Introduction

Clonorchiasis is an important food-borne parasitic disease caused by Clonorchis sinensis infection. Thirty-five million people have been infected globally, of whom approximately 15 million are in China (Lun et al. 2005). Opportunistic infection of human being occurs by ingestion of raw or improperly cooked freshwater fish containing live encysted metacercariae (Sripa et al. 2010). The dwelling of C. sinensis adult worm in bile ducts results in a series of pathological changes such as pyogenic cholangitis, cholelithiasis, cholecystitis and hepatic fibrosis, even cholangiocarcinoma (Fried and Abruzzi 2010; Fried et al. 2011). Excretory–secretory products of C. sinensis (CsESPs) were considered to play important roles in the pathophysiologic responses (Pak et al. 2009) and parasite survival in the host bile ducts, where was a very hostile environment (Salazar-Calderon et al. 2003). Characterization of each component of ESPs and identification of accurate roles in the pathogenesis of C. sinensis infection may provide clues to find new strategies in prevention and therapy of clonorchiasis (Ma et al. 2007; Hu et al. 2009).

Protein disulfide isomerase (PDI) is an essential catalyst in different biological systems showing formation and reshuffling of disulfide bonds in the endoplasmic reticulum (ER) (Gilbert 1998). As molecular chaperones and foldases, PDI belongs to a member of thioredoxin superfamily to modulate the protein secretory pathway in eukaryotes (Freedman et al. 1994). The classical structure of PDIs involves two homologous catalytic domains, a and a′, comprising each an active site with a CxxC motif, separated by two homologous non-catalytic domains, b, b′, and a highly acidic C-terminal extension c (Byrne et al. 2009; Kozlov et al. 2010). Although PDIs were originally identified in the lumen of the ER, previous studies suggested that they also presented on the cell surface and modulated cellular interactions in a number of pathogens (Wilkinson and Gilbert 2004). It is well known that many major components of proteins related to adhesion and invasion of parasites are cysteine-rich and dependent on correct folding via disulfide bond formation of cysteines (Naguleswaran et al. 2005). In Neospora caninum and Toxoplasma gondii, both NcPDI and TgPDI were reported to play the important role in the parasite–host cell interaction (Liao et al. 2006; Robinson and Roy 2006). Moreover, PDI could also protect adult parasite against oxidative stress, which could be a relevant survival mechanism in Fasciola hepatica (Salazar-Calderon et al. 2003). Thus, PDIs have been considered to play significant roles in parasite–host cell interaction.

In the present study, we isolated a full-length cDNA of CsPDI from the adult C. sinensis cDNA library and did sequence analysis, cloning, expression, and immunolocalization of CsPDI, as well as its transcription levels in C. sinensis different stages.

Materials and methods

Sequence analysis of CsPDI

A full-length cDNA (clone number Cs17f06) encoding CsPDI was identified from a cDNA plasmid library of adult C. sinensis using bioinformatics tool provided by NCBI web site (http://www.ncbi.nlm.nih.gov). The physicochemical parameters, signal peptide, and structure characters of the deduced amino acid sequence were analyzed by using ExPASy (http://ca.expasy.org/tools). Vector NTI suite 8.0 was used to analyze the homology of the target sequence.

Cloning, expression, and purification of the recombinant CsPDI (rCsPDI)

The polymerase chain reaction (PCR) was employed to amplify the target gene using sense primer 5′-CGCCGAATTCCAGGTTGAGGTGATG-3′ (EcoR I, underlined) and antisense primer 5′-GCGCCTCGAGCTACAAGTCGCGTTT-3′ (Xho I, underlined). PCR amplification condition was 94 °C for 1 min, 61 °C for 1 min, 72 °C for 1 min for 35 cycles, and then 72 °C for 10 min. The purified PCR products were purified and then digested by the corresponding restriction enzymes (EcoR I + Xho I), and cloned into the prokaryotic expression vector pET28a (+) (Novagen, USA). The recombinant plasmid was confirmed by DNA sequencing, and then transformed into Escherichia coli BL21/DE3 (Promega, USA). The protein expression was induced with 1 mM isopropyl-β-d-thiogalactopyranoside (IPTG) at 37 °C for 4 h in Luria-Bertani medium (LB). The bacteria were harvested by centrifuging at 4 °C, and the recombinant protein was purified by His·Bind Resin Kit (Novagen, USA). The eluted protein was dialyzed in phosphate buffered saline (PBS, pH 7.4). The purity of the rCsPDI was identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE, 12 % gel). The final concentration of rCsPDI was detected by the BCA Assay Kit (Novagen, USA).

