuclease complex to produce long-term effects (Tavernarakis et al., 2000). Because of the potential wide utility of RNAi for
Schistosomiasis affects approximately 280 million people worldwide and is a significant cause of morbidity in many countries (Montressor et al., 2002). The schistosomes are exposed to oxidative stress in the veins of their final host because of the presence of reactive oxygen species (ROS). However, antioxidants produced by the worms might help to remove ROS and thus increase their survival in the bloodstream (Kazura et al., 1981; Butterworth, 1984; LoVerde, 1998). Schistosomes lack catalase (Mkoji et al., 1988), which is a major hydrogen peroxide (H2O2)-neutralizing enzyme in many organisms, but H2O2 may be controlled by glutathione peroxidases (GPx) despite their poor reactivity toward H2O2 (Williams et al., 1992; Maiorino et al., 1996). Given the absence of catalase, we sought to identify additional antioxidant enzymes that could neutralize H2O2. The identification and characterization of these enzymes could aid in the development of an anti-schistosome vaccine or chemotherapy targeting the parasite’s redox defenses (Sayed and Williams, 2004). Peroxiredoxin (Prx), also known as thioredoxin peroxidase (TPx), is an important antioxidant recently described in a wide variety of organisms (McGonigle et al., 1998; Rhee et al., 2001; Hofmann et al., 2002), and the unique enzyme thioredoxin glutathione reductase (TGR) has also been described (Alger et al., 2002; Kuntz et al., 2007). Williams and colleagues (Kuntz et al., 2007) used RNA interference (RNAi) in Schistosoma mansoni and screened inhibitory compounds, including potassium antimonyl tartrate (PAT) and oltipraz (OPZ), which are no longer used to treat schistosomiasis, to test the hypothesis that TGR could be an essential parasite protein and a potentially important drug target. They found that TGR was essential for parasite survival and that auranofin was an efficient inhibitor of pure TGR. Furthermore, PAT and OPZ also inhibited TGR activity, suggesting that TGR was a key target of those compounds. These results validated parasite TGR as a schistosome drug target, based on a combination of genetic and biochemical approaches. Received 12 September 2013; revised 26 February 2014; accepted 10 March 2014. * Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Key Laboratory of Animal Parasitology, Ministry of Agriculture of China, Shanghai, 200241, P.R. China. DOI: 10.1645/13-397.1 463
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TABLE I. Sequences of siRNAs and primers. Name S1 siRNA S2 siRNA S3 siRNA S4 siRNA Control siRNA
Sequence
Target regions of SjTGR
Sense 5 0 -GUGGUUUCGAUCAACAAAUTT-3 0 Antisense 5 0 -AUUUGUUGAUCGAAACCACTT-3 0 Sense 5 0 -CCGGUAGAUACUGAGAAUATT-3 0 Antisense 5 0 -UAUUCUCAGUAUCUACCGGTT-3 0 Sense 5 0 -GGGCUAUAAAGUUUCACUATT-3 0 Antisense 5 0 -UAGUGAAACUUUAUAGCCCTT-3 0 Sense 5 0 -GGAGUCAAGUUCGCAAAGUTT-3 0 Antisense 5 0 -ACUUUGCGAACUUGACUCCTT-3 0 Sense 5 0 -GCGACGAUCUGCCUAAGAUdTdT-3 0 Antisense 5 0 -GCGACGAUCUGCCUAAGAUdTdT-3 0
963–983 1,067–1,089 628–649 1,016–1,038 No
Note: We tested the 5 siRNAs for 3 replicates to knockdown the expression of SjTGR. S3 siRNA, and S4 siRNA were ineffective in the experiments.
schistosome gene analysis, we studied the function of the SjTGR gene using RNAi to knockdown its expression using 2 siRNAs and examined the DNA-protection and peroxidase activities of recombinant SjTGR. MATERIALS AND METHODS
transfection efficiency in parasites in vitro. Parasites were collected 3 hr post-treatment in the dark, washed with fresh RPMI 1640, cut into small slivers, and then digested for 30 min with trypsinogen (0.25%) at 37 C. After centrifugation for 5 min, the precipitates were collected and suspended in RPMI 1640 (Bayne et al., 1994). The parasite cells were analyzed by flow cytometry (FCM) to determine the ratio of fluorescent cells. Untreated parasites were used as controls.
