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Characterization of thioredoxin glutathione reductase in Schiotosoma japonicum
Parasitology International 61 (2012) 475–480
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Characterization of thioredoxin glutathione reductase in Schiotosoma japonicum Yanhui Han a, b, Min Zhang a, Yang Hong a, Zhu Zhu a, Dong Li a, Xiangrui Li b, Zhiqiang Fu a,⁎, Jiaojiao Lin a,⁎⁎ a b
Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Key Laboratory of Animal Parasitology, Ministry of Agriculture of China, Shanghai 200241, PR China College of Veterinary Medicine, Nanjing Agricultural University, Nanjing, Jiangsu, 210095, PR China
a r t i c l e
i n f o
Article history: Received 4 January 2012 Received in revised form 22 March 2012 Accepted 23 March 2012 Available online 30 March 2012 Keywords: Schistosoma japonicum Thioredoxin glutathione reductase Clone Vaccine
a b s t r a c t Schistosomiasis is one of the most prevalent and serious parasitic diseases in the world and remains an important public health problem in China. Screening and discovery of an effective vaccine candidate or new drug target is crucial for the control of this disease. In this study, we cloned a cDNA encoding Schistosoma japonicum (S. japonicum) thioredoxin glutathione reductase (SjTGR) from the cDNA of 42-day-old adult worms. The open reading frame (ORF) of the gene was 1791 base pairs (bp) encoding a protein of 596 amino acids. SjTGR was subcloned into pET-32a (+) and expressed in Escherichia coli (E. coli) BL21 (DE3). The recombinant protein rSjTGR exhibited enzymatic activity of 5.13 U/mg with DTNB as the substrate, and showed strong immunogenecity. Real-time PCR results indicated that SjTGR was expressed at a higher level in 35-day-old schistosome worms in transcript. We vaccinated BALB/c mice with rSjTGR in combination with MONTANIDE™ ISA 206 VG (ISA 206) and observed a 33.50% to 36.51% (P b 0.01) decrease in the adult worm burden and a 33.73%to 43.44% (P b 0.01) decrease in the number of eggs counted compared to the ISA 206 or blank control groups in two independent vaccination tests. ELISA analysis demonstrated that rSjTGR induced a high level of SjTGR-specific IgG, IgG1, and IgG 2a antibodies and induced elevated production of IFN-γ. This study provides the basis for further investigations into the biological function of SjTGR and further evaluation of the potential use of this molecule as a vaccine candidate or new drug target is warranted. © 2012 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Schistosomiasis is one of the most prevalent and serious parasitic diseases globally, with an estimated 200 million people infected in 76 countries and territories over the past decade, and it remains an important public health problem in some developing countries [1]. A recent report indicated that the morbidity due to schistosomiasis was underestimated, and resulted in an estimated 280,000 deaths in subSaharan Africa alone every year [2]. No vaccine has been developed against the disease. Praziquantel is the only drug extensively utilized to cure schistosomiasis, no new drugs have been introduced after praziquantel [3]. In developing countries, constant reinfection of individuals living in close proximity makes drug treatment alone inefficient [4]. The best way to control schistosomiasis is through immunization with an anti-schistosomiasis vaccine combined with drug treatment [5]. A vaccine that could induce even a partial reduction in worm burden could potentially limit parasite transmission [6]. Thus, to screen and identify an effective vaccine candidate or new drug target is urgent for the control of this disease. There are two redox systems in mammals: one is based on glutathione (GSH), and the other is based on the protein thioredoxin ⁎ Corresponding author. Tel.: + 86 21 3429 3618; fax: + 86 21 3429 3619. ⁎⁎ Corresponding author. Tel.: + 86 21 3429 3440; fax: + 86 21 5408 1818. E-mail addresses: [email protected] (Z. Fu), [email protected] (J. Lin). 1383-5769/$ – see front matter © 2012 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.parint.2012.03.005
(Trx). Both major systems transfer electrons from NADPH via the enzymes glutathione reductase (GR) and thioredoxin reductase (TrxR) [7,8]. In a different manner from mammals, it was recently discovered that a unique multifunctional enzyme, thioredoxin glutathione reductase (TGR), replaced these two enzymes in Schistosoma mansoni (S. mansoni) [9]. Further studies revealed that this enzyme was a selenoprotein exhibiting a carboxylterminal active site motif—GCUC. “U” was selenocysteine (Sec), and was able to confer unique properties to selenoprotein because of its high reactivity. Angela N. Kuntz et al. [8] have demonstrated that S. mansoni TGR was the first validated, key antischistosomiasis drug target. The partial sequence encoding the S. japonicum TGR gene has been previously reported [10], but the biological function of this gene remains unclear. S. japonicum is a species of Asian schistosome that exhibits a significant effect on human health. The parasites survive in the veins of the final host and escape from the reactive oxygen species (ROS) [11], which are released from the effective host cells that adhere to the antibody-coated worms. In this study, the full-length cDNA encoding SjTGR was cloned and expressed in E. coli. The expression and localization of SjTGR in schistosome worms were analyzed, the protective efficacy induced by the recombinant SjTGR was evaluated, and the relevant immuno-mechanisms were investigated. This study provides the basis for biological functionality studies of the SjTGR gene, and for the potential evaluation of SjTGR as a vaccine candidate or drug target against schistosome infection in the future.
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2. Materials and methods
2.5. Thioredoxin reductase (TrxR) activity assay of recombinant SjTGR
2.1. Parasite and animals
The 5, 5′-diothiobis (2-nitrobenzoic acid) (DTNB) (Sigma, USA) reduction assay was used to determine the TrxR activity of rSjTGR at room temperature (25 °C). The reaction mix contained 900 μl working buffer (100 mM potassium phosphate, PH 7.0, 10 mM ethylene diamine tetraacetic acid (EDTA), and 240 μM β-Nicotinamide adenine dinucleotide phosphate (NADPH) (Sigma, USA)), 60 μl assay buffer (100 mM potassium phosphate, pH 7.0, 10 mM EDTA), 30 μl DTNB (40 mg/ml), and 10 μl (0.76 mg/ml or 0.103 nmol) recombinant protein. The reaction was initiated upon addition of DTNB. The absorbance at 412 nm was measured using ultraviolet spectrophotometry. One unit of TrxR activity was defined as the production of 1 μmol of 2-nitro-5thiobenoic acid (TNB) per minute. The mouse liver Thioredoxin reductase and the plasmid pET-32a(+) expression protein (~19 kDa) were used as positive and negative controls, respectively.
The S. 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 (CAAS). 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 as a vaccine challenge. Worms that were 7, 14, 21, 28, 35 and 42 days old were obtained by perfusion of New Zealand rabbits artificially infected with schistosome. We separated the 42-day-old worms manually as males or females. Worms were washed with PBS and stored in liquid nitrogen until use. Male 6–8 week old BALB/c mice were purchased from Shanghai Experimental Animal Centre, 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. 2.2. Cloning and molecular characterization of S. japonicum TGR Primers were designed according to the nucleotide sequences of the clone S. japonicum (GenBank accession no. EU938325), and a bacterialtype SECIS element [12] was added to the antisense primer. The forward and backward oligonucleotides, 5′-GCGGGATCCATTTCATTCAACATGCC3′ and 5′-CGCCTCGAGGGCCGCATAGGCTAACGATTGGTGCAGACCTGCAACCGATTATTAACCTCAGCAACCGGT-3′, were used to amplify the complete open reading frame (ORF) of SjTGR. The underlined sequences represent the sites of BamHIand XhoI (Takara, Japan), respectively. PCR amplification was carried out using the cDNA of 42day-old adult worms of S. japonicum as a template. The PCR product was subcloned into the pMD19-T vector (Takara, Japan) according to the manufacturer's instructions and positive clones were selected for sequencing. 2.3. Phylogenetic and sequence analysis Blast and PSI-Blast searches against the National Center for Biotechnology Information (NCBI) non-redundant protein sequence database, using SjTGR as a query, were used to identify orthologs of SjTGR. The molecular weight (MW) and isoelectric point (pI) of SjTGR were calculated using the Compute pI/Mw tool (http://www.expasy. ch/tools/pi tool.html). The signal peptide was predicted using the SignalP 3.0 server (http://www.cbs.dtu.dk/services/SignalP/). 2.4. Expression and purification of recombinant SjTGR (rSjTGR) The PCR product was digested with the corresponding enzymes, purified by agarose gel electrophoresis, and subcloned into pET-32a (+) vectors (Novagen, USA) for expression, which was verified by sequencing. The recombinant protein was expressed in E. coli strain BL21 (DE3) cells (Invitrogen, USA). Production of the recombinant protein was induced in Luria–Bertani broth (LB) containing kanamycin (1 mM) at 20 °C with shaking by the addition of Isopropyl-Dthiogalactopyranoside (IPTG) to 1 mM for 8 h. The cells were collected by centrifugation at 4 °C, and 12,000 rpm/min for 20 min. The pellet was resuspended with 15–20 ml phosphate buffered saline (PBS), then frozen/thawed alternatively approximately, sonicated at 4 °C for 15 min, and centrifugated for 20 min at 12,000 rpm/min. The supernatant was then filtered through a nickel affinity column (Novagen, USA). Recombinant protein was eluted in 60 mM imidazole and dialyzed against PBS for 2 h. The concentration was determined using a Bradford assay kit (Sangon, China). The purified protein was assessed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and stored at − 80 °C.
