Parasitology International 63 (2014) 359–365

Contents lists available at ScienceDirect

Parasitology International journal homepage: www.elsevier.com/locate/parint

Expression and characterization of cathepsin L-like cysteine protease from Philasterides dicentrarchi Sang Phil Shin a,1, Sang Yoon Han a,1, Jee Eun Han a, Jin Woo Jun a, Ji Hyung Kim b, Se Chang Park a,⁎ a b

Laboratory of Aquatic Biomedicine & Research Institute for Veterinary Science, College of Veterinary Medicine, Seoul National University, Seoul 151–742, Republic of Korea Korea Institute of Ocean Science and Technology, Ansan, Republic of Korea

a r t i c l e

i n f o

Article history: Received 3 June 2013 Received in revised form 6 November 2013 Accepted 4 December 2013 Available online 19 December 2013 Keywords: Philasterides dicentrarchi Site direct mutagenesis Cathepsin L like protease E. coli expression system Cystein proteases

a b s t r a c t Philasterides dicentrarchi is a causative agent of scuticociliatosis in olive flounder Paralichthys olivaceus, aquaculture in Korea. In this study, a cDNA encoding a cathepsin L-like cysteine protease (PdCtL) of P. dicentrarchi (synonym Miamiensis avidus) was identified. To express the PdCtL recombinant protein in a heterologous system, 10 codons were redesigned to conform to the standard eukaryotic genetic code using polymerase chain reaction (PCR)-based site-directed mutagenesis. The recombinant P. dicentrarchi procathepsin L (proPdCtL) was expressed at high levels in E. coli Rosetta (DE3) pLysS with a pPET21a vector, and successfully refolded, purified, and activated into a functional and enzymatically active form. The optimal pH for protease activity was 5. Similar to other cysteine proteases, enzyme activity was inhibited by E64 and leupeptin. Immunogenicity of recombinant PdCtL was assessed by enzyme-linked immunosorbent assay, western blot, and specific anti-recombinant PdCtL antibodies were detected. Our results suggest that the biochemical characteristics of the recombinant ciliate proPdCtL protein are similar to those of the cathepsin L-like cysteine protease, that the PCR-based site-direct mutated ciliate gene was successfully expressed in a biochemically active form, and that the recombinant PdCtL acted as a specific epitope in olive flounder. © 2013 Elsevier Ireland Ltd. All rights reserved.

1. Introduction Several scuticociliate species belonging to the genera Uronema, Miamiensis, and Philasterides have been recognized as important opportunistic pathogens or environmental scavengers in marine fish. [1–4]. These ciliates have been reported from farmed sea bass, Dicentrarchus labrax, in the Mediterranean [1], olive flounder, Paralichthys olivaceus [2,3], and indo-pacific seahorse, Hippocampus kuda [4]. Among the ciliates, three scuticociliate species such as Pseudocohnilembus persalinus, U. marinum and P. dicentrarchi are responsible for scuticociliatosis in cultured olive flounder in Korea [5,6]. In particular, scuticociliatosis caused by P. dicentrarachi is recognized as one of the most important parasite problems affecting the cultured olive flounder fish farming industry in Korea [7]. The presence of P. dicentrarachi in fish tissues has been associated with various pathological changes, including bleeding cutaneous ulcers, dystrophic and necrotic effects in muscle, hypochromic anemia, and encephalitis associated with softening or liquefaction of brain tissues [7]. Although little is currently known regarding the process by which P. dicentrarchi invades host tissue or the mechanisms by which this parasite evades the host's defense response, it has been hypothesized that cysteine proteases might be involved in the invasion and pathogenicity ⁎ Corresponding author. Tel.: +82 2 880 1282; fax: +82 2 880 1213. E-mail address: [email protected] (S.C. Park). 1 The authors contributed equally to this work. 1383-5769/$ – see front matter © 2013 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.parint.2013.12.007