Parasites and animals
qRT-PCR analysis
Schistosoma japonicum (Chinese strain) life-cycle was maintained in Oncomelania hupensis snails and New Zealand rabbits at the Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences. Cercariae were collected by exposing infected snails to light, and the numbers and viability of cercariae were determined under a light microscope prior to use for challenge. Male BALB/c mice (6-wk-old) and rabbits (2.0–2.5 kg body weight) were purchased from the Shanghai Experimental Animal Center, Chinese Academy of Sciences (China). Animal care and all procedures involving animals were conducted according to the principles of Shanghai Veterinary Research Institute for the Care and Use of Laboratory Animals.
At 2 days post-siRNA treatment, total parasite RNA was extracted using Trizol reagent (Invitrogen, Carlsbad, California) and cDNA was synthesized using a PrimeScript RT Reagent Kit With gDNA Eraser (Takara, Shiga, Japan). qRT-PCR was performed in a reaction mixture of 20 ll containing 0.8 ll primers (10 lM), 1 ll cDNA, 8.2 ll EASY Dilution Buffer (Takara), and 10 ll 23SYBR Green PCR Premix Taq (Takara) in a Mastercycler ep Realplex (Eppendorf, Hamburg, Germany). The thermal cycling profiles were as follows: 95 C for 2 min followed by 40 cycles of amplification (95 C for 15 sec, 60 C for 15 sec, 72 C for 20 sec). Primers for SjTGR were as follows: forward primer: 5 0 -CTACTGGCGAGCGTCCAAAATACC-30 , reverse primer: 5 0 -AACATCACCGCCCAAACTGACAAG-3 0 . The amplification fragment was 170 bp long. Shistosoma japonicum a-tubulin (Sjatubulin) was used as an internal control for normalization. Primers for the Sja-tubulin gene were: sense: 5 0 -CTGATTTTCCATT-CGTTTG-30 ; antisense: 5 0 -GTTGTCTACCATGTTGGCA-3 0 , which amplified a product of 213 bp.
Preparation of siRNA Two specific siRNAs (S1 and S2) targeting different regions of the S. japonicum TGR gene (GenBank EU938325) were designed (Table I), and these and a non-schistosome control siRNA (NC-L) were chemically synthesized by GenePharma Company (Shanghai, China). Preparation of recombinant SjTGR Purified recombinant SjTGR was prepared as described by Han et al. (2012). Recombinant SjTGR (rSjTGR) was eluted in 60 mM imidazole and dialyzed against phosphate-buffered saline (PBS). The concentration was determined using a Bradford assay kit (Sangon, Shanghai, China). rSjTGR was stored at 80 C until use. Treatment of parasites with siRNAs by soaking in vitro Rabbits were infected with approximately 6,000 cercariae and the parasites were artificially perfused at 11 days post-infection. Isolated parasites were cultured in RPMI 1640 medium with 10% rabbit serum (Gibco, Grand Island, New York), 0.1% lactoalbumin hydrolysate (Sigma, St. Louis, Missouri), 0.2 U/ml insulin (Sigma), 1 lM hydrocortisone (Sigma), 0.5 lM hypoxanthine (Sigma), 1 lM 5-hydroxytryptamine (Sigma), and antibiotics (100 U/ml penicillin and 100 lg/ml streptomycin) at 37 C in an atmosphere of 5% CO2, 95% air in 6-well plates (150 6 10 parasites per well). The parasites were treated with siRNAs by soaking for 2 or 3 days at a final concentration of 200 nM (Cheng et al., 2009). The knockdown effect was observed using quantitative real-time polymerase chain reaction (qRT-PCR) and western blotting.