2.6. Preparation of the total RNA and cDNA Total RNA was isolated from each developmental stage of the parasite using TRIzol reagent (Invitrogen), digested with RNase-free DNaseI (Takara) to remove genome DNA, and then purified using an RNeasy Mini kit (QIAGEN, USA). Complementary cDNA was synthesized with Superscript II reverse transcriptase (Invitrogen) in 40 μl of reaction mixture using random hexamer oligonucleotides (Takara) as primers. Total mRNA was stored at − 80 °C until used. 2.7. Real-time PCR analysis The cDNA of the different stages of parasite was applied as a PCR template. The specific primers for SjTGR were as follows: forward primer: 5′-CTACTGGCGAGCGTCCAAAATACC-3′, reverse primer: 5′AACATCACCGCCCAAACTGACAAG-3′. The amplification fragment was 170 bp. The primers targeting the S. japonicum tubulin gene (forward primer: 5′-CTGATTTTCCATTCGTTTG-3′; reverse primer: 5′-GTTGTCTACCATGTTGGCA-3′) amplified a product of 213 bp, used as an internal standard. Real-time PCR was performed in a reaction mixture of 20 μl containing 0.8 μl primers (10 μM), 1 μl cDNA, 8.2 μl EASY Dilution Buffer (Takara), and 10 μl 2 × SYBR Green PCR Premix Taq (Takara). Real-time PCR was performed in a Mastercycler ep Realplex (Eppendorf, Germany). The thermal cycling profiles were as follows: 95 °C for 2 min, followed by 40 cycles of amplification (95 °C for 15 s, 60 °C for 15 s, 72 °C for 20 s). 2.8. Immunolocalization of SjTGR in worms of S. japonicum Frozen sections (8 μm in size) of schistosome adult worms were prepared and fixed with freezing acetone for 30 min at −20 °C, blocked for 2 h at room temperature with 10% goat serum, then incubated overnight at 4 °C with the mouse serum specific to recombinant SjTGR at a concentration of 1:100. After washing with PBST, the sections were probed at room temperature with 1:1000 green fluorescence conjugated goat anti-mouse IgG (H+ L) (Invitrogen, USA), subsequently stained with 4, 6-diamidino-2-phenylindole (DAPI) (Takara), and finally detected using a fluorescence microscope (Nikon, Japan). 2.9. Evaluating the protective efficacy of recombinant SjTGR 2.9.1. Mouse vaccination and challenge A total of 30 mice were randomly divided into three groups, and each mouse per group was subcutaneously injected with either 20 μg of recombinant protein SjTGR mixed with ISA 206 (Seppic, France), ISA 206 alone, or PBS only, respectively, three times over an interval of 2 weeks. One week after the last vaccination, the mice were challenged percutaneously with 40 ± 1 cercariae. Forty-two days post-challenge, the worms were perfused and counted. The worm
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reduction rates were calculated using the following formula: percentage reduction in worm burden = (mean worm burden of control group − mean worm burden of vaccinated group) / mean worm burden of control group × 100%. The weight of each liver tissue was measured and homogenized in 10 ml PBS, and 1 ml homogenate was mixed with 1 ml 10% NaOH and incubated at 56 °C for 1 h. An average of three counts per 100 μl of mixture was performed to calculate the number of eggs, and this count was converted into eggs per gram (EPG). The egg reduction rates were calculated using the formula: Percentage reduction in liver egg count = (mean EPG from control group − mean EPG from vaccinated group) / mean EPG from control group × 100%. 2.9.2. Enzyme linked immunosorbant assays Serum samples were collected from each mouse prior to vaccination, 1 week after each immunization, and before perfusion, and then sorted at − 80 °C until used. ELISA was performed to detect the titer of specific IgG, IgG 1, and IgG 2a antibodies against recombinant SjTGR. A 96-well ELISA plate (Costar, USA) was coated with 100 μl of recombinant SjTGR at a concentration of 10 μg/ml in carbonate-bicarbonate buffer (PH = 9.6) per well overnight at 4 °C. The plate was washed with PBST and blocked with 1.5% bovine serum albumin (BSA) in PBST for 1 h at 37 °C. After washing, 100 μl of each serum sample at a 1:100 dilution was added and incubated for 1 h at 37 °C. Plate-bound antibody was detected with peroxidaseconjugated goat-antimouse IgG (1:1000) (Takara), IgG 1 (1:4000), and IgG 2a (1:4000) (Biolegend, USA), respectively. After 1 h incubation at 37 °C, 3,3′5,5′-tetramethyl benzidine dihydrochloride (TMB) (Sigma, USA) was added as a substrate solution. Finally, the reaction was stopped with 2 M H2SO4 (50 μl/well), and the absorbance was measured at 450 nm. 2.9.3. Cytokine analysis Splenocytes were isolated from spleens of each experimental mouse 10 days after the third immunization using the EZ-Sep Mouse 1× Lymphocyte separation medium according to the manufacturer's instructions with five mice per group. The splenocytes were cultured in RPMI 1640 medium (Gibco, USA) supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin G sodium, 100 μg/ml streptomycin sulfate, and 250 ng/ml amphotericin B. For cytokine assays, 30 μg/ml polymyxin B was added to the cultures to abrogate the cytokine response to LPS, as previously described [13]. Splenocytes were adjusted to 1 × 10 6 cells per well and maintained in this culture with medium alone or stimulated with rSjTGR (25 μg/ml) in a 96-well plate for IFN-γ and IL-4 assays [14,15]. The detection of cytokines was performed using the Duoset ELISA kit (R&D, Diagnostic) according to the manufacturer's directions.
