Cloning, expression, and characterization of Schistosoma japonicum ...

Parasitol Res (2012) 110:775–786 DOI 10.1007/s00436-011-2552-8

ORIGINAL PAPER

Cloning, expression, and characterization of Schistosoma japonicum tegument protein phosphodiesterase-5 Min Zhang & Yanhui Han & Zhu Zhu & Dong Li & Yang Hong & Xiujuan Wu & Zhiqiang Fu & Jiaojiao Lin

Received: 18 January 2011 / Accepted: 6 July 2011 / Published online: 22 July 2011 # Springer-Verlag 2011

Abstract The tegument proteins of schistosomes are regarded as potential vaccine candidates and drug targets to control schistosomiasis. Nucleotide pyrophosphatase/ phosphodiesterase-5 (NPP-5), which belongs to a multigene family of nucleotide pyrophosphatase/phosphodiesterases (NPPs), is important in the hydrolysis of pyrophosphate or phosphodiester bonds in nucleotides and their derivatives. In the present study, SjNPP-5, identified as one of the tegument proteins of Schistosoma japonicum in our previous proteomic studies, was cloned on a fragment of 1,371 bp and expressed as a recombinant protein of 69 kDa. Real-time RT-PCR analysis showed that SjNPP-5 was up-regulated at 21–42 days, and the expression level in 42-day-old male worms was almost nine times higher than that in females. Western blot analysis revealed that rSjNPP-5 had good antigenicity. Immunofluorescence analysis found that SjNPP-5 was a membrane-associated antigen mainly distributed on the surface of the male adult worm of S. japonicum. BALB/c mice vaccinated with rSjNPP-5 three times showed a 29.90% worm reduction (P0.05). Immunization with rSjNPP-5 induced a mixed Th1/Th2 response in which Th1 was dominant. The response was characterized by a reduced IgG1/IgG2a ratio and elevated production of cytokines IFN-γ and IL-4. This study suggested that SjNPP-5 may be important in schistosome development, and further

M. Zhang : Y. Han : Z. Zhu : D. Li : Y. Hong : X. Wu : Z. Fu : J. Lin (*) Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Key Laboratory of Animal Parasitology, Ministry of Agriculture, 518 Ziyue road, Minhang, Shanghai 200241, People’s Republic of China e-mail: [email protected]

investigations are required to fully understand the function of this molecule.

Introduction Schistosomes are parasitic blood helminths that infect approximately 200 million people in tropical and subtropical countries (Wang et al. 2008) and result in about 280,000 deaths annually (van der Werf et al. 2003). In China, schistosomiasis, caused by infection with Schistosoma japonicum, affects 365,700 individuals, with more than 67.6 million at risk of infection in 2009, as reported by China Health Statistics 2010. Although widespread chemotherapy with praziquantel can effectively reduce the morbidity associated with schistosomiasis (WHO 2002), its effects are not sustainable and do not prevent re-infection (Loukas et al. 2007). It is suggested that the combined use of chemotherapy and vaccination is the basis for a novel, more versatile method to control schistosomiasis (Bergquist et al. 2005). The identification of appropriate schistosome antigens which could reduce pathology and limit parasite transmission is particularly important in the case of schistosomiasis (Rofatto et al. 2009). The entire schistosome surface is covered by a syncytial layer, the tegument, composed of the basal membrane, the outer plasma membrane, and the matrix (Braschi et al. 2006). The outer plasma membrane is trilaminate in the cercaria, but has a heptalaminate appearance in the adult worm interpreted as a normal plasma membrane overlain by a secreted bilayer termed as membranocalyx (Hockley and McLaren 1973; Wilson and Barnes 1974). The tegument surface membrane forms the schistosome–host interface involved in nutrition, immune evasion and modulation, excretion, osmoregulation, sensory reception,