Preparation of CsESPs and antiserum

C. sinensis adult worms were isolated from the bile ducts of parasite-infected cats. The CsESPs were prepared as described previously (Hu et al. 2007) and stored at −80 °C. The purified rCsPDI (200 μg) and CsESPs (200 μg) were respectively emulsified with equal volume of complete Freund's adjuvant and subcutaneously injected into two 6-week-old male Sprague–Dawley (SD) rats. Meanwhile, the naive serum was taken before injection. Three booster injections were given with 100 μg purified protein containing incomplete Freund's adjuvant at 2 weeks interval. The antisera were collected 2 weeks after the last injection. The titers of the antisera were determined by enzyme-linked immunosorbent assay (ELISA).

Western blotting analysis

The purified rCsPDI and CsESPs were subjected to SDS-PAGE (12 %) and subsequently electrotransferred onto the polyvinylidene difluoride (PVDF) membrane (Whatman, USA) at 100 V for 1.5 h. The PVDF membrane was blocked with PBS (pH 7.4) containing 5 % skim milk for 2 h at room temperature, washed five times with PBS (pH 7.4), and then incubated with anti-CsPDI rat serum (1:100 dilutions), naive rat serum (1:100 dilutions), C. sinensis-infected rat serum (1:100 dilutions), His·Tag monoclonal antibody (mAb-His) (Novagen, USA, 1:2,000 dilutions), and anti-CsESPs rat serum (1:100 dilutions) for overnight at 4 °C, respectively. The membrane was incubated with horseradish peroxidase (HRP)-conjugated goat anti-rat/anti-mouse IgG (ProteinTech Group, USA, 1:2,000 dilutions) for 2 h at room temperature after washing. The color was developed with diaminobenzidine (DAB) substrate solution.

Quantitative real-time PCR analysis of CsPDI gene expression

The C. sinensis adult worms, eggs, and metacercariae were collected as previously described (Chen et al. 2011). Total RNA was extracted from them using TRIZOL (Invitrogen, USA) according to the manufacturer's instructions. The sense and antisense primers for CsPDI were 5′-TCAGTCCGATTGTCGTAACCA-3′, 5′-TCGTGAATACCCTCCGTGCTA-3′, and primers of Csβ-actin (GenBank accession number EU109284.1), as internal control (Yoo et al. 2009), were 5′-ACCGTGAGAAGATGACGCAGA-3′ and 5′-GCCAAGTCCAAACGAAGAATT-3′, respectively. The condition of real-time PCR was 95 °C for 10 min, and then 95 °C for 15 s, 60 °C for 10 s, and 72 °C for 60 s for 40 cycles; the reaction was finally ended up with a cooling step to 40 °C using SYBR Premix ExTaq™ II (Takara, Japan). The reaction was conducted in LightCyclery™ system (LC480), and the data were analyzed according to the 2−ΔΔCt method (Pfaffl 2001).

Immunohistochemical localization of CsPDI

The C. sinensis adult worm, metacercariae, and the liver tissue of C. sinensis-infected cat were fixed in 4 % paraformaldehyde, embedded with paraffin, and sliced into sections of 4–5 μm in thickness. The sections were deparaffinized by dimethylbenzene three times and dehydrated by gradient alcohol. Then, the sections were blocked with normal goat serum for 2 h at room temperature, and incubated with anti-CsPDI rat serum (1:200 dilutions in 0.1 % BSA-PBS) and naive rat serum (1:200 dilutions) for overnight at 4 °C, respectively. The sections were incubated with Cy™ 3-conjugated AffiniPure goat anti-rat IgG (1:400 dilutions) for 1 h at room temperature in darkness and quenched for 15 min, then imaged under fluorescence microscope (Carl Zeiss, Germany).

Results

Sequence analysis of CsPDI

Open reading frame (ORF) of CsPDI was identified from the adult C. sinensis cDNA library. The ORF contained 1,317 bp encoding 438 amino acids. The predicted molecular mass (MW) of CsPDI was 52 kDa, and the isoelectric point (PI) was 6.57. No signal peptide or transmembrane domain was found. Two thioredoxin superfamily domains were found in the position of aa36–135 (a domain) and aa164–267 (a′ domain). These domains comprised thioredoxin family conserved sites (aa55–73 and aa184–202), which were homologous to bacterial thioredoxin and contained the putative redox site WCGHCK (Salazar-Calderon et al. 2003) (Fig. 1). BLASTx analysis showed that the deduced amino acid sequence was homologous with PDI of Bos taurus, Homo sapiens, Schistosoma mansoni, Trypanosoma brucei, and Plasmodium falciparum with 53 %, 49 %, 43 %, 36 %, and 25 % identities, respectively (Fig. 1).