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Furthermore, the N-terminal extension contained a glutaredoxin (Grx) domain comprised of approximately 110 amino acids, including an active site CPFC. The protein was predicted to be a selenoprotein with TGA encoding the SeCys residue. The true terminator was a TAA box. The sequence analysis also demonstrated that SjTGR did not possess a signal peptide. 3.2. Expression and purification of recombinant SjTGR Most of the recombinant S. japonicum TGR was expressed in E. coli as a soluble His-tagged fusion protein when bacterial growth occurred at 20 °C. The recombinant protein was purified by Niaffinity chromatography. SDS-PAGE analysis revealed that the recombinant protein had a molecular weight of approximately 74 kDa. An enzyme activity assay indicated that the TrxR activity of the purified recombinant SjTGR, the rat liver TrxR, and plasmid pET-32a (+) expression protein was 5.13 U/mg, 10 U/mg, and 0.83 U/mg, respectively. These findings revealed that recombinant SjTGR exhibited the specific activity of TrxR. 3.3. SjTGR transcription analyses in worms at different developmental stages using real-time PCR The transcription of SjTGR in 7, 14, 21, 28, and 35-day-old worms, as well as 42-day-old female and male worms, was detected by realtime quantitative RT-PCR analysis using α-tubulin as an internal control (Fig. 1). The results indicated that SjTGR mRNA was expressed in all stages investigated, and the expression levels in 35-day-old worms and 42-day-old female worms were approximately seven times higher than the 7- and 14-day-old worms, and over two times higher than 21- and 28-day-old worms and 42-day-old male worms. 3.4. Immunolocalization of SjTGR in the schistosome We used an immunofluorescence assay to determine the distribution of SjTGR in the 42-day-old schistosome adult worms, and specific staining was primarily present in the tegument and parenchyma of worms probed with SjTGR-specific serum but not on the section probed with normal mouse serum (Fig. 2). 3.5. Evaluation of the protective efficacy against schistosome infection induced by rSjTGR in mice The mice were perfused at 42 days post-infection, and the reductions in worm burden and reductions in liver egg count were
2.10. Statistical analysis The data were subjected to the Student's t-test to calculate the level of significance of the differences between the experiment group and the control group using the software package GraphPad Prism. 3. Results 3.1. Cloning and bioinformatics analysis of SjTGR The full-length sequence of SjTGR was obtained by RT-PCR from the mRNA of 42-day-old adult worms. The ORF of SjTGR was 1791 bp encoding a protein of 596 amino acids with a predicted molecular weight of 64.940 kDa and an isoelectric point of 6.30. Bioinformatics analysis revealed that the schistosome TGRs contained sequences of NADH-binding and FAD-binding domains, a thioldisulfide redox active center, -Cys-Val-Asn-Val-Gly-Cys-, and a dimer interface domain of pyridine nucleotide disulfide oxidoreductases.
Fig. 1. Analysis of SjTGR expression in different development stages of S. japonicum by Real-time PCR. 7-d, 14-d, 21-d, 28-d, 35-d, represent 7-day, 14-day, 21-day, 28-day, 35-day-old worms, respectively. 42d F and 42d M, represent female and male adult worms at 42 days. The expression of the gene encoding α-tubulin was used as a control.
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Fig. 2. The localization analysis of SjTGR by immunofluorescence. (A) Probed with SjTGR specific mouse serum. (B) Probed with the normal mouse serum. Arrows indicate the localization of SjTGR.
calculated and listed in Table 1. The results indicated that the mice immunized with rSjTGR induced a 33.5–36.51% decrease in the adult worm burden and a 33.73–43.44% decrease in eggs counted compared to the PBS control group in two independent vaccination tests. Partial worm reduction was also seen in mice injected with adjuvant alone in the two experiments. The preliminary results indicated that SjTGR induced partial protection against S. japonicum infection. 3.6. Detection of SjTGR specific IgG antibodies The levels of rSjTGR specific IgG antibodies prior to vaccination and 1 week post the 1st, 2nd, and 3rd vaccination, and 6 weeks postinfection were measured. No markedly rSjTGR specific IgG antibodies were detected in the adjuvant and PBS control groups, and no significant differences were observed between the two groups. However, the SjTGR specific IgG antibody level increased significantly after the 1st immunization, and was maintained continuously at a high level until the mice were perfused at 6 weeks post-infection. The results revealed that rSjTGR stimulated a strong, specific antibody response. The rSjTGR specific IgG1 and IgG2a antibody levels were also evaluated and the results are shown in Table 2. In rSjTGR vaccinated mice, both the rSjTGR specific IgG1 and IgG2a antibody levels increased following the first vaccination, and continuously gradually increased at the 2nd and 3rd vaccination. The titer of the specific IgG1 antibody was higher compared to the IgG2a antibody, and the IgG 1/ IgG 2a ratio was significantly elevated at the 1st immunization and
Table 1 Protection level induced by BABL/c mice immunized with SjTGR. Groups Experiment 1 TGR–adjuvant (n = 15) ISA 206 (n = 15) PBS (n = 11) Experiment 2 TGR–adjuvant (n = 10) ISA 206 (n = 11) PBS (n = 10)
Worm burden (mean ± S.E.) Liver eggs (mean ± S.E.) (% reduction) (% reduction) 14.13 ± 4.13 (33.50%**)
26079 ± 10970.53 (33.73%**)
21.13 ± 3.39 (0.60%) 21.27 ± 4.83
42297.08 ± 10850.34 (0) 39356.81 ± 12408.98
16.33 ± 5.83 (36.51%**)
38990.05 ± 15124.91 (43.44%**)
23.10 ± 5.70 (10.21%) 25.73 ± 7.90
78733.33 ± 30119.43 (0) 68939.39 ± 19249.14
Each mouse was infected with 40 ± 1 carceriae and perfused at 42 days post-infection. Data are expressed as mean ± S.E. **Statistically significant compared to PBS control group (P b 0.01).
then decreased at the 2nd immunization. No significant changes in the IgG1 and IgG2a antibody levels were observed in both mice from the adjuvant control and blank control group. 3.7. Detection of IFN-γ and IL-4 in supernatants of splenocyte culture in vitro The cytokines IFN-γ and IL-4, produced by splenocytes from rSjTGR vaccinated mice, were detected. The results indicated that cells stimulated with rSjTGR produced statistically significantly higher levels of IFN-γ compared with cells grown in medium alone, and the level of IL-4 was not found to be significantly different in rSjTGR treated cells compared to untreated controls. 4. Discussion In China, significant progress has been made in the control of schistosomiasis japonica over the past 50 years [16], but the disease remains an important public health problem. To screen and identify effective vaccine candidates or new drug targets is urgent for the control of this disease. A recent study revealed that S. mansoni schistosome died within 4 days after silencing TGR expression in vitro, and a TGR inhibitor killed the parasites rapidly in culture and partially cured schistosome-infected mice, suggesting that TGR is potentially an essential parasite protein and a potentially important drug target for schistosome [8]. In this study, the S. japonicum TGR gene with a bacterial-type SECIS element was inserted into a reverse primer and was successfully cloned and expressed in E. coli. Real-time PCR analysis demonstrated that SjTGR transcripts were expressed in all worm experimental groups from different developmental stages, with a higher expression level observed in 35-day-old adult worms and 42-day-old female worms. Immmunofluorescence analysis revealed that this protein was primarily expressed in the tegument and the parenchyma of S. japonicum worms, and two independent mouse vaccination tests demonstrated that significant worm and egg reduction was observed when mice were immunized with rSjTGR. These findings, taken together with the finding from SmTGR, indicated that SjTGR was essential for the development of S. japonicum. As a schistosome protein that may correspond with the maintenance of cellular redox oxygen and the evasion of reactive oxygen species generated by the host's immune response, further studies to elucidate the biological function of SjTGR and to evaluate the potential of SjTGR as vaccine candidate or new drug target for the control of S. japonicum are warranted. Previously studies have reported that Th1-type antigens potentially induce partial protection against schistosome infection, whereas Th2-type antigens potentially reduce liver pathologic damage
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Table 2 IgG1 and IgG2a immune profile induced by vaccination with recombinant SjTGR. Times
Groups IgG1
0 1 2 3
IgG2a
IgG1/IgG2a
SjTGR–adjuvant
ISA 206
PBS
SjTGR–adjuvant
ISA 206
PBS
SjTGR–adjuvant
0.076 ± 0.013 0.361 ± 0.077* 0.481 ± 0.066* 0.590 ± 0.034*
0.074 ± 0.009 0.073 ± 0.010 0.094 ± 0.057 0.082 ± 0.002
0.086 ± 0.003 0.081 ± 0.013 0.080 ± 0.005 0.086 ± 0.012
0.096 ± 0.008 0.166 ± 0.077* 0.296 ± 0.112* 0.313 ± 0.063*
0.086 ± 0.009 0.087 ± 0.006 0.086 ± 0.011 0.083 ± 0.006
0.090 ± 0.005 0.086 ± 0.012 0.084 ± 0.006 0.092 ± 0.013
0.792 2.181 1.626 1.888
0, 1, 2 and 3 represent pre-immunization, 1st, 2nd and 3rd immunization. Data are expressed as mean ± S.E. *(P b 0.01) indicates statistical significance compared to the group of mice immunized with ISA 206.