characteristics of P. dicentrarchi [8,9]. However, these proteases have not been completely cloned and characterized at the molecular level, and the genetic code of ciliates differs from that of other eukaryotes [10]. The standard UAA and UAG stop codons specify glutamine (Gln, Q) in Scuticociliatida and UGA is the only stop codon [10]. Therefore, expression of the ciliate gene in a heterologous protein expression systems remains rather limited [11]. The parasite cathepsin L-like cysteine protease family is important in parasite development and pathogenesis [12,13]. These family proteins also allow the parasite to evade the host's immune system [12] and are virulence factors in some pathogenic organisms [13]. Therefore, the cathepsin L-like cysteine protease of parasites has gained attention as targets for development of new anti-parasitic drugs and vaccines [12,14]. In the present study, we describe cloning and enzymatic characterization of the cathepsin L-like protease from P. dicentrarchi. 2. Materials and methods 2.1. Parasites and total RNA isolation Ciliates were isolated from indo-pacific seahorse and identified as reported previously [4]. P. dicentrarchi was then propagated in olive flounder and was reisolated from skin lesion and ascites of the fish. The ciliates were washed and concentrated by dilution of the samples with Hanks Balanced Salt Solution (HBSS) containing antibiotics (100 U/ml penicillin, 100 μg/ml streptomycin) followed by centrifugation

360

S.P. Shin et al. / Parasitology International 63 (2014) 359–365

for 5 min. This process was repeated until clean P. dicentrarchi was obtained. The ciliates were cultured in sterile artificial sea water (20‰ salinity) supplemented with 0.1% yeast extract and antibiotics. Total RNA was extracted using an RNA extraction kit with TRIzol reagent (Invitrogen, Carlsbad, CA, USA) in accordance with the manufacturer's instructions. 2.2. cDNA synthesis and 3′rapid amplification cDNA ends (RACE)-polymerase chain reaction (PCR) A gene specific primer (GSP) was designed based on the partial region of cathepsin L-like protease sequences from M. avidus (GenBank accession no. DR981402). To obtain a full length copy of P. dicecntrarchi Cathepsin L (PdCtL) like protease, RACE PCR was performed using a SMATerTM RACE cDNA Amplication Kit (Clontech, Palo Alto, CA, USA) according to the manufacturer's instructions. The 3′ RACE-PCR product was amplified by PCR using a PdCtL-specific primer (Sense primer: GSP1, 5-ATG AAA GCC GCT TTA ATA TTA-3). New primers were designed to confirm the obtained sequence and to amplify the full-length PdCtL cDNA PCR (Sense primer: GSP2, 5-GAC AAT GTG GAT CTT GCT G-3; Antisense primer: GSP3, 5-CAG CAA GAT CCA CAT TGT C-3; Antisense primer: GSP4, 5- TGT TAC AAA TGA ATA TAT TTT GCT-3). 2.3. Sequence and phylogenetic analysis The PCR amplification products were cloned into a plasmid vector using a TOPO cloner PCR cloning kit (Enzynomics, Seoul, Korea), following the manufacturer's instructions. The cloned products were sequenced using universal primers (M13F: 5-GTA AAA CGA CGG CCA GT-3; M13RpUC: 5-CAG GAA ACA GCT ATG AC-3) and a terminator cycle sequencing kit (Applied Biosystems, Foster City, CA, USA). Electrophoresis of the sequencing reactions was performed using an automatic DNA analyzer (Applied Biosystems). Sequencing data were compared with the database using the BLAST program. The position of the putative signal peptide cleavage site was predicted using the SignalP 3.0. Alignment of the predicted amino acid sequences of PdCtL and protozoan cathepsin L-like proteases was carried out using ClustalW and was edited for presentation using Boxshade 3.21 software. Amino acid sequences of the complete preproenzyme of PdCtL and other parasite cathepsin L-like proteases were analyzed phylogenetically using MEGA4 software to prepare a phylogenetic tree. The tree was constructed using the neighbor-joining method with 1000 bootstrap replicates and 92,441 seeds. Gaps were introduced to maximize sequence identity. The molecular mass and predicted pI of the deduced protein were determined by Compute pI/Mw software. 2.4. Site-direct mutagenesis of PdCtL Mutagenic oligonucleotides were used to convert 10 UAA stop codons in the coding sequence to CAA glutamine codons using PCRbased site directed mutagenesis to over express PdCtL in E. coli (Supplementary Table S1). Mutagenesis was modified as described previously [14]. First, GSP1-337-338R and GSP4-337-338 F were utilized as two primer sets for the conversion of the stop codons at amino acid positions 337 and 338. The first step was carried out in a T-personal 48 thermocycler (Biometra, Goettingen, Germany) with the following parameters: an initial denaturation step of 94 °C, 3 min; 35 serial cycles of denaturation at 94 °C, 30 s, annealing at 55 °C, 30 s extension at 72 °C, 1 min; and final extension step of 72 °C, 10 min with GSP1337-338R and GSP4-337-338 F primer sets using i-pfu DNA polymerase PCR preMix (Intronbio, Seoul, Korea), respectively. The PCR products were purified with a Plus PCR Purification Kit (Nucleogen, Seoul, Korea). One microliter of purified PCR product was used for efficient annealing and extension in the following step. The second step for annealing and extension was performed as follows: annealing at 60 °C, 30 s, extension at 72 °C, 50 s, and denaturation at 94 °C, 30 s, for 20 cycles