776

and signal transduction (Jones et al. 2004). In addition, during the migration and growth of the schistosome in the final host, the tegument surface membrane maintains a dynamic turnover, and its structure differentiates at different developmental stages of the parasite, along with constant changes in its biological function. However, all of these changes are attributed to the alteration of tegumental surface proteins, and therefore, the study of these proteins is an effective method for the discovery of vaccines and drug targets. S. japonicum nucleotide pyrophosphatase/phosphosdiesterase-5 (SjNPP-5), identified as a surface-exposed protein, belongs to a multigene family of nucleotide pyrophosphatase/phosphodiesterases (NPPs) which contains seven structurally related ecto-enzymes. NPPs hydrolyze pyrophosphate or phosphodiester bonds in a variety of extracellular compounds including nucleotides, (lyso)phospholipids, and choline phosphate esters (Stefan et al. 2005) and are therefore involved in various physiological processes including nucleotide recycling, modulation of purinergic receptor signaling, regulation of extracellular pyrophosphate levels, cell proliferation, differentiation and motility, platelet aggregation, and possibly in the regulation of insulin receptor signaling and the activity of ecto-kinases (Goding et al. 2003; Rofatto et al. 2009). According to sequence homology with the catalytic site of NPPs, NPP-5 was detected as a type-I transmembrane metalloenzyme with an extracellular domain containing a conserved catalytic site, but little is known about its function (Goding et al. 2003). In this study, we report the cloning and expression of a putative SjNPP-5 and evaluation of its protective efficacy against schistosome infection in murine models.

Materials and methods Parasites and animals Parasites of a Chinese strain of S. japonicum (Anhui isolate) at 7, 13, 21, 28, 35, and 42 days were obtained by perfusion of artificially infected New Zealand rabbits. Partial 42-day-old worms were manually separated into males and females. The different developmental stages of schistosomes and both male and female worms were collected for real-time reverse transcription polymerase chain reaction (real-time RT-PCR) analysis. Male, 6–8week-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 the Shanghai Veterinary Research Institute for the Care and Use of Laboratory Animals.

Parasitol Res (2012) 110:775–786

Cloning and molecular characterization of SjNPP-5 We isolated and identified the tegument proteins of S. japonicum at five different developmental stages. After analysis of the data, we found that SjNPP-5 (FN316919) was similar to one of the surface proteins (AAX27926.2) of S. japonicum at 7, 21, and 42 days. Primers were designed according to the nucleotide sequence of clone SJFCA3969 (FN316919) of S. japonicum. The forward and reverse oligonucleotides, 5′-ATGGATCCATGAGTGAGGAGATGC3′ and 5′-GCGTCGACTTAACAGTGTAAATTA-3′, were used to amplify the complete open reading frame (ORF) of SjNPP-5. PCR amplification was carried out using schistosomula cDNA as a template. The amplification conditions involved an initial denaturation step at 94°C for 5 min, then 32 cycles at 94°C for 30 s, 55°C for 30 s, and 72°C for 2 min, and a post-PCR step at 72°C for 10 min. The obtained PCR fragment was subcloned into the pMD19-T vector (Takara) and sequenced. Phylogenetic and sequence analysis BLAST and PSI-BLAST searches against the NCBI nonredundant protein sequence database, using SjNPP-5 as a query, were used to identify orthologues of SjNPP-5. For phylogenetic analysis, the amino acid sequence of SjNPP-5 and other NPP-5 sequences obtained from the GenBank database were analyzed using the neighbor-joining method and plotted with the MEGA (Kumar et al. 2004). The molecular weight (MW) and isoelectric point (pI) of SjNPP-5 were calculated using the Compute pI/Mw tool (http://www.expasy.ch/tools/pitool.html). The signal peptide was predicted using the SignalP 3.0 server (http://www.cbs. dtu.dk/services/SignalP/); transmembrane helices were analyzed using TMHMM, version 2.0 (http://www.cbs.dtu. dk/services/TMHMM-2.0/), and N-glycosylation sites were analyzed using NetNGlyc 1.0 (http://www.cbs.dtu. dk/services/NetNGlyc/). Real-time RT-PCR analysis of SjNPP-5 transcription at different developmental stages of S. japonicum Total RNA was extracted from the different development stages of worms, discussed above, using Trizol (Invitrogen, USA). The mRNAs were purified with an RNeasy Mini Kit and quantitated by spectrophotometry (Biophotometer, Eppendorf, Germany). Five micrograms of RNA, random hexamer primers, and Superscript III reverse transcriptase (Invitrogen) were added to the reverse transcription reaction. The primers designed for SjNPP-5 (forward primer: 5′-TGACCCAGACAACAAAGAAATG-3′; reverse primer: 5′-TGGACGCACACGACAAAC-3′) amplified a product of 178 bp. Real-time PCR was performed in a