caused by this parasite in a murine host [17]. In our study, a high level of rSjTGR specific IgG and IgG1, a moderate level of IgG2a, and a reduced IgG1/IgG2a ratio were observed following the first immunization. Cytokine analysis revealed that IFN-γ levels, but not IL-4, significantly increased when splenocytes from rSjTGR vaccinated mice were stimulated with rSjTGR. Our findings suggested that the rSjTGR vaccination potentially induces a mixed Th1/Th2 but polarized Th1 immune response. The involvement of IFN-γ in protective immunity to schistosomiasis is well documented in a murine model [18]. In the irradiated cercariae vaccination model that induced a high level of protection, treatment with monoclonal anti-IFN-γ antibody totally abrogated the protective immunity achieved [19]. Similar results were obtained when IFN-γ knock-out mice were exposed to the radiation-attenuated vaccine, confirming the essential role of IFNγ in protective immunity against murine schistosomiasis [20]. The high level of IFN-γ induced following vaccination with rSjTGR may be potentially the reason that partial protection was obtained in recombinant protein immunized mice in this study. Moreover, increased expression of IL-4 in rSjTGR vaccinated mice potentially correlates with the egg-induced Th2 type immune response [1]. Recently, a quantitative high throughput screen (qHTS) was devised, based on the activity of the S.mansoni redox pathway, and a chemical library consisting of 71,028 compounds was identified via qHTS; some compounds exhibited IC50 (50% inhibitory concentration) values in a low nanomolar range. A furoxan was identified, and it was very effective against all stages of S. mansoni, adult worms of S. japonicum, and S. haematobium [21]. A putative drug-lead was presented by David L. Williams and his group regarding the redox-detoxification pathway of schistosomes [2]. Additionally, several oxidoreductases, such as S. mansoni thioredoxin glutathione reductase (SmTGR) [7,8], S. mansoni thioredoxin peroxidase (SmTPx) [22], and S. japonicum peroxiredoxin-1 (SjPrx-1) [11] have been investigated and reported. These studies have provided an important basis for research into anti-schistosomiasis drug screening and development, and our study has accumulated additional useful information in this research area. SjTGR plays an important role in host–parasite interplay regarding escaping the host immune response as an oxidoreductase. The results of this study indicate that rSjTGR potentially induces a partially protective effect against schistosome infection in BALB/c mice, and further studies investigating the biological function of this molecule and evaluating the potential as a vaccine candidate or new drug target are warranted.
Acknowledgements We thank Dr. Williams for his helpful suggestions in the study. We would like to thank Yaojun Shi from Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences for technical assistance with parasite collection. This work was supported by the National Basic Research Program of China (No. 2007CB513108), State-level public welfare scientific research courtyard basic scientific
research operation cost 2010JB10 and Special found for Agroscientific Reserch in the Public Insterest (No. 200903036).
References [1] Lopes DO, Paiva LF, Martins MA, Cardoso FC, Rajao MA, Pinho JM, et al. Sm21.6 a novel EF-hand family protein member located on the surface of Schistosoma mansoni adult worm that failed to induce protection against challenge infection but reduced liver pathology. Vaccine Jun 24 2009;27(31):4127–35. [2] Cioli D, Valle C, Angelucci F, Miele AE. Will new antischistosomal drugs finally emerge? Trends in Parasitology Sep 2008;24(9):379–82. [3] Fenwick A, Savioli L, Engels D, Robert Bergquist N, Todd MH. Drugs for the control of parasitic diseases: current status and development in schistosomiasis. Trends in Parasitology Nov 2003;19(11):509–15. [4] Bergquist NR. Schistosomiasis vaccine development: progress and prospects. Memórias do Instituto Oswaldo Cruz 1998;93(Suppl. 1):95–101. [5] Bergquist NR. Schistosomiasis: from risk assessment to control. Trends in Parasitology Jul 2002;18(7):309–14. [6] Chitsulo L, Loverde P, Engels D. Schistosomiasis. Nature Reviews Microbiology Jan 2004;2(1):12–3. [7] Alger HM, Williams DL. The disulfide redox system of Schistosoma mansoni and the importance of a multifunctional enzyme, thioredoxin glutathione reductase. Molecular and Biochemical Parasitology Apr 30 2002;121(1):129–39. [8] Kuntz AN, Davioud-Charvet E, Sayed AA, Califf LL, Dessolin J, Arner ES, et al. Thioredoxin glutathione reductase from Schistosoma mansoni: an essential parasite enzyme and a key drug target. PLoS Medicine Jun 2007;4(6):e206. [9] Alger HM, Sayed AA, Stadecker MJ, Williams DL. Molecular and enzymatic characterisation of Schistosoma mansoni thioredoxin. International Journal of Parasitology Sep 2002;32(10):1285–92. [10] Xie SY, Yin XY, Hua WQ, Zeng XJ, Chen HG, Liang YS, et al. Cloning and sequence analysis of thioredoxin glutathione reductase gene of Schistosoma japonicum. Chinese Journal of Schistosomiasis Control 2008;6(20):427–30. [11] Kumagai T, Osada Y, Ohta N, Kanazawa T. Peroxiredoxin-1 from Schistosoma japonicum functions as a scavenger against hydrogen peroxide but not nitric oxide. Molecular and Biochemical Parasitology Mar 2009;164(1):26–31. [12] Arner ES, Sarioglu H, Lottspeich F, Holmgren A, Bock A. High-level expression in Escherichia coli of selenocysteine-containing rat thioredoxin reductase utilizing gene fusions with engineered bacterial-type SECIS elements and co-expression with the selA, selB and selC genes. Journal of Molecular Biology Oct 8 1999;292(5):1003–16. [13] Cardoso LS, Araujo MI, Goes AM, Pacifico LG, Oliveira RR, Oliveira SC. Polymyxin B as inhibitor of LPS contamination of Schistosoma mansoni recombinant proteins in human cytokine analysis. Microbial Cell Factories 2007;6:1. [14] Fonseca CT, Brito CF, Alves JB, Oliveira SC. IL-12 enhances protective immunity in mice engendered by immunization with recombinant 14 kDa Schistosoma mansoni fatty acid-binding protein through an IFN-gamma and TNF-alpha dependent pathway. Vaccine Jan 2 2004;22(3–4):503–10. [15] Pacifico LG, Fonseca CT, Chiari L, Oliveira SC. Immunization with Schistosoma mansoni 22.6 kDa antigen induces partial protection against experimental infection in a recombinant protein form but not as DNA vaccine. Immunobiology 2006;211(1–2):97–104. [16] Wu ZD, Lu ZY, Yu XB. Development of a vaccine against Schistosoma japonicum in China: a review. Acta Tropica Nov-Dec 2005;96(2–3):106–16. [17] Sher A, Coffman RL, Hieny S, Scott P, Cheever AW. Interleukin 5 is required for the blood and tissue eosinophilia but not granuloma formation induced by infection with Schistosoma mansoni. Proceedings of the National Academy of Sciences of the United States of America Jan 1990;87(1):61–5. [18] Hewitson JP, Hamblin PA, Mountford AP. Immunity induced by the radiationattenuated schistosome vaccine. Parasite Immunology Jul–Aug 2005;27(7–8): 271–80. [19] Smythies LE, Coulson PS, Wilson RA. Monoclonal antibody to IFN-gamma modifies pulmonary inflammatory responses and abrogates immunity to Schistosoma mansoni in mice vaccinated with attenuated cercariae. Journal of Immunology Dec 1 1992;149(11):3654–8. [20] Wilson RA, Coulson PS, Betts C, Dowling MA, Smythies LE. Impaired immunity and altered pulmonary responses in mice with a disrupted interferon-gamma
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receptor gene exposed to the irradiated Schistosoma mansoni vaccine. Immunology Feb 1996;87(2):275–82. [21] Sayed AA, Simeonov A, Thomas CJ, Inglese J, Austin CP, Williams DL. Identification of oxadiazoles as new drug leads for the control of schistosomiasis. Nature Medicine Apr 2008;14(4):407–12.
[22] Pushpamali WA, De Zoysa M, Kang HS, Oh CH, Whang I, Kim SJ, et al. Comparative study of two thioredoxin peroxidases from disk abalone (Haliotis discus discus): cloning, recombinant protein purification, characterization of antioxidant activities and expression analysis. Fish & Shellfish Immunology Mar 2008;24(3): 294–307.