except for the primers. The resulting product was then used in the third step. The third step was performed for 35 cycles of denaturation (94 °C, 30 s), annealing (55 °C, 30 s), and extension (72 °C, 50 s), with the GSP1-GSP4 primer set. In this manner, the stop codons 337 and 338 of the P. dicentrarchi cathepsin L were converted, and were then usable as the template for the next mutagenesis. The above method, from the first to third step was reiterated to convert the remaining stop codons in serial order. Finally, this process generated a synthetic allele, which mutated scuticociliate preprocathepsin L. 2.5. Expression, affinity purification and refolding of recombinant PdCtL The sequence encoding the predicted proPdCtL was amplified from the obtained plasmid clone by PCR using NdeI-proPdCtL-F (5-CGC CAT ATG ATC TTC ATG ATG AGC AAC AAC CAA AC-3) and XhoI-proPdCtL-R (5-CCG CTC GAG GTA GAG GGG GTA GGC AAC G-3) primers to which NdeI and XhoI restriction sites had been added to assist cloning. The underline codon ATC is specified as start codon for expression of proPdCtL. The PCR product was cloned into the pPET21aHISTag expression vector and designated pHIST-proPdCtL. The ligation product was used to transform chemically competent E. coli, Rosetta (DE3) pLysS (Novagen, Gibbstown, NJ, USA) and protein expression was induced with 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) for 24 h at 37 °C. The bacterial lysate was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and the expressed fusion protein was identified by Coomassie Blue staining and Western blot using anti6Xhis tag antibody. The inclusion bodies containing the recombinant protein were isolated from bacterial lysates by centrifugation, and solubilized with 50 mL of denaturation buffer (8 M urea, 20 mM Tris HCl, pH 8.0, 0.5 M NaCl, 5 mM imidazole, and 1 mM β-mercaptoethanol) and stirred for 16 h at room temperature. The recombinant protein present in the suspension was purified by metal-affinity chromatography in Chelating-Sepharose® Fast Flow resin (GE Healthcare, Piscataway, NJ, USA), previously charged with 300 mM NiSO4 and equilibrated with denaturation buffer. The column was washed with ten volumes of denaturation buffer (8 M urea, 20 mM Tris HCl, pH 8.0, 0.5 M NaCl, 5 mM imidazole, and 1 mM β-mercaptoethanol). This procedure was followed by three additional wash steps: ten volumes of a buffer (20 mM Tris HCl, 0.5 M NaCl, pH 8.0) containing 6 M urea and 20 mM imidazole in the first wash; ten volumes of the same buffer containing 4 M urea and 40 mM imidazole in the second wash; and ten volumes of the same buffer containing 3 M urea and 60 mM imidazole in the third wash. The recombinant protein was eluted with five volumes of the elution buffer containing 20 mM Tris HCl, 0.5 M NaCl (pH 8.0), 3 M urea, and 1 M imidazole. Several dialysis steps were also performed; first against a solution containing 20 mM Tris HCl, 0.5 M NaCl (pH 8.0), 2 M urea, 0.1% glycine, 10 mM EDTA and 0.5 M imidazole; second, against a solution containing 20 mM Tris HCl, 0.5 M NaCl, (pH 8.0), 2 M urea, 0.1% glycine, and 10 mM EDTA. The last dialysis was against phosphatebuffered saline solution (PBS), 0.1% glycine, and 0 M urea. Purified protein samples were analyzed by 12% SDS-PAGE. The concentration of the purified fusion protein was determined using a BCA kit (Pierce, Rockford, IL, USA). 2.6. Autoprocessing and enzymatic characterization of recombinant PdCtL Purified proPdCtL (5 μg) was incubated in 0.1 M sodium acetate buffer (pH 4) containing 10 mM cysteine with 30 μg/mL of dextran sulfate for 30 min and then recombinant proPdCtL fluorometric assays were carried out as described previously [14]. In brief, hydrolysis of the substrates containing the 7-amino-4-methyl coumarin (AMC) fluorophore was conducted in microtitre plate format using a Microplate Fluorometer (Packard Co., Palo Alto, CA, USA) (excitation wavelength: 355 nm and emission wavelength: 460 nm). The synthetic