Parasitol Res (2012) 110:775–786

reaction mixture of 20 μl containing 0.8 μl primers (10 μM), 1 μl cDNA, 8.2 μl EASY Dilution Buffer (TaKaRa, China), and 10 μl 2× SYBR Green PCR Premix Taq (TaKaRa). The cycling protocol was 95°C for 2 min, followed by 40 cycles of 95°C for 15 s, 60°C for 15 s, and 72°C for 20 s in a Mastercycler Ep Reaplex (Eppendorf). 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. A negative control which did not include cDNA as a template was included in each PCR run. Expression and purification of rSjNPP-5 The full-length cDNA of SjNPP-5 was amplified by PCR with the sense primer (5′-ATGGATCCATGAGT G AG G AG ATGC - 3′) and antisen se prim er (5′ GCGTCGACTTAACAGTGTAAATTA-3′). The SjNPP-5 cDNA fragment was purified and digested with BamHI and SalI to generate inserts with overhanging ends that were ligated into the multiple cloning sites present in the pET32a(+) expression vector (Invitrogen) to produce a recombinant plasmid pET32a(+)-SjNPP-5. The plasmids were transformed into BL21(DE3) Escherichia coli cells, and transformants were confirmed by PCR, restriction enzyme digestion, and sequence analysis. For protein expression, transformed BL21(DE3) E. coli cells were grown in 500 ml of Luria–Bertani medium plus ampicillin (100 μg/ml) at 37°C until the OD600 =0.6. Then, cultures were induced with 1 mM isopropylthio-β-D-galactoside (IPTG) for 5–6 h at 37°C. The cells were harvested by centrifugation at 10,000×g for 15 min. The supernatants were removed, and the cell pellets were resuspended in 4 ml of ice-cold 1× binding buffer (500 mM NaCl, 20 mM Tris–HCl, 5 mM imidazole, pH 7.9) per 100 ml culture volume. The samples were sonicated on ice and freeze-thawed three times until they were no longer viscous. The lysates were centrifuged at 12,000×g for 15 min to collect inclusion bodies and cellular debris while leaving other soluble substances. The pellets were resuspended in 5 ml of 1× binding buffer plus 6 M urea per 100 ml culture, then incubated on ice for 1 h to completely solubilize the protein. The recombinant protein SjNPP-5 with the His-tag was expressed in an inclusion body form after analysis by SDS–PAGE. It was then purified by Ni2+ affinity chromatography using HisBind-Resin Chromatography (Novagen, USA) under denaturing conditions according to standard protocols. The purified recombinant protein was refolded through successive dialysis against phosphate-buffered saline (PBS), pH 7.4, containing decreasing concentrations of 6, 4, 3, 2, and 1 M urea, and PBS only.