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Parasitology International journal homepage: www.elsevier.com/locate/parint
Characterization of thioredoxin glutathione reductase in Schiotosoma japonicum Yanhui Han a, b, Min Zhang a, Yang Hong a, Zhu Zhu a, Dong Li a, Xiangrui Li b, Zhiqiang Fu a,⁎, Jiaojiao Lin a,⁎⁎ a b
Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Key Laboratory of Animal Parasitology, Ministry of Agriculture of China, Shanghai 200241, PR China College of Veterinary Medicine, Nanjing Agricultural University, Nanjing, Jiangsu, 210095, PR China
a r t i c l e
i n f o
Article history: Received 4 January 2012 Received in revised form 22 March 2012 Accepted 23 March 2012 Available online 30 March 2012 Keywords: Schistosoma japonicum Thioredoxin glutathione reductase Clone Vaccine
a b s t r a c t Schistosomiasis is one of the most prevalent and serious parasitic diseases in the world and remains an important public health problem in China. Screening and discovery of an effective vaccine candidate or new drug target is crucial for the control of this disease. In this study, we cloned a cDNA encoding Schistosoma japonicum (S. japonicum) thioredoxin glutathione reductase (SjTGR) from the cDNA of 42-day-old adult worms. The open reading frame (ORF) of the gene was 1791 base pairs (bp) encoding a protein of 596 amino acids. SjTGR was subcloned into pET-32a (+) and expressed in Escherichia coli (E. coli) BL21 (DE3). The recombinant protein rSjTGR exhibited enzymatic activity of 5.13 U/mg with DTNB as the substrate, and showed strong immunogenecity. Real-time PCR results indicated that SjTGR was expressed at a higher level in 35-day-old schistosome worms in transcript. We vaccinated BALB/c mice with rSjTGR in combination with MONTANIDE™ ISA 206 VG (ISA 206) and observed a 33.50% to 36.51% (P b 0.01) decrease in the adult worm burden and a 33.73%to 43.44% (P b 0.01) decrease in the number of eggs counted compared to the ISA 206 or blank control groups in two independent vaccination tests. ELISA analysis demonstrated that rSjTGR induced a high level of SjTGR-specific IgG, IgG1, and IgG 2a antibodies and induced elevated production of IFN-γ. This study provides the basis for further investigations into the biological function of SjTGR and further evaluation of the potential use of this molecule as a vaccine candidate or new drug target is warranted. © 2012 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Schistosomiasis is one of the most prevalent and serious parasitic diseases globally, with an estimated 200 million people infected in 76 countries and territories over the past decade, and it remains an important public health problem in some developing countries [1]. A recent report indicated that the morbidity due to schistosomiasis was underestimated, and resulted in an estimated 280,000 deaths in subSaharan Africa alone every year [2]. No vaccine has been developed against the disease. Praziquantel is the only drug extensively utilized to cure schistosomiasis, no new drugs have been introduced after praziquantel [3]. In developing countries, constant reinfection of individuals living in close proximity makes drug treatment alone inefficient [4]. The best way to control schistosomiasis is through immunization with an anti-schistosomiasis vaccine combined with drug treatment [5]. A vaccine that could induce even a partial reduction in worm burden could potentially limit parasite transmission [6]. Thus, to screen and identify an effective vaccine candidate or new drug target is urgent for the control of this disease. There are two redox systems in mammals: one is based on glutathione (GSH), and the other is based on the protein thioredoxin ⁎ Corresponding author. Tel.: + 86 21 3429 3618; fax: + 86 21 3429 3619. ⁎⁎ Corresponding author. Tel.: + 86 21 3429 3440; fax: + 86 21 5408 1818. E-mail addresses: [email protected] (Z. Fu), [email protected] (J. Lin). 1383-5769/$ – see front matter © 2012 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.parint.2012.03.005
(Trx). Both major systems transfer electrons from NADPH via the enzymes glutathione reductase (GR) and thioredoxin reductase (TrxR) [7,8]. In a different manner from mammals, it was recently discovered that a unique multifunctional enzyme, thioredoxin glutathione reductase (TGR), replaced these two enzymes in Schistosoma mansoni (S. mansoni) [9]. Further studies revealed that this enzyme was a selenoprotein exhibiting a carboxylterminal active site motif—GCUC. “U” was selenocysteine (Sec), and was able to confer unique properties to selenoprotein because of its high reactivity. Angela N. Kuntz et al. [8] have demonstrated that S. mansoni TGR was the first validated, key antischistosomiasis drug target. The partial sequence encoding the S. japonicum TGR gene has been previously reported [10], but the biological function of this gene remains unclear. S. japonicum is a species of Asian schistosome that exhibits a significant effect on human health. The parasites survive in the veins of the final host and escape from the reactive oxygen species (ROS) [11], which are released from the effective host cells that adhere to the antibody-coated worms. In this study, the full-length cDNA encoding SjTGR was cloned and expressed in E. coli. The expression and localization of SjTGR in schistosome worms were analyzed, the protective efficacy induced by the recombinant SjTGR was evaluated, and the relevant immuno-mechanisms were investigated. This study provides the basis for biological functionality studies of the SjTGR gene, and for the potential evaluation of SjTGR as a vaccine candidate or drug target against schistosome infection in the future.
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2. Materials and methods
2.5. Thioredoxin reductase (TrxR) activity assay of recombinant SjTGR
2.1. Parasite and animals
The 5, 5′-diothiobis (2-nitrobenzoic acid) (DTNB) (Sigma, USA) reduction assay was used to determine the TrxR activity of rSjTGR at room temperature (25 °C). The reaction mix contained 900 μl working buffer (100 mM potassium phosphate, PH 7.0, 10 mM ethylene diamine tetraacetic acid (EDTA), and 240 μM β-Nicotinamide adenine dinucleotide phosphate (NADPH) (Sigma, USA)), 60 μl assay buffer (100 mM potassium phosphate, pH 7.0, 10 mM EDTA), 30 μl DTNB (40 mg/ml), and 10 μl (0.76 mg/ml or 0.103 nmol) recombinant protein. The reaction was initiated upon addition of DTNB. The absorbance at 412 nm was measured using ultraviolet spectrophotometry. One unit of TrxR activity was defined as the production of 1 μmol of 2-nitro-5thiobenoic acid (TNB) per minute. The mouse liver Thioredoxin reductase and the plasmid pET-32a(+) expression protein (~19 kDa) were used as positive and negative controls, respectively.
The S. 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 (CAAS). 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 as a vaccine challenge. Worms that were 7, 14, 21, 28, 35 and 42 days old were obtained by perfusion of New Zealand rabbits artificially infected with schistosome. We separated the 42-day-old worms manually as males or females. Worms were washed with PBS and stored in liquid nitrogen until use. Male 6–8 week old BALB/c mice were purchased from Shanghai Experimental Animal Centre, 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. 2.2. Cloning and molecular characterization of S. japonicum TGR Primers were designed according to the nucleotide sequences of the clone S. japonicum (GenBank accession no. EU938325), and a bacterialtype SECIS element [12] was added to the antisense primer. The forward and backward oligonucleotides, 5′-GCGGGATCCATTTCATTCAACATGCC3′ and 5′-CGCCTCGAGGGCCGCATAGGCTAACGATTGGTGCAGACCTGCAACCGATTATTAACCTCAGCAACCGGT-3′, were used to amplify the complete open reading frame (ORF) of SjTGR. The underlined sequences represent the sites of BamHIand XhoI (Takara, Japan), respectively. PCR amplification was carried out using the cDNA of 42day-old adult worms of S. japonicum as a template. The PCR product was subcloned into the pMD19-T vector (Takara, Japan) according to the manufacturer's instructions and positive clones were selected for sequencing. 2.3. Phylogenetic and sequence analysis Blast and PSI-Blast searches against the National Center for Biotechnology Information (NCBI) non-redundant protein sequence database, using SjTGR as a query, were used to identify orthologs of SjTGR. The molecular weight (MW) and isoelectric point (pI) of SjTGR were calculated using the Compute pI/Mw tool (http://www.expasy. ch/tools/pi tool.html). The signal peptide was predicted using the SignalP 3.0 server (http://www.cbs.dtu.dk/services/SignalP/). 2.4. Expression and purification of recombinant SjTGR (rSjTGR) The PCR product was digested with the corresponding enzymes, purified by agarose gel electrophoresis, and subcloned into pET-32a (+) vectors (Novagen, USA) for expression, which was verified by sequencing. The recombinant protein was expressed in E. coli strain BL21 (DE3) cells (Invitrogen, USA). Production of the recombinant protein was induced in Luria–Bertani broth (LB) containing kanamycin (1 mM) at 20 °C with shaking by the addition of Isopropyl-Dthiogalactopyranoside (IPTG) to 1 mM for 8 h. The cells were collected by centrifugation at 4 °C, and 12,000 rpm/min for 20 min. The pellet was resuspended with 15–20 ml phosphate buffered saline (PBS), then frozen/thawed alternatively approximately, sonicated at 4 °C for 15 min, and centrifugated for 20 min at 12,000 rpm/min. The supernatant was then filtered through a nickel affinity column (Novagen, USA). Recombinant protein was eluted in 60 mM imidazole and dialyzed against PBS for 2 h. The concentration was determined using a Bradford assay kit (Sangon, China). The purified protein was assessed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and stored at − 80 °C.