S.P. Shin et al. / Parasitology International 63 (2014) 359–365

substrates carbobenzoxy-phenylalanyl-arginyl-AMC (Z-Phe-Arg-AMC) (Sigma, St. Louis, MO, USA) was used as substrates for the activity assay or for the cathepsin L protease inhibitor assay. Activity assays were performed at 37 °C in a total volume of 100 μl (100 mM sodium acetate/pH 5 or at another pH as required and 0.2 mM DTT). Substrates and processed recombinant proPdCtL were added to a final concentration of 0.1 mM and 1 μg/ml, respectively. The effects of various inhibitors (Pepstatin A, PMSF, EDTA, 1, 10-phenanthroline, E64, leupeptin) were evaluated by measuring residual enzyme activity at pH 5 as described previously [15]. All assays were carried out in triplicate.

361

2.7. Immunization with recombinant PdCtL and detection of specific antibodies Ten days before the start of the immunization experiment, fish were moved to a quarantine unit and acclimatized to 20 °C. Five fish were immunized twice within a 2 week interval with an intraperitoneal injection (i.p.) of 50 μg of recombinant PdCtL mixed 1:1 with Freund's incomplete adjuvant (200 μl). Control fish (n = 5) were injected i.p. (200 μl), following the same schedule, using PBS mixed 1:1 with Freund's incomplete adjuvant (Sigma). Two weeks after the second