Fig. 1
figure 1

Amino acid sequence alignment of PDI from Clonorchis sinensis with that of PDI from other species. C. sinensis (C. s), Bos taurus (B. t, NP_001193274.1), Homo sapiens (H. s, CAA89996.1), Schistosoma mansoni (S. m, XP_002576968.1), Trypanosoma brucei (T. b, XP_845754.1), and Plasmodium falciparum (P. f, XP_001348023.1). The conserved site (aa55–73 and aa184–202) of thioredoxin family was indicated with bold line, the redox sites WCGHCK (aa62–67 and aa191–196) were indicated with gray frame, and the proposed delimitations of a, a′ (catalytic domains) and b (non-catalytic domains) were showed by a line

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Expression and purification of rCsPDI

The purified rCsPDI presented a single band at around 52 kDa, which coincided with the prediction of bioinformatics analysis (Fig. 2). The final concentration of recombinant protein was 200 μg/ml.

Fig. 2
figure 2

SDS-PAGE analysis of expressed product of pET-28a (+)-CsPDI in Escherichia coli BL21/DE3. Protein molecular weight marker (M); pET-28a(+) transformants without IPTG induction (lane 1); pET-28a(+) transformants with IPTG induction (lane 2); pET-28a(+)-CsPDI transformants without IPTG induction (lane 3); pET-28a(+)-CsPDI transformants with IPTG induction (lane 4); supernatant of the lysate of pET-28a(+)-CsPDI transformants (lane 5); precipitation of the lysate of pET-28a(+)-CsPDI transformants (lane 6); the purified recombinant CsPDI protein (lane 7)

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Western blotting analysis

The rCsPDI was recognized by mAb-His, anti-CsPDI rat serum, and C. sinensis-infected rat serum, while no corresponding band was detected by naive rat serum (Fig. 3a). The rCsPDI was probed by anti-CsESPs serum, and CsESPs was probed by anti-CsPDI serum, while no reaction was detected by naive serum control (Fig. 3b).

Fig. 3
figure 3

Western blotting analysis of CsPDI. a Western blotting analysis of recombination CsPDI. Purified rCsPDI was probed with mAb-His, anti-rCsPDI rat serum, and serum from Clonorchis sinensis-infected rat (lanes 1–3, respectively); no reaction was detected in the naive serum control (lanes 4–6). b Identification of CsPDI as an excretory–secretory product. Anti-CsESPs serum was detected by the rCsPDI (lane 1); anti-rCsPDI serum was detected by ESPs (lane 2); no reaction was detected by the naive serum control (lanes 3–4)

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Transcript analysis of CsPDI at the development stages of C. sinensis

The transcripts of CsPDI were detected in the development stages of adult worm, metacercaria, and egg. Normalized by Csβ-actin, the transcription level of CsPDI in the metacercaria stage was six times higher than that in adult worm, whereas the expression level of CsPDI mRNA in the egg stage was one and a half times higher than that in adult worm (Fig. 4).

Fig. 4
figure 4

Quantitative real-time PCR analysis of transcripts of CsPDI normalized by Csβ-actin. The data were analyzed by software package SPSS13.0. *p value ≤ 0.05; **p value ≤ 0.01

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Immunohistochemical localization of CsPDI in the adult worm, metacercaria, and the liver tissue of C. sinensis-infected cat

CsPDI was detected in the sections of adult worm and metacercaria. Specific fluorescence was detected in the intestine, vitellarium, and intrauterine eggs of adult worm, as well as in the vitellarium and cyst wall of metacercaria. No specific fluorescence was observed in the adult worm and metacercaria when treated with naive serum (Fig. 5). In the liver tissue of C. sinensis-infected cat, specific fluorescence signal was observed on the wall of bile duct and in the lumen of liver tissue (Fig. 6).

Fig. 5
figure 5

Immunolocalization of CsPDI in the adult worm and metacercaria of Clonorchis sinensis. a, c, e, g Adult worm and metacercaria under optical microscope. b, d, f, h The same part under fluorescence microscope. d, h Probed with anti-CsPDI rat serum. b, f Probed with naive serum as negative controls. The images were magnified at ×50 for adult worm and ×400 for metacercaria, respectively. I intestine, V vitellarium, E egg, C cyst wall

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Fig. 6
figure 6

Immunolocalization of CsPDI in the liver tissue of Clonorchis sinensis-infected cat. a, c Adult worm and its surrounding bile duct under optical microscope. b, d The same part under fluorescence microscope. b Probed with anti-CsPDI rat serum. d Probed with naive serum as a negative control. The images were magnified at ×50. E wall of bile duct, L lumen of bile duct

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Discussion

In this study, we isolated a full-length cDNA of CsPDI from cDNA plasmid library of adult C. sinensis. The protein showed high similarities with PDIs of other organisms. The CsPDI comprised two catalytic thioredoxin motifs CxxC, which were characteristic domains of thioredoxin superfamily. The purified rCsPDI with 6 × His Tag was obtained in our studies. Analysis of quantitative real-time PCR indicated that CsPDI was expressed across all development stages of the parasite which occur in the definitive host, i.e., metacercaria, adult worm, and egg. It was also clarified in the immunolocalization assay. CsPDI was confirmed as a component of CsESPs by western blotting and immunohistochemical localization of the liver tissue of C. sinensis-infected cat in this study.