Western blotting At 3 days post-treatment with siRNA, soluble proteins from differentlytreated parasites were extracted using ProteoJET Mammalian Cell Lysis Reagent (Fermentas, Ontario, Canada), supplemented with a cocktail of protease inhibitors and measured using a Bradford assay kit (Sangon). The proteins were then fractionated by 10% sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) and electrotransferred to a 0.45-lm pore size nitrocellulose membrane (Whatman, Lower Saxony, Germany) at 260 mA for 2 hr at 4 C. Non-specific protein–protein interactions were blocked with 5% nonfat dry milk in PBS (pH 7.4) containing 0.1% Tweent 20 (Sigma) (PBST). The membrane was incubated for 1 hr at room temperature with mouse anti-SjTGR serum or anti-a-tubulin (Beyotime, Shanghai, China) at 1:100 dilutions. The membrane was then washed 3 times for 10 min each with PBST and incubated with horseradish peroxidase-conjugated goat anti-mouse IgG (HþL) (1:1,000) (Takara) for 1.5 hr. The result was finally detected using an HRP-DAB visualization Kit (Tiangen, Shanghai, China) according to the manufacturer’s instructions. The results of western blots were converted into a histogram by measuring the optical density of the bands using Image J2x software (National Institutes of Health [NIH], Bethesda, Maryland). Determination of TrxR activity
Evaluation of siRNA transfection efficiency in vitro A fluorescein-labeled siRNA (final concentration 200 nM) was chemically synthesized by GenePharma and used to examine the
In vitro, soluble proteins were extracted from untreated, control, and siRNA-treated worms. Protein concentrations were determined using a Bradford assay kit (Sangon) according to the manufacturer’s instructions.
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FIGURE 1. Efficiency of siRNA transfection analyzed by FCM. Fluorescein-labeled siRNA was marked by FITC. Gray region indicates background cells. Black region (J region) indicates fluorescent cells. (A) represents cells of treated parasites, (B) represents cells of untreated parasites. All data are expressed as the mean 6 SE of triplicate experiments.
The enzymatic assay was performed at 25 C in 0.1 M potassium phosphate (pH 7.5) containing 10 mM ethylenediaminetetra-acetic acid. 5, 5 0 dithiobis (2-nitrobenzoic acid) (DTNB) (Sigma) was used as a substrate to determine TrxR activity. The enzymatic assay contained 100 lM bnicotinamide adenine dinucleotide phosphate (NADPH) (Sigma) and 3 mM DTNB, and the initial increase in A412 was recorded during the first 2 min by the addition of DTNB. One unit of TrxR was defined as that required for the NADPH-dependent production of 2 lM of 2-nitro-5thiobenzoic acid per min using e412 nm ¼ 13.6 mM1cm1. DNA-protection activity of recombinant SjTGR The antioxidant activity of SjTGR was investigated using a DNAcleavage (nicking) assay to assess protection from DNA damage caused by a metal-catalyzed oxidation (MCO) system, as previously described (Kim et al., 1988; Li et al., 2004). Briefly, 100 ll of reaction mixture containing 33 lM FeCl3, 3.3 mM dithiothreitol (DTT), and a final concentration of purified SjTGR fusion protein ranging from 100–600 lg/ml were incubated for 2 hr at 37 C. pUC19 super-coiled plasmid DNA (300 ng) was then added into each reaction mixture and incubated at 37 C for 2.5 hr. Finally, the mixtures were examined by subjecting 20-ll samples to agarose gel electrophoresis with ethidium bromide to assess DNA protection. Bovine serum albumin (BSA) was assayed as a control. Peroxidase activity of recombinant SjTGR The ability of recombinant SjTGR to reduce H2O2 was determined using the method described by Lim and colleagues (Lim et al., 1993). Briefly, a reaction mixture (100 ll) containing different concentrations (0– 50 lg/ml) of rSjTGR and 50 mM reaction buffer (Tris-HCl, pH 8.0) was incubated for 30 min at 37 C. H2O2 was added to a final concentration of 30 lM and incubated for an additional 30 min. Subsequently, 0.9 ml (8% v/v) of trichloroacetic acid was added to terminate the reaction, and protein was then removed by centrifugation. Finally, 0.2 ml ferrous ammonium sulfate (10 mM) and 0.1 ml potassium thiocyanate (2.5 M) were added into the mixture and the absorbance was measured at 480 nm. Reduced H2O2 was determined using a known amount of H2O2 as a standard. Statistical analysis The data were analyzed to compare the differences among groups using Student’s t-tests.