A

Fig. 1. Alignment of the predicted amino acid sequence of P. dicentrarchi cathepsin L with those of other ciliate cathepsin L and phylogram of the predicted PdCtL protein sequence and selected sequences of other eukaryote cathepsin L proteases. (A), Identical amino acid residues are darkly shaded, similar amino acids are lightly shaded, unrelated residues have a white background, and the amino acid numbers are shown on the left. The catalytic triad residues are marked with asterisks, and the gaps (−) are introduced to maximize alignment. Closed arrow and arrowheads indicate putative cleavage sites for the signal sequence and propeptide of P. dicentrarchi cathepsin L, respectively. The ERFNIN and the intramolecular processing GNFD motifs are shown by underline. The cathepsin L proteases aligned with P. dicentrarchi (PhdCTL, JR673412) are from Uronema marinum (UroCTL, AAX51228), Tetrahymena pyriformis (TepCTL, BAA31161), Tetrahymena thermophila (TetCTL, AAA30114), and Paramecium tetraurelia (PatCTL, CAA62869). (B), The neighbor-joining tree was constructed with MEGA4 software. Scale bar represents 0.2 amino acid substitutions per site. The cathepsin L proteases aligned with P. dicentrarchi cathepsin L (JR673412) were from Caenorhabditis elegans cathepsin L (NP507199), Dictyocaulus viviparus cathepsin L (AAK77918), Haemonchus contortus cathepsin L (AAF86584), Meloidogyne incognita cathepsin L (CAD89795), Rotylenchulus reniformis cathepsin L (AAY45870), Heterodera glycines cathepsin L (CAA70693), Globodera pallida cathepsin L (AAY46196), Gnathostoma spinigerum cathepsin L (ABY28387), Uronema marinum cathepsin L (AAX51228), Taenia solium cathepsin L (AAS00027), Fasciola hepatica cathepsin L (AAF76330), Fasciola gigantica cathepsin L (AAF44676), Brugia pahangi cathepsin L (O17473), Brugia malayi cathepsin L (BAD11762), Tetrahymena pyriformis cathepsin L (BAA31161), Cryptobia salmositica cathepsin L (AAU14993), Leishmania major cathepsin L (AAB48120), Leishmania mexicana cathepsin L (CAA78443), Trypanosoma brucei cathepsin L (CAA38238), Trypanosoma congolense cathepsin L (CAA81061), Trypanosoma cruzi cathepsin L (AAA30181), Paramecium tetraurelia cathepsin L (CAA62869), Tetrahymena thermophila cathepsin L (AAA30114), Trypanosoma rangeli cathepsin L (2117247A), Trypanoplasma borrelli cathepsin L (ABQ23398), Uronema marinum cathepsin B (AAR19103), Taenia pisiformis cathepsin L (AEG19548), Taenia asiatica cathepsin L (BAH03397), Taenia saginata cathepsin L (BAH03396), Echinococcus multilocularis cathepsin L (BAF02517), Clonorchis sinensis cathepsin L (GAA56666), and Schistosoma japonicum cathepsin L (CAX72171).

362

S.P. Shin et al. / Parasitology International 63 (2014) 359–365

Fig. 1 (continued).

immunization, serum samples were collected and checked for specific antibodies against recombinant PdCtL by enzyme-linked immunosorbent assay (ELISA). The ELISA was conducted using the ELISA Starter kit (Komabiotech, Seoul, Korea) following the manufacturer's protocol. Briefly, the ELISA plate was coated overnight at 4 °C with 100 μl of recombinant PdCtL (5 μg/ml) per well in coating buffer at pH 9.6. All subsequent incubation steps were performed for 1 h at room temperature with shaking (150 rpm). The plate was blocked with 200 μl of 5% skim milk in PBS. Sera were diluted 200 times with 1% skim milk in PBS including 0.1% Tween-20 (PBST) and incubated in a 100 μl volume. Further incubation steps were with 100 μl mouse anti olive flounder IgM (1:33, Aquatic Diagnostics) and subsequently with 100 μl goat anti-mouse-HRP (1:2000, Abcam, Cambridge, UK). Plates were washed with PBST between all incubation steps. After the last wash, the plates were incubated for 10 min at room temperature in the dark with 100 μl of 3,3′,5,5′-tetramethylbenzidine and H2O2 used as substrates. The reaction was stopped with 2 M H2SO4 and the OD was measured at 450 nm.

with PBST, then incubated with antisera against the recombinant PdCtL (1:200 dilution) for overnight. After washing, the membrane was incubated with mouse anti-olive flounder IgM (1:1000) for 1 h. Further incubation was with goat anti-mouse-HRP (1:2000) for 1 h. Detection was performed using the Supersignal West Pico Chemiluminescent Substrate (Thermo Scientific, IL, USA) and was captured with a Chemidoc chemiluminescence imaging system (Bio-Rad, Hercules, CA).