The members of the PDIs were expressed in the lumen of ER to participate in the folding and maturation of newly synthesized proteins (Hong and Soong 2008). The C-terminus sequence of PDI in mammal functioned as a retention signal for luminal proteins in ER, which prevented them from being exported through the secretory pathway (Finken et al. 1994). However, the C-terminus sequence could be deleted from the polypeptide of PDIs without affecting any of its major functions (Freedman et al. 2002). In this study, we found that CsPDI contained a typical redox-active site a (aa58–67, FYAPWCGHCK) as an N-terminal signal peptide (Rubotham et al. 2005), but not possessed the C-terminus sequence c (HEEL), which was an ER retention motif found in PDIs of other species. Although CsPDI did not have the ER-retention signal, it was located, at least in part, in the ER and was supposed to play a critical role in the secretory pathway, just like in Leishmania (Padilla et al. 2003). Moreover, the identification of CsPDI as a component of CsESPs in our study will help corroborate our speculation.

As a multifunctional protein, another role of PDI was in catalyzing oxidation of disulfides of nascent polypeptides as it was usually abundantly expressed in the lumen of ER. However, in N. caninum (Naguleswaran et al. 2005), T. gondii (Meek et al. 2002a, b), and Giardia (Gillin et al. 1990), PDIs were found to be expressed on the surface of parasites. In S. mansoni, PDI was identified in the gastrodermis and protonephridia (Finken et al. 1994). In addition, PDI of F. hepatica has been reported as a component of ESPs to protect the parasite in oxidative stress environment (Salazar-Calderon et al. 2003). The ESPs of parasites often contained antioxidants which were shed from tegument, and proteases which were secreted from intestine (Berasain et al. 1997; Robinson et al. 2009). In C. sinensis, the CsPDI was located in the wall of intestine, which might help to explain why CsPDI was present in the CsESPs. Moreover, the specific localization of CsPDI was observed in the lumen of bile duct of the cat infected with C. sinensis, which might serve as another evidence of its role as a component of CsESPs.

The PDI of Giardia lamblia expressed on the surface was reported to play a role in protecting this protozoan in oxidative stress environment, which was considered to be one of mechanisms for the parasite surviving (Gillin et al. 1990). PDIs were expressed on the surface of N. caninum tachyzoites and T. gondii tachyzoites to functionally influence adhesive/invasive capacities of the tachyzoites (Lekutis et al. 2001; Naguleswaran et al. 2005). Recently, it has been shown that PDI was abundantly present in Plasmodium micronema and essential for parasite invasion (Lal et al. 2009). Therefore, further investigations are required to demonstrate whether CsPDI plays an important role in C. sinensis invasion and survival.

Immunohistochemical assay of CsPDI showed that it was localized in the vitellarium of adult worm and metacercaria. The vitellarium was a nutritive organ during sexual development, which was of great significance for the growth of C. sinensis. Study in S. mansoni showed that PDI may take part in the nutrition supply to the germ cells, gastrodermis of gut, wall cells of protonephridia, and sustentacular cells of testes (Finken et al. 1994). It suggested that CsPDI may also be involved in C. sinensis growth and development.

Quantitative real-time PCR analysis revealed that the transcription level of CsPDI in the adult stage was lower than that in metacercaria and egg. It was possibly due to the fact that a large amount of CsPDI was lost as a component of CsESPs in the procedure of collecting adult worms. In this case, CsPDI was reduced in the total RNA of adult worms. On the other hand, the expression level of CsPDI mRNA in metacercaria presented the highest in three stages. It supposed that the protein played an important role in the growth and development of metacercaria, especially in the parasite invasion.

In this study, CsPDI was cloned, expressed, and purified using E. coli BL21 expression system. CsPDI was characterized as a novel molecule from CsESPs, and the protein may be of great importance in the parasite invasion and survival. Our studies will be the cornerstone of future researches on biological characterization, the role in host–parasite interplay, and the potential value as a therapy target in C. sinensis infection.