2.6. Preparation of the total RNA and cDNA Total RNA was isolated from each developmental stage of the parasite using TRIzol reagent (Invitrogen), digested with RNase-free DNaseI (Takara) to remove genome DNA, and then purified using an RNeasy Mini kit (QIAGEN, USA). Complementary cDNA was synthesized with Superscript II reverse transcriptase (Invitrogen) in 40 μl of reaction mixture using random hexamer oligonucleotides (Takara) as primers. Total mRNA was stored at − 80 °C until used. 2.7. Real-time PCR analysis The cDNA of the different stages of parasite was applied as a PCR template. The specific primers for SjTGR were as follows: forward primer: 5′-CTACTGGCGAGCGTCCAAAATACC-3′, reverse primer: 5′AACATCACCGCCCAAACTGACAAG-3′. The amplification fragment was 170 bp. The primers targeting the S. japonicum tubulin gene (forward primer: 5′-CTGATTTTCCATTCGTTTG-3′; reverse primer: 5′-GTTGTCTACCATGTTGGCA-3′) amplified a product of 213 bp, used as an internal standard. Real-time PCR was performed in a reaction mixture of 20 μl containing 0.8 μl primers (10 μM), 1 μl cDNA, 8.2 μl EASY Dilution Buffer (Takara), and 10 μl 2 × SYBR Green PCR Premix Taq (Takara). Real-time PCR was performed in a Mastercycler ep Realplex (Eppendorf, Germany). The thermal cycling profiles were as follows: 95 °C for 2 min, followed by 40 cycles of amplification (95 °C for 15 s, 60 °C for 15 s, 72 °C for 20 s). 2.8. Immunolocalization of SjTGR in worms of S. japonicum Frozen sections (8 μm in size) of schistosome adult worms were prepared and fixed with freezing acetone for 30 min at −20 °C, blocked for 2 h at room temperature with 10% goat serum, then incubated overnight at 4 °C with the mouse serum specific to recombinant SjTGR at a concentration of 1:100. After washing with PBST, the sections were probed at room temperature with 1:1000 green fluorescence conjugated goat anti-mouse IgG (H+ L) (Invitrogen, USA), subsequently stained with 4, 6-diamidino-2-phenylindole (DAPI) (Takara), and finally detected using a fluorescence microscope (Nikon, Japan). 2.9. Evaluating the protective efficacy of recombinant SjTGR 2.9.1. Mouse vaccination and challenge A total of 30 mice were randomly divided into three groups, and each mouse per group was subcutaneously injected with either 20 μg of recombinant protein SjTGR mixed with ISA 206 (Seppic, France), ISA 206 alone, or PBS only, respectively, three times over an interval of 2 weeks. One week after the last vaccination, the mice were challenged percutaneously with 40 ± 1 cercariae. Forty-two days post-challenge, the worms were perfused and counted. The worm
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reduction rates were calculated using the following formula: percentage reduction in worm burden = (mean worm burden of control group − mean worm burden of vaccinated group) / mean worm burden of control group × 100%. The weight of each liver tissue was measured and homogenized in 10 ml PBS, and 1 ml homogenate was mixed with 1 ml 10% NaOH and incubated at 56 °C for 1 h. An average of three counts per 100 μl of mixture was performed to calculate the number of eggs, and this count was converted into eggs per gram (EPG). The egg reduction rates were calculated using the formula: Percentage reduction in liver egg count = (mean EPG from control group − mean EPG from vaccinated group) / mean EPG from control group × 100%. 2.9.2. Enzyme linked immunosorbant assays Serum samples were collected from each mouse prior to vaccination, 1 week after each immunization, and before perfusion, and then sorted at − 80 °C until used. ELISA was performed to detect the titer of specific IgG, IgG 1, and IgG 2a antibodies against recombinant SjTGR. A 96-well ELISA plate (Costar, USA) was coated with 100 μl of recombinant SjTGR at a concentration of 10 μg/ml in carbonate-bicarbonate buffer (PH = 9.6) per well overnight at 4 °C. The plate was washed with PBST and blocked with 1.5% bovine serum albumin (BSA) in PBST for 1 h at 37 °C. After washing, 100 μl of each serum sample at a 1:100 dilution was added and incubated for 1 h at 37 °C. Plate-bound antibody was detected with peroxidaseconjugated goat-antimouse IgG (1:1000) (Takara), IgG 1 (1:4000), and IgG 2a (1:4000) (Biolegend, USA), respectively. After 1 h incubation at 37 °C, 3,3′5,5′-tetramethyl benzidine dihydrochloride (TMB) (Sigma, USA) was added as a substrate solution. Finally, the reaction was stopped with 2 M H2SO4 (50 μl/well), and the absorbance was measured at 450 nm. 2.9.3. Cytokine analysis Splenocytes were isolated from spleens of each experimental mouse 10 days after the third immunization using the EZ-Sep Mouse 1× Lymphocyte separation medium according to the manufacturer's instructions with five mice per group. The splenocytes were cultured in RPMI 1640 medium (Gibco, USA) supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin G sodium, 100 μg/ml streptomycin sulfate, and 250 ng/ml amphotericin B. For cytokine assays, 30 μg/ml polymyxin B was added to the cultures to abrogate the cytokine response to LPS, as previously described [13]. Splenocytes were adjusted to 1 × 10 6 cells per well and maintained in this culture with medium alone or stimulated with rSjTGR (25 μg/ml) in a 96-well plate for IFN-γ and IL-4 assays [14,15]. The detection of cytokines was performed using the Duoset ELISA kit (R&D, Diagnostic) according to the manufacturer's directions.
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Furthermore, the N-terminal extension contained a glutaredoxin (Grx) domain comprised of approximately 110 amino acids, including an active site CPFC. The protein was predicted to be a selenoprotein with TGA encoding the SeCys residue. The true terminator was a TAA box. The sequence analysis also demonstrated that SjTGR did not possess a signal peptide. 3.2. Expression and purification of recombinant SjTGR Most of the recombinant S. japonicum TGR was expressed in E. coli as a soluble His-tagged fusion protein when bacterial growth occurred at 20 °C. The recombinant protein was purified by Niaffinity chromatography. SDS-PAGE analysis revealed that the recombinant protein had a molecular weight of approximately 74 kDa. An enzyme activity assay indicated that the TrxR activity of the purified recombinant SjTGR, the rat liver TrxR, and plasmid pET-32a (+) expression protein was 5.13 U/mg, 10 U/mg, and 0.83 U/mg, respectively. These findings revealed that recombinant SjTGR exhibited the specific activity of TrxR. 3.3. SjTGR transcription analyses in worms at different developmental stages using real-time PCR The transcription of SjTGR in 7, 14, 21, 28, and 35-day-old worms, as well as 42-day-old female and male worms, was detected by realtime quantitative RT-PCR analysis using α-tubulin as an internal control (Fig. 1). The results indicated that SjTGR mRNA was expressed in all stages investigated, and the expression levels in 35-day-old worms and 42-day-old female worms were approximately seven times higher than the 7- and 14-day-old worms, and over two times higher than 21- and 28-day-old worms and 42-day-old male worms. 3.4. Immunolocalization of SjTGR in the schistosome We used an immunofluorescence assay to determine the distribution of SjTGR in the 42-day-old schistosome adult worms, and specific staining was primarily present in the tegument and parenchyma of worms probed with SjTGR-specific serum but not on the section probed with normal mouse serum (Fig. 2). 3.5. Evaluation of the protective efficacy against schistosome infection induced by rSjTGR in mice The mice were perfused at 42 days post-infection, and the reductions in worm burden and reductions in liver egg count were
2.10. Statistical analysis The data were subjected to the Student's t-test to calculate the level of significance of the differences between the experiment group and the control group using the software package GraphPad Prism. 3. Results 3.1. Cloning and bioinformatics analysis of SjTGR The full-length sequence of SjTGR was obtained by RT-PCR from the mRNA of 42-day-old adult worms. The ORF of SjTGR was 1791 bp encoding a protein of 596 amino acids with a predicted molecular weight of 64.940 kDa and an isoelectric point of 6.30. Bioinformatics analysis revealed that the schistosome TGRs contained sequences of NADH-binding and FAD-binding domains, a thioldisulfide redox active center, -Cys-Val-Asn-Val-Gly-Cys-, and a dimer interface domain of pyridine nucleotide disulfide oxidoreductases.