2.8. Western blotting against recombinant protease and parasite lysate

The 1182 bp PdCtL cDNA contained an open reading frame (ORF) of 1038 bp from the first ATG start codon through the TGA stop codon, flanked by 144 bp of the 3′-untranslated region. The PdCtL sequence was deposited in the GenBank database under accession no. JQ673412. The PdCtL harbored a 16-residue putative signal peptide, a 111residue propeptide, and the 218-residue mature enzyme. The molecular weights of the putative prepro-, pro, and mature-forms of PdCtL were 37,330, 35,761, and 22,942 Da, respectively. PdCtL exhibited a degree of identity with U. marinum cathepsin L-like protease (41%). The prodomain contained an ERFNIN like motif and a GNFD motif, both typical of cathepsin L-like cysteine proteases (Fig. 1A). The theoretical

The P. dicentrarchi lysate was prepared as reported previously [8]. Briefly, the ciliates were washed 3 times by centrifugation at 650 g for 5 min in 0.25 M sucrose at 4 °C. The cells were then disrupted ultrasonically. The homogenate was centrifuged at 15,000 g for 15 min and the resulting supernatant fraction stored in aliquots at −80 °C. The recombinant PdCtLs (0.1, 0.5, 1, 2, and 4 μg per lane) and parasite lysates (stock, 1/2, and 1/4 dilution) were subjected to SDS-PAGE and transferred onto polyvinylidene fluoride (PVDF) membrane. The membrane was blocked with 5% skim milk in PBST at room temperature for 1 h, washed 5 times

2.9. Statistical analysis Statistical analyses were performed using SPSS 16.0 software (SPSS Inc, Chicago, IL, USA). The results of ELISA assay were compared using Student's t-test and differences were considered significant at P b 0.05. 3. Results 3.1. Molecular cloning and characterization of a cDNA encoding a cysteine protease from P. dicentrarchi

S.P. Shin et al. / Parasitology International 63 (2014) 359–365

363

cleavage site of the prodomain between the aspartic acid at position 127 and the leucine at position 128 was determined by homology comparison. The proline residue (position 129) in the N-terminal domain of the catalytic region (position 2 in the mature protein), which may function to prevent N-terminal proteolysis, was conserved. PdCtL contained the characteristic cysteine, histidine, and asparagine active site residues. The mature enzyme contained the cysteine protease catalytic triad, Cys152, His290, and Asn310 as well as the six cysteine residues involved in formation of the disulfide bonds (Cys149–190, Cys183–225, and Cys284–334). Additionally, two potential N-glycosylation sites were identified (positions 23 and 228), one of which was located in the proregion (Asn–Gln–Thr) and the other in the mature region (Asn–Gln–Ser). It is corresponding with the N-glycosylation site of U. marinum cathepsin L [14]. A phylogenetic analysis of 30 cathepsin L-like proteases of parasites revealed that PdCtL was more closely related to those of ciliophora (Fig. 1B).

3.2. Heterologous expression and enzymatic characterization of recombinant PdCtL In PdCtL contained 10 UAA universal stop codons, which code for glutamine (Q) in P. dicentrarchi. The mutagenic oligonucleotides were used to convert 10 UAA stop codons in the coding sequence to CAA glutamine codons by PCR-based site-directed mutagenesis. The expected band of the PdCtL protein (35.76 kDa) was observed on 12% SDSPAGE of bacterial cell lysates from induced cultures (Fig. 2; lanes 1 and 2). Based on the Western blotting, the recombinant proPdCtL was present in the bacterial cell lysate pellet in an insoluble form as inclusion bodies (Fig. 2; lanes 3 and 4). The recombinant proteins present in the inclusion bodies were isolated, solubilized and purified (Fig. 2; lane 5). The purified recombinant protein yield was approximately 2.4 mg/ml and was activated by sodium acetate buffer (pH 4) containing cysteine and dextran sulfate. The purified recombinant PdCtL was enzymatically active against the substrate (Z-Phe-Arg-AMC) at pH 5 or 6.0 (Fig. 3). Proteolytic activity was completely inhibited by adding of the cysteine protease specific inhibitors E64 and leupeptin. The effect of the inhibitors on protease activity of PdCtL using Z-Phe-Arg-AMC as the substrate is shown in Table 1.