777

Antigenicity analysis of rSjNPP-5 by western blotting Purified rSjNPP-5 was subjected to SDS–PAGE and then transferred electrophoretically onto a 0.45-μm pore size nitrocellulose membrane (Whattman, Germany) at 260 mA for 2 h at 4°C. Membranes were blocked with 5% nonfat dry milk in phosphate-buffered saline Tween 20 (PBST) (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 2 mM KH2PO4, 0.05% Tween 20) for 1 h at 37°C. Membranes were then washed three times with PBST and probed with anti-SWAP (an antigen preparation from adult S. japonicum worms) rabbit serum, antirSjNPP-5 mice serum or naïve rabbit serum, diluted 1:100 in PBST overnight at 4°C. The membranes were washed three times and probed with goat anti-mouse or anti-rabbit IgG conjugated to horseradish peroxidase (HRP) (Sigma, USA) diluted 1:2,500 in PBST for 1 h at room temperature. After three washes, the membranes were visualized using precipitation-type TMB Substrate Solution (Sigma) and imaged using an Image Quant 300 (GE Healthcare, USA). Immunolocalization of SjNPP-5 in male adult worms of S. japonicum Frozen sections (8 μm in size) of male adult worms were prepared and fixed with freezing acetone for 30 min at −20°C. The sections were washed three times using PBST and blocked with 10% goat serum in PBST for 2 h at room temperature. The slices were washed three times and incubated with mouse serum specific to rSjNPP-5 or naive mouse serum diluted 1:100 in blocking buffer overnight at 4°C. Samples were washed three times again and probed with CY3-conjugated anti-mouse IgG (Rockland, USA), diluted 1:1,000 in blocking buffer for 1 h at room temperature. After three washes, parasite nuclei were stained with 2-(4-amidinophenyl)-6-indolecarbamidine dihydrochloride (DAPI) at a final concentration of 10 μg/ml for 5 min at room temperature. Parasites were visualized by fluorescence microscopy (Nikon, Japan). All of the parameters and microscope settings were maintained throughout the process. Vaccination of mice Thirty mice were divided randomly into three groups of ten mice each. Mice were injected subcutaneously with rSjNPP-5 in 206 adjuvant (20 μg/100 μl/mouse) on days 0, 15, and 30. The animals in the adjuvant control and blank control groups were injected with 206 adjuvant in PBS (100 μl/mouse) and PBS only (100 μl/mouse), respectively, according to the same immunization protocol.

778

Parasitol Res (2012) 110:775–786

Detection of antibodies specific for SjNPP-5 On the tenth day after each immunization, the sera of ten mice from each experimental group were collected by retroorbital bleeding. Detection of specific IgG, IgG1, and IgG2a antibodies against SjNPP-5 was performed by direct ELISA. Ninety-six-well microtiter plates (Costar, USA) were coated overnight with 100 μl of rSjNPP-5 diluted to a concentration of 10 μg/ml in carbonate bicarbonate buffer (pH 9.6). The plates were washed three times in PBST, then blocked with 3% BSA in PBST for 1 h at 37°C. The plates were again washed three times. Test sera were diluted 1:100 in PBST, added to the wells (100 μl/well), and incubated for 2 h at 37°C. After washing three times, goat anti-mouse IgG conjugated to HRP (Sigma) was diluted 1:1,000 in PBST, and HRP-conjugated goat anti-mouse IgG1 and IgG2a (AbD Serotec, UK) were diluted 1:4,000 in PBST and added at 100 μl/well. After 1 h incubation at 37°C, the plates were washed three times, and the substrate, 3,3′5, 5′-tetramethyl benzidine dihydrochloride, was added (100 μl/well). The plates were incubated for 10 min at room temperature in the dark, and the reaction was stopped using 2 M H2SO4 (50 μl/well). The absorbance was read at 450 nm using a microplate reader (BioTek, USA). Cytokine analysis Cytokine detection was performed using serum from five mice 10 days after the third immunization with rSjNPP-5 plus 206 adjuvant and 206 adjuvant in PBS as a control. The BioLegend LEGEND MAX™ ELISA kit with precoated plates was used to quantify cytokines in the serum (Wang et al. 2010). The concentrations of IFN-γ and IL-4 in the test sera were analyzed using the mouse IFN-γ ELISA kit (cat. no. 430807, Biolegend, USA) and the mouse IL-4 ELISA kit (cat. no. 431007, Biolegend), respectively, according to the manufacturer's procedures. All standards and samples were run in triplicate as recommended. Evaluation of immune protective efficacy against S. japonicum challenge Two weeks after the last vaccination, all mice were challenged with 40±1 viable cercaria percutaneously via a wet glass lid. Forty-two days after challenge, worms were collected by perfusion from the hepatic portal system and then counted. The worm reduction rates were calculated using the 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%. To count the number of eggs in the liver, 1.0-g samples of liver tissue from each test mouse were homogenized in 10 ml of 5% NaOH, incubated at 56°C