RESULTS Efficiency of siRNAs transfection The efficiency of siRNA transfection was determined by soaking treated schistosomes with fluorescent siRNAs and analyzing the results by FCM. The proportion of fluorescent cells in the treated group was 6.25% 6 1.28 (Fig. 1A) compared with 0.64% 6 0.16 in the untreated group (Fig. 1B). Effect of RNA interference on SjTGR in vitro at the transcript level The knockdown efficiencies of the 4 SjTGR-specific and control siRNAs on SjTGR transcripts in vitro were analyzed by qRTPCR (Fig. 2). SjTGR mRNA was reduced by 9.66% by the S1 siRNA and 46.01% by the S2 siRNA compared with untreated parasites, while levels remained almost unchanged by treatment with the S3 siRNA, S4 siRNA, and the control siRNA. These results indicate that the S2 siRNA sequence affected the targetgene expression in vitro. Effect of RNAi on SjTGR in vitro at the protein level The effect of siRNA knockdown on SjTGR at the protein level in vitro was examined by western blotting. SjTGR protein expression was inhibited in parasites at 3 days post-S2 siRNA treatment (Fig. 3A). Western blot results were normalized and converted into a histogram using Image J2x software (NIH) (Fig. 3B). The results of western blotting were similar to those seen at the mRNA level. TrxR activity in the S2 siRNA group was 1.16 U/mg, which was significantly lower (P , 0.01) than in the untreated, control siRNA, and S1 siRNA groups (1.51 U/mg, 1.55 U/mg, and 1.51 U/mg, respectively).
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FIGURE 2. Effects of RNAi on SjTGR transcription in vitro analyzed by qRT-PCR. The value in each column is the ratio of the optical density of SjTGR to that of Sja-tubulin. All data are expressed as the mean 6 SE of triplicate experiments.
Super-coiled DNA-protection activity and peroxidase activity of SjTGR The activity of recombinant TGR was detected in the same way as for native TrxR (Han et al., 2012). Recombinant SjTGR was defrosted from 80 C and analyzed by SDS-PAGE to check that it was not degraded. A DNA-nicking assay was used to determine the DNAprotection activity of SjTGR using pUC19 super-coiled plasmid DNA and the MCO system. Oxygen free radicals produced by the MCO system caused nicking of pUC19 super-coiled plasmid DNA. pUC19 super-coiled plasmid DNA was measured by gelmobility shift after treatment. Separately incubated MCO components did not damage pUC19 super-coiled plasmid DNA. DNA nicking was prevented by the addition of purified recombinant SjTGR, but not in the absence of rSjTGR or by addition of the control protein, BSA, under the same conditions. Furthermore, the protective effect of rSjTGR was concentration dependent, and decreasing levels of rSjTGR in the reaction reduced plasmid protection from oxidative cleavage. The lowest concentration of purified rSjTGR that conferred protection was 100 lg/ml, and almost 100% of DNA-nicking was prevented by rSjTGR at a concentration of 600 lg/ml after 2.5 hr incubation at 37 C (Fig. 4). To determine the antioxidant properties of purified rSjTGR, peroxidase activity was measured using H2O2 as the substrate. The reduction of peroxide was monitored based on the remaining H2O2 in the ferrithiocyanate system. rSjTGR failed to remove H2O2 even with increasing concentrations (Fig. 5), indicating that rSjTGR did not possess the peroxidase activity required to remove H2O2 directly. DISCUSSION Schistosomes can survive in the bloodstream of their final host for 5–30 yr. Several survival mechanisms have been proposed,
FIGURE 3. Effect of siRNA knockdown on SjTGR protein levels in vitro by western blotting analysis. Worms were collected at 3 days posttreatment. (A) The soluble proteins were extracted from the treated worms. Sja-tubulin was used as a loading control. (B) The results of western blots were converted into a histogram by measuring the optical densities of the bands. All data are expressed as the mean 6 SE of triplicate experiments. *Statistically significant compared with untreated group (P , 0.05), **(P , 0.01).