Fig. 3. pH activity profile of recombinant PdCtL. The activity of PdCtL against Z-Phe-ArgAMC substrate was measured at different pHs. The activities are shown relative to that at pH 5 being 100%. The results are mean values from triplicate experiments ± standard deviation.

3.3. Induced specific antibody titer and western blot The absorbance on the ELISA for specific anti-recombinant PdCtL antibodies in the immunized fish was higher than that of control fish 2 weeks post immunization (Fig. 4), indicating that immunizing with recombinant PdCtL induced a specific immune reaction. A significant difference (P b 0.05) was observed in mean absorbance in the ELISA of immunized fish (1.323) compared to that of control fish (0.176). Western blot analysis of the olive flounder antiserum against recombinant proPdCtL and P. dicentrarchi lysates showed that the antiserum reacted with proteins at the molecular masses of 36 and 23 kDa, respectively (Fig. 5). 4. Discussion The gene coding for a cathepsin L-like cysteine protease of P. dicentrarchi was cloned and sequenced. When the amino acid sequence was aligned with cathepsin L like cysteine proteases of various ciliates, PdCtL was homologous to other cathepsins L in the proximity of the core catalytic triad residues but showed less homology in the proregion (Fig. 1A). The ERFNIN motif is characteristic for the propeptide region of non-cathepsin B papain-like proteases and has been suggested to play a role inhibiting proteolytic activity [16]. This ERFNIN motif is found in cathepsin L of other ciliates [14,16]. The GNFD, which may be involved in pH-dependent intramolecular processing [17], was also found in the PdCtL proregion. This motif was also conserved, although alanine was substituted for asparatic acid in PdCtL (Fig. 1A). The phylogenetic analysis showed that PdCtL is more closely related to the cathepsin L of ciliates than to other parasites cathepsin L (Fig. 1B). Table 1 Inhibition assay of proPdCtL by various inhibitors.

Fig. 2. Analysis of the recombinant PdCtL expressed from IPTG-induced E. coli Rosetta (DE3) pLysS by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Bands were visualized by Coomassie Blue staining. Lane M: molecular mass protein markers; lane 1: supernatant after centrifugation (soluble fraction); lane 2: inclusion of body pellet after centrifugation (insoluble fraction); lane 3: western blotting of the reactivity of mouse anti-his-proPdCtL in supernatant; lane 4: western blotting of the reactivity of mouse antihis-proPdCtL in inclusion body pellet; lane 5: protein purified from insoluble fraction.

Inhibitors

Specificity

Concentration (mM)

E64 Leupeptin

Cystein proteases Cystein proteases and trypsin like serine proteases Serineprotease Asparatic proteases Metalloproteases Metalloproteases

0.1 0.01

PMSF Pepstatin 1,10-Phenanthroline EDTA

1 0.1 1 1

Enzyme activity (% control)a 2±3 0±3

98 97 93 95

± ± ± ±

4 4 1 4

a Enzyme activity was estimated using N-carbobenzoxy-phenylalanyl-arginyl-AMC (Z-Arg-Arg-AMC) as a substrate. The results are mean values from triplicate experiments ± standard deviation.

364

S.P. Shin et al. / Parasitology International 63 (2014) 359–365

Cut off

Fig. 4. Enzyme-linked immunosorbent assay analysis for recombinant PdCtL specific antibody in the immunized and control group. The dotted line at OD 450 nm = 0.23 indicates the cut-off value of the assay, i.e. the average OD of control group sera plus three times the standard deviation of these sera.