Fig. 1 The recombinant plasmid was identified by enzyme digestion. Lane 1 the blank plasmid pET32a(+), lane 2 the recombinant plasmid pET32a(+)–SjNPP-5, lane 3 the recombinant plasmid pET32a(+)– SjNPP-5 digested with BamHI and SalI restriction enzymes, M Marker DL2000 DNA Ladder

for 1 h, and then mixed thoroughly. An average of three counts per 20 μl of mixture were taken to estimate the number of eggs, and this count was converted to 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%.

Results Molecular cloning and bioinformatic analysis of SjNPP-5 The full-length sequence of the S. japonicum cDNA encoding SjNPP-5 was obtained by PCR amplification from a transcriptome cDNA clone of 18-day-old schistosomula using specific oligonucleotides that were designed using the corresponding mRNA clone SJFCA39699 (GenBank ID: FN316919.1) (Fig. 1). The resulting full-length cDNA contained an ORF of 1,371 bp, encoding a protein of 456 amino acids with a predicted molecular mass of approximately 52.2 kDa and an isoelectric point of 8.37. Two amino acid sequence differences were found at amino acid positions 274 (asparagine instead of aspartic acid) and 322 (arginine instead of histidine) when comparing the protein sequence obtained in this study with the published sequence (Protein ID: CAX72650.1). Fig. 2 Clustal X alignment of the derived amino acid sequences of„ SjNPP-5 (FN316919), SmNPP-5 (ACB70464), RnNPP-5 (NP_001012762), MmNPP-5 (NP_114392), HsNPP-5 (NP_067547). The regions with high identity and similarity among NPP-5 sequences are shown as black and gray columns, according to the Clustal X algorithm. Conserved domain is indicated by the continuous box

Parasitol Res (2012) 110:775–786

779

780

Parasitol Res (2012) 110:775–786

Fig. 3 Phylogenetic tree of S. japonicum NPP-5, S. mansoni NPP-5, and all seven R. norvegicus, M. musculus, and H. sapiens NPPs using the neighbor-joining method and plotted with MEGA. The sequence accession numbers are RnNPP-1 (AAL26912.1), RnNPP-2 (NP_476445.2), RnNPP-3 (NP_062243.2), RnNPP-4 (NP_001100362.1), RnNPP-6 (XP_224853.3) and RnNPP-7 (NP_001012484.1), MmNPP-1 (NP_032839.3), MmNPP-2

(NP_056559.2), MmNPP-3 (NP_598766.2), MmNPP-4 (NP_950181.2), MmNPP-6 (NP_796278.1), MmNPP-7 (NP_001025462.1), HsNPP-1 (NP_006199.1), HsNPP-2 (NP_006200.3), HsNPP-3 (NP_005012.2), HsNPP-4 (NP_055751.1), HsNPP-6 (NP_699174.1), and HsNPP-7 (NP_848638.2) (the accession numbers of the other members are cited in the legend of Fig. 1)

BLASTP comparisons of the deduced S. japonicum protein sequence to those in GenBank showed that the best match (E value=0.0) was SmNPP-5, with 72% identity and 83% similarity. The closest orthologues of SjNPP-5 were the NPP-5 sequence from Rattus norvegicus (E value= 8e−66), followed by the sequences from Mus musculus

(E value=7e−65) and Homo sapiens (E value=2e−64) (Fig. 2). SjNPP-5 contained the NPP signature sequence (residues 37–363) and was recognized as part of the Pfam Type I phosphodiesterase/nucleotide pyrophosphatase family (PF01663) with an E value of 3e−63. The N-terminal sequence of SjNPP-5 contained a putative signal peptide (residues 1–26) and a transmembrane domain (residues 9–31). Four high-probability N-glycosylation sites were found at Asn59, Asn113, Asn175, and Asn258. The phylogenetic tree of the NPP superfamily is shown in Fig. 3. The results indicated that SjNPP-5 was most closely related to SmNPP-5 and grouped with the NPP-5 and NPP-4 sequences in a branch. mRNA expression analysis of SjNPP-5 in worms from different developmental stages and gender