including the production of protective antioxidant proteins to neutralize oxidative damage resulting from the host’s immune response and from self-generated oxygen radicals (LoVerde, 1998). These antioxidant proteins have been an important focus of research with regard to schistosomiasis. Three types of Prxs, classified as novel antioxidant proteins, have recently been cloned and sequenced from S. mansoni and S. japonicum. These proteins work predominantly to remove H2O2 in schistosomes (Kwatia et al., 2000; Williams et al., 2001; Sayed and Williams, 2004; Kumagai et al., 2006). Another antioxidant protein, TGR, has also been cloned and characterized from S. japonicum, and the protective efficacy of SjTGR against schistosome infection in mice has been examined (Han et al., 2012; Song et al., 2012). Immunization with recombinant SjTGR induced a 33.5–36.51% decrease in worm burden. TGR is thus an important vaccine candidate for the treatment of schistosomiasis, and we speculated that it might therefore also represent an important protein in terms of parasite survival.
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FIGURE 4. Protection of super-coiled DNA cleavage by rSjTGR in an MCO system. Lanes (1–6) pUC19 plasmid þ FeCl3 þ DTT þ recombinant SjTGR (600, 500, 400, 300, 200, 100 lg/ml, respectively); (7) pUC19 plasmid þ FeCl3; (8) pUC19 plasmid þ DTT; (9) pUC19 plasmid þ FeCl3 þ DTT; (10) pUC19 plasmid þ FeCl3 þ DTT þ 600 lg/ml BSA; (11) pUC19 plasmid. NF, nicked form of pUC19 plasmid; SF, super-coiled form of pUC19 plasmid.
In the present study, we studied the biological function of SjTGR using 4 siRNAs (S1, S2, S3, and S4) based on regions of the SjTGR transcript. The results showed that S2 siRNA could partially knockdown SjTGR expression in schistosomes in vitro at both the transcript and protein levels. The different knockdown efficacies of these 4 siRNAs may correlate with their distinct locations in the coding sequence of the SjTGR mRNA. We used a simple soaking method to transfect the siRNAs into the parasites. This is an effective and convenient method that causes minimal physical damage to the schistosomes (Kuntz et al., 2007; Ndegwa et al., 2007; Cheng et al., 2009). Electroporation and Lipofectaminet have also been used to transfect siRNAs; the former has proven to be an effective delivery method but the latter was shown to be toxic to the cultured parasites, leading to parasite death (Correnti et al., 2005; Krautz-Peterson et al., 2007). Both soaking and electroporation were found to be effective methods for knockdown of schistosome genes with siRNA (KrautzPeterson et al., 2007). We compared the efficacies of these two methods for gene knockdown in S. japonicum and found that electroporation was associated with a higher mortality, especially in larval schistosomes. We previously determined that 200 nM was the optimal concentration of siRNA for inducing gene suppression (Cheng et al., 2009). We therefore used the soaking method and a concentration of 200 nM was chosen in the present study. RNAi has previously been used to identify gene function in other parasites. In the first study of RNAi in vitro in an animal parasite, Samarasinghe et al. (2011) found that genes expressed at sites accessible to the environment were likely to be susceptible to RNAi in the sheep gastrointestinal nematode Haemonchus contortus. In addition, knockdown of the H. contortus H11 gene prior to infection reduced the worm burden in sheep by 40% and egg output by 57%. RNAi has been reported in the trematodes F. hepatica, in which successful knockdown of the cysteine proteases cathepsin B and L in the infective stage resulted in marked reductions in target transcript levels and in the expression of the encoded proteins in the gut (McGonigle et al., 2008). In the first report of targeted manipulation of endogenous gene expression, researchers reported that dsRNA treatment of S. mansoni sporocysts resulted in disrupted expression of the genes encoding the GAPDH and the glucose transporter protein 1 (SGTP1) in the human blood fluke stage (Boyle et al., 2003). The transforming growth factor-b (TGF-b) type II receptor (SmTbR II) was suppressed by RNAi in adult worms. That study was the first to used RNAse III to digest a long dsRNA encoding a portion of SmTbR II into smaller fragment siRNAs (Osman et al., 2006). Kuntz et al. (2007) found that TGR was essential for survival of S. mansoni. Using RNAi and selecting a long double-stranded
FIGURE 5. Peroxidase activity of rSjTGR protein. Peroxidase activity was assayed using purified SjTGR fusion protein and H2O2 (30 lM) in a concentration-dependent manner. Remaining H2O2 was measured spectrophotometrically and calculated as the percentage of H2O2 removed by rSjTGR. Values are the mean 6 SE from 3 similar experiments. Error bars represent SE (n ¼ 3).