Fig. 1. Analysis of SjTGR expression in different development stages of S. japonicum by Real-time PCR. 7-d, 14-d, 21-d, 28-d, 35-d, represent 7-day, 14-day, 21-day, 28-day, 35-day-old worms, respectively. 42d F and 42d M, represent female and male adult worms at 42 days. The expression of the gene encoding α-tubulin was used as a control.
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Fig. 2. The localization analysis of SjTGR by immunofluorescence. (A) Probed with SjTGR specific mouse serum. (B) Probed with the normal mouse serum. Arrows indicate the localization of SjTGR.
calculated and listed in Table 1. The results indicated that the mice immunized with rSjTGR induced a 33.5–36.51% decrease in the adult worm burden and a 33.73–43.44% decrease in eggs counted compared to the PBS control group in two independent vaccination tests. Partial worm reduction was also seen in mice injected with adjuvant alone in the two experiments. The preliminary results indicated that SjTGR induced partial protection against S. japonicum infection. 3.6. Detection of SjTGR specific IgG antibodies The levels of rSjTGR specific IgG antibodies prior to vaccination and 1 week post the 1st, 2nd, and 3rd vaccination, and 6 weeks postinfection were measured. No markedly rSjTGR specific IgG antibodies were detected in the adjuvant and PBS control groups, and no significant differences were observed between the two groups. However, the SjTGR specific IgG antibody level increased significantly after the 1st immunization, and was maintained continuously at a high level until the mice were perfused at 6 weeks post-infection. The results revealed that rSjTGR stimulated a strong, specific antibody response. The rSjTGR specific IgG1 and IgG2a antibody levels were also evaluated and the results are shown in Table 2. In rSjTGR vaccinated mice, both the rSjTGR specific IgG1 and IgG2a antibody levels increased following the first vaccination, and continuously gradually increased at the 2nd and 3rd vaccination. The titer of the specific IgG1 antibody was higher compared to the IgG2a antibody, and the IgG 1/ IgG 2a ratio was significantly elevated at the 1st immunization and
Table 1 Protection level induced by BABL/c mice immunized with SjTGR. Groups Experiment 1 TGR–adjuvant (n = 15) ISA 206 (n = 15) PBS (n = 11) Experiment 2 TGR–adjuvant (n = 10) ISA 206 (n = 11) PBS (n = 10)
Worm burden (mean ± S.E.) Liver eggs (mean ± S.E.) (% reduction) (% reduction) 14.13 ± 4.13 (33.50%**)
26079 ± 10970.53 (33.73%**)
21.13 ± 3.39 (0.60%) 21.27 ± 4.83
42297.08 ± 10850.34 (0) 39356.81 ± 12408.98
16.33 ± 5.83 (36.51%**)
38990.05 ± 15124.91 (43.44%**)
23.10 ± 5.70 (10.21%) 25.73 ± 7.90
78733.33 ± 30119.43 (0) 68939.39 ± 19249.14
Each mouse was infected with 40 ± 1 carceriae and perfused at 42 days post-infection. Data are expressed as mean ± S.E. **Statistically significant compared to PBS control group (P b 0.01).
then decreased at the 2nd immunization. No significant changes in the IgG1 and IgG2a antibody levels were observed in both mice from the adjuvant control and blank control group. 3.7. Detection of IFN-γ and IL-4 in supernatants of splenocyte culture in vitro The cytokines IFN-γ and IL-4, produced by splenocytes from rSjTGR vaccinated mice, were detected. The results indicated that cells stimulated with rSjTGR produced statistically significantly higher levels of IFN-γ compared with cells grown in medium alone, and the level of IL-4 was not found to be significantly different in rSjTGR treated cells compared to untreated controls. 4. Discussion In China, significant progress has been made in the control of schistosomiasis japonica over the past 50 years [16], but the disease remains an important public health problem. To screen and identify effective vaccine candidates or new drug targets is urgent for the control of this disease. A recent study revealed that S. mansoni schistosome died within 4 days after silencing TGR expression in vitro, and a TGR inhibitor killed the parasites rapidly in culture and partially cured schistosome-infected mice, suggesting that TGR is potentially an essential parasite protein and a potentially important drug target for schistosome [8]. In this study, the S. japonicum TGR gene with a bacterial-type SECIS element was inserted into a reverse primer and was successfully cloned and expressed in E. coli. Real-time PCR analysis demonstrated that SjTGR transcripts were expressed in all worm experimental groups from different developmental stages, with a higher expression level observed in 35-day-old adult worms and 42-day-old female worms. Immmunofluorescence analysis revealed that this protein was primarily expressed in the tegument and the parenchyma of S. japonicum worms, and two independent mouse vaccination tests demonstrated that significant worm and egg reduction was observed when mice were immunized with rSjTGR. These findings, taken together with the finding from SmTGR, indicated that SjTGR was essential for the development of S. japonicum. As a schistosome protein that may correspond with the maintenance of cellular redox oxygen and the evasion of reactive oxygen species generated by the host's immune response, further studies to elucidate the biological function of SjTGR and to evaluate the potential of SjTGR as vaccine candidate or new drug target for the control of S. japonicum are warranted. Previously studies have reported that Th1-type antigens potentially induce partial protection against schistosome infection, whereas Th2-type antigens potentially reduce liver pathologic damage
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Table 2 IgG1 and IgG2a immune profile induced by vaccination with recombinant SjTGR. Times
Groups IgG1
0 1 2 3
IgG2a
IgG1/IgG2a
SjTGR–adjuvant
ISA 206
PBS
SjTGR–adjuvant
ISA 206
PBS
SjTGR–adjuvant
0.076 ± 0.013 0.361 ± 0.077* 0.481 ± 0.066* 0.590 ± 0.034*
0.074 ± 0.009 0.073 ± 0.010 0.094 ± 0.057 0.082 ± 0.002
0.086 ± 0.003 0.081 ± 0.013 0.080 ± 0.005 0.086 ± 0.012
0.096 ± 0.008 0.166 ± 0.077* 0.296 ± 0.112* 0.313 ± 0.063*
0.086 ± 0.009 0.087 ± 0.006 0.086 ± 0.011 0.083 ± 0.006
0.090 ± 0.005 0.086 ± 0.012 0.084 ± 0.006 0.092 ± 0.013
0.792 2.181 1.626 1.888
0, 1, 2 and 3 represent pre-immunization, 1st, 2nd and 3rd immunization. Data are expressed as mean ± S.E. *(P b 0.01) indicates statistical significance compared to the group of mice immunized with ISA 206.