Fig. 4 Stage and gender differential expression of SjNPP-5 in S. japonicum by real-time RT-PCR. Data were normalized against amplification of an internal housekeeping control gene Sjtubulin

The expression of SjNPP-5 at the mRNA level was determined in S. japonicum in 7-, 13-, 18-, 23-, 32-, and

Parasitol Res (2012) 110:775–786

Fig. 5 Expression analysis of the rSjNPP-5 protein using SDS–PAGE (12%). Lanes 1 and 2 total extract from BL21 before and after induction with 1 mM IPTG, lane 3 and 4 total extract from pET32a(+) before and after induction with 1 mM IPTG, lanes 5 and 6 total extract from rSjNPP-5 before and after induction with 1 mM IPTG, lane M molecular mass markers

42-day-old worms, as well as in 42-day-old female and male worms using real-time RT-PCR analysis, with tubulin as the internal control (Fig. 4). The results revealed that mRNA expression was around five times higher in the schistosomes at 21, 28, 35, and 42 days than at 7 and 14 days. In addition, expression levels in the male worms were almost nine times higher than in the females at 42 days.

781

Fig. 7 Western blotting analysis of the antigenicity of rSj-NPP-5. Lane M molecular mass markers, lanes 1, 2, and 3 purified rSjNPP-5 was probed with anti-rSjNPP-5 mice serum (the positive control), antiSWAP rabbit serum (the experiment group), and naive rabbit serum (the negative control)

Expression, purification, and antigenicity analysis of rSjNPP-5 The gene was cloned into the pET32a(+) expression vector, and the recombinant protein was successfully expressed as a His fusion protein with an expected size of 69 kDa (Fig. 5). The transformed bacterial cells were sonicated, and the lysates were separated into soluble and insoluble fractions. The inclusion bodies contained the majority of the recombinant protein, which was mostly soluble, by extraction with 8 M urea. The protein was purified by affinity chromatography using His binding columns under denaturing conditions, and was then refolded by dialysis against PBS containing successive decreasing concentrations of urea. The purity of the preparation was accessed by SDS–PAGE (Fig. 6). The recombinant protein was identified further by western blotting using anti-SWAP rabbit serum (for the experimental group), anti-rSjNPP-5 mouse serum (for the positive control), or naive rabbit serum (for the negative control). A positive band of 69 kDa was observed in the experimental group and positive control but not in the negative control, which revealed that rSjNPP-5 had good antigenicity (Fig. 7). Immunolocalization of SjNPP-5 in male adult worms

Fig. 6 Purification analysis of the rSjNPP-5 protein using SDS–PAGE (12%). Lane M molecular mass markers, lane 1 inclusion bodies of pET32a(+)-SjNPP-5 after lysis, lane 2 rSjNPP-5 purified through Ni2 + -charged column chromatography and after dialysis

The localization of SjNPP-5 was observed by immunofluorescence with anti-rSjNPP-5 mouse serum and naive mouse serum. Specific staining was clearly observed in the section probed with SjNPP-5-specific serum but not in

782

Parasitol Res (2012) 110:775–786

Fig. 8 Immunolocalization of SjNPP-5 in male adult worm of S. japonicum. Secondary antibody CY3-conjugated anti-mouse IgG (red) were used for fluorescence detection of SjNPP-5 on male adult worm

sections. DAPI (blue) was used to stain parasite nuclei. a The section was probed with anti-rSjNPP-5 mice serum. b The section was probed with naive mice serum (the negative control)

the section probed with normal mouse serum (Fig. 8). The results revealed that SjNPP-5 was a membrane-associated antigen mainly presented on the surface of the male adult worm of S. japonicum.

levels were observed before or after vaccination. The results revealed that rSjNPP-5 may stimulate a strong antibody response (P