(ds) RNA in vitro, 92% of parasites died within 4 days after knockdown of TGR expression. Cercariae were mechanically transformed to schistosomula by vortexing, and the schistosomula were then cultured with 54 lg/ml of each dsRNA after separating the bodies from tails. After 3 days of TGR dsRNA treatment, TGR protein activity (using DTNB as substrate) was reduced by 63.5%, and a marked decrease in TGR mRNA was detected by RT-PCR. In our study, we collected 11-day-old schistosomula from infected rabbits and cultured them with different siRNAs. Early stages of schistosomula are commonly used for siRNA analysis, but the parasites are difficult to collect before 10 days post-infection, and schistosomula around 11 days are therefore usually used for siRNA studies in our lab. After 3 days, TGR activity was reduced by 25.16% in vitro. A partial decrease in the expression of TGR was also seen. siRNAs have recently been applied instead of dsRNAs to avoid inducing the stress (toxic) response while retaining the ability to induce genespecific knockdown (Elbashir et al., 2001; Harborth et al., 2001). However, siRNAs can also induce toxicity, though this is generally slower and less severe than that associated with dsRNAs (Chopra et al., 2002). RNAi offers great potential for in vitro target validation and as a potentially novel therapeutic strategy based on the efficient knockdown of a target gene (Aigner, 2006). The effects of RNAi in this study were less than expected. TGR is an antioxidant protein in schistosomes, and we hypothesized that TGR could reduce H2O2, oxygen free radicals, or both. We therefore used a DNA-nicking assay to determine the antioxidant activity of SjTGR and showed that SjTGR could protect schistosome nucleic acids from oxidative damage by reducing oxygen free radicals. We simultaneously determined the peroxidase activity of SjTGR, and found no change in reduced H2O2 with increasing concentrations of rSjTGR, suggesting that SjTGR could scavenge oxygen free radicals to protect the schistosomes but could not directly reduce H2O2. H2O2 is reduced directly by GPx and Prxs, which are downstream enzymes of TGR and regulated by TGR. GPx combined with Prxs could thus reduce H2O2 to H2O in
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schistosomes in the absence of catalase (Mkoji et al., 1988; Simurda et al., 1988; Maiorino et al., 1996). However, GPx and Prxs are themselves maintained in the reduced, active state by GSH, Trx, or both. Electrons from NADPH are transferred to GSH and Trx by the unique parasite reductase, TGR (Sayed and Williams, 2004). Knockdown of SjTGR, therefore, did not completely block the actions of GPx and Prx, which could still eliminate ROS. This may help to explain why knockdown of TGR with S2 siRNA only reduced the TGR activity by 25.16% in vitro. In summary, the present investigation identified and characterized SjTGR, which may play an important role in the clearance of oxygen free radicals and protection of S. japonicum parasites against oxidative damage. RNAi targeting SjTGR partially knockdown its expression at both the transcript and protein levels. However, SjTGR could directly remove oxygen free radicals but not H2O2, which might help to explain the observed effects of RNAi. ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation of China (31172315), the Special Fund for Agro-Scientific Research in the Public Interest (200903036), the basic scientific research operation cost of state-level public welfare scientific research countryard (2013JB18), the Science and Technology Commission of Shanghai Municipality (12140902700), and the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD).
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