caused by this parasite in a murine host [17]. In our study, a high level of rSjTGR specific IgG and IgG1, a moderate level of IgG2a, and a reduced IgG1/IgG2a ratio were observed following the first immunization. Cytokine analysis revealed that IFN-γ levels, but not IL-4, significantly increased when splenocytes from rSjTGR vaccinated mice were stimulated with rSjTGR. Our findings suggested that the rSjTGR vaccination potentially induces a mixed Th1/Th2 but polarized Th1 immune response. The involvement of IFN-γ in protective immunity to schistosomiasis is well documented in a murine model [18]. In the irradiated cercariae vaccination model that induced a high level of protection, treatment with monoclonal anti-IFN-γ antibody totally abrogated the protective immunity achieved [19]. Similar results were obtained when IFN-γ knock-out mice were exposed to the radiation-attenuated vaccine, confirming the essential role of IFNγ in protective immunity against murine schistosomiasis [20]. The high level of IFN-γ induced following vaccination with rSjTGR may be potentially the reason that partial protection was obtained in recombinant protein immunized mice in this study. Moreover, increased expression of IL-4 in rSjTGR vaccinated mice potentially correlates with the egg-induced Th2 type immune response [1]. Recently, a quantitative high throughput screen (qHTS) was devised, based on the activity of the S.mansoni redox pathway, and a chemical library consisting of 71,028 compounds was identified via qHTS; some compounds exhibited IC50 (50% inhibitory concentration) values in a low nanomolar range. A furoxan was identified, and it was very effective against all stages of S. mansoni, adult worms of S. japonicum, and S. haematobium [21]. A putative drug-lead was presented by David L. Williams and his group regarding the redox-detoxification pathway of schistosomes [2]. Additionally, several oxidoreductases, such as S. mansoni thioredoxin glutathione reductase (SmTGR) [7,8], S. mansoni thioredoxin peroxidase (SmTPx) [22], and S. japonicum peroxiredoxin-1 (SjPrx-1) [11] have been investigated and reported. These studies have provided an important basis for research into anti-schistosomiasis drug screening and development, and our study has accumulated additional useful information in this research area. SjTGR plays an important role in host–parasite interplay regarding escaping the host immune response as an oxidoreductase. The results of this study indicate that rSjTGR potentially induces a partially protective effect against schistosome infection in BALB/c mice, and further studies investigating the biological function of this molecule and evaluating the potential as a vaccine candidate or new drug target are warranted.
Acknowledgements We thank Dr. Williams for his helpful suggestions in the study. We would like to thank Yaojun Shi from Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences for technical assistance with parasite collection. This work was supported by the National Basic Research Program of China (No. 2007CB513108), State-level public welfare scientific research courtyard basic scientific
research operation cost 2010JB10 and Special found for Agroscientific Reserch in the Public Insterest (No. 200903036).
References [1] Lopes DO, Paiva LF, Martins MA, Cardoso FC, Rajao MA, Pinho JM, et al. Sm21.6 a novel EF-hand family protein member located on the surface of Schistosoma mansoni adult worm that failed to induce protection against challenge infection but reduced liver pathology. Vaccine Jun 24 2009;27(31):4127–35. [2] Cioli D, Valle C, Angelucci F, Miele AE. Will new antischistosomal drugs finally emerge? Trends in Parasitology Sep 2008;24(9):379–82. [3] Fenwick A, Savioli L, Engels D, Robert Bergquist N, Todd MH. Drugs for the control of parasitic diseases: current status and development in schistosomiasis. Trends in Parasitology Nov 2003;19(11):509–15. [4] Bergquist NR. Schistosomiasis vaccine development: progress and prospects. Memórias do Instituto Oswaldo Cruz 1998;93(Suppl. 1):95–101. [5] Bergquist NR. Schistosomiasis: from risk assessment to control. Trends in Parasitology Jul 2002;18(7):309–14. [6] Chitsulo L, Loverde P, Engels D. Schistosomiasis. Nature Reviews Microbiology Jan 2004;2(1):12–3. [7] Alger HM, Williams DL. The disulfide redox system of Schistosoma mansoni and the importance of a multifunctional enzyme, thioredoxin glutathione reductase. Molecular and Biochemical Parasitology Apr 30 2002;121(1):129–39. [8] Kuntz AN, Davioud-Charvet E, Sayed AA, Califf LL, Dessolin J, Arner ES, et al. Thioredoxin glutathione reductase from Schistosoma mansoni: an essential parasite enzyme and a key drug target. PLoS Medicine Jun 2007;4(6):e206. [9] Alger HM, Sayed AA, Stadecker MJ, Williams DL. Molecular and enzymatic characterisation of Schistosoma mansoni thioredoxin. International Journal of Parasitology Sep 2002;32(10):1285–92. [10] Xie SY, Yin XY, Hua WQ, Zeng XJ, Chen HG, Liang YS, et al. Cloning and sequence analysis of thioredoxin glutathione reductase gene of Schistosoma japonicum. Chinese Journal of Schistosomiasis Control 2008;6(20):427–30. [11] Kumagai T, Osada Y, Ohta N, Kanazawa T. Peroxiredoxin-1 from Schistosoma japonicum functions as a scavenger against hydrogen peroxide but not nitric oxide. Molecular and Biochemical Parasitology Mar 2009;164(1):26–31. [12] Arner ES, Sarioglu H, Lottspeich F, Holmgren A, Bock A. High-level expression in Escherichia coli of selenocysteine-containing rat thioredoxin reductase utilizing gene fusions with engineered bacterial-type SECIS elements and co-expression with the selA, selB and selC genes. Journal of Molecular Biology Oct 8 1999;292(5):1003–16. [13] Cardoso LS, Araujo MI, Goes AM, Pacifico LG, Oliveira RR, Oliveira SC. Polymyxin B as inhibitor of LPS contamination of Schistosoma mansoni recombinant proteins in human cytokine analysis. Microbial Cell Factories 2007;6:1. [14] Fonseca CT, Brito CF, Alves JB, Oliveira SC. IL-12 enhances protective immunity in mice engendered by immunization with recombinant 14 kDa Schistosoma mansoni fatty acid-binding protein through an IFN-gamma and TNF-alpha dependent pathway. Vaccine Jan 2 2004;22(3–4):503–10. [15] Pacifico LG, Fonseca CT, Chiari L, Oliveira SC. Immunization with Schistosoma mansoni 22.6 kDa antigen induces partial protection against experimental infection in a recombinant protein form but not as DNA vaccine. Immunobiology 2006;211(1–2):97–104. [16] Wu ZD, Lu ZY, Yu XB. Development of a vaccine against Schistosoma japonicum in China: a review. Acta Tropica Nov-Dec 2005;96(2–3):106–16. [17] Sher A, Coffman RL, Hieny S, Scott P, Cheever AW. Interleukin 5 is required for the blood and tissue eosinophilia but not granuloma formation induced by infection with Schistosoma mansoni. Proceedings of the National Academy of Sciences of the United States of America Jan 1990;87(1):61–5. [18] Hewitson JP, Hamblin PA, Mountford AP. Immunity induced by the radiationattenuated schistosome vaccine. Parasite Immunology Jul–Aug 2005;27(7–8): 271–80. [19] Smythies LE, Coulson PS, Wilson RA. Monoclonal antibody to IFN-gamma modifies pulmonary inflammatory responses and abrogates immunity to Schistosoma mansoni in mice vaccinated with attenuated cercariae. Journal of Immunology Dec 1 1992;149(11):3654–8. [20] Wilson RA, Coulson PS, Betts C, Dowling MA, Smythies LE. Impaired immunity and altered pulmonary responses in mice with a disrupted interferon-gamma
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Y. Han et al. / Parasitology International 61 (2012) 475–480
receptor gene exposed to the irradiated Schistosoma mansoni vaccine. Immunology Feb 1996;87(2):275–82. [21] Sayed AA, Simeonov A, Thomas CJ, Inglese J, Austin CP, Williams DL. Identification of oxadiazoles as new drug leads for the control of schistosomiasis. Nature Medicine Apr 2008;14(4):407–12.
[22] Pushpamali WA, De Zoysa M, Kang HS, Oh CH, Whang I, Kim SJ, et al. Comparative study of two thioredoxin peroxidases from disk abalone (Haliotis discus discus): cloning, recombinant protein purification, characterization of antioxidant activities and expression analysis. Fish & Shellfish Immunology Mar 2008;24(3): 294–307.