The cathepsin L gene is related with the adaption of host, preference of infection site, and developmental stage of parasite (including parasite and intermediate host interaction). Especially, P. dicentarchi cathepsin L gene is closed to U. marinum cathepsin L gene and both of two species use marine fish as host. The molecular characteristics of the P. dicentrarchi cathepsin L-like protease sequence supported its classification and host preference after parasite speciation. An interesting feature of this ciliate is their use of alternative nuclear genetic codes [10]. Such codons result in premature polypeptide chain termination when corresponding transgenes are expressed as recombinant proteins in bacteria, yeast, or insect host cell lines. The PdCtL contained 10 UAA universal stop codons, which code for glutamine (Q) in P. dicentrarchi. Mutagenic oligonucleotides were used to convert 10 UAA stop codons in the coding sequence to the CAA glutamine codon by PCR-based site directed mutagenesis for overexpressing PdCL in E. coli. The part of the gene for PdCtL that encodes for the proenzyme was cloned into the E. coli expression vector, pPET21a. The cell extracts

M

1

2

3

4

5

6

7

8

50kDa 37kDa

20kDa

Fig. 5. Western blot analysis with anti-proPdCtL antibodies. Lane M: molecular mass protein markers; lane 1–5: proPdCtL (4, 2, 1, 0.5, and 0.1 μg, respectively); lane 6–8: P. dicentrarchi lysate (stock, 1/2, and 1/4 dilutions, respectively).

from proPdCtL transformed E. coli cultures induced with IPTG, showed a major protein band of about 36 kDa on SDS-PAGE (Fig. 2) which was consistent with the predicted size for the fusion protein. The fusion proPdCtL protein was solubilized in 8 M urea, and refolded as described above. Proteolytic activity of recombinant proPdCtL was determined using a fluorogenic substrate. The recombinant proPdCtL showed optimal activity at pH 5 using the synthetic substrate Z-Phe-Arg-AMC (Fig. 3). Reagents known to specifically inhibit serine, aspartic, or metalloproteases exerted little detectable influence on PdCtL activity. In contrast, PdCtL enzymatic activity was greatly reduced or blocked completely with all of the tested cysteine protease inhibitors, including E64 and leupeptin. Leupeptin was the most potent inhibitor of those tested, and 0.01 mM resulting in 100% inhibition. In the presence of 0.1 mM E64, a cysteine protease-specific inhibitor, 98% inhibition was observed (Table 1). These results support the conclusion that proPdCtL exhibits cysteine protease activity. Although several attempts have been made to treat the disease with various chemotherapeutants [18,19], no chemotherapeutants are available, particularly for systemic infections due to the high virulence and endoparasitic habitat of the ciliate. In addition, some agents (such as niclosamide and oxyclozanide), which are effective against P. dicentrarchi, are toxic to fish [20] and formalin (saturated by 37% formaldehyde) is a known carcinogen [21]. Thus, vaccination is an attractive alternative to chemotherapeutic treatments to effectively prevent this disease. Iglesias et al. [22] reported that immunizing turbot with P. dicentrarchi lysate plus adjuvant or with formalin-fixed ciliates induces synthesis of agglutinating antibodies and confers a degree of protection against a challenge infection, suggesting the usefulness of vaccine. In addition, the highest level of protection was achieved in animals immunized with a purified protein from E. coli, suggesting that even the inactive form of the protein can provide significant epitopes that contribute to protection against disease [23,24]. We demonstrated that immunization of recombinant PdCtL plus adjuvant in olive flounder induced a specific antibody (Fig. 4). Based on western blotting, the specific antibody reacted with P. dicentrarchi lysate as well as recombinant proPdCtL at 23 kDa and 36 kDa respectively (Fig. 5). This appears to indicate that the specific antibody induced in the olive flounder after

S.P. Shin et al. / Parasitology International 63 (2014) 359–365

immunization with the recombinant proPdCtL-adjuvant recognizes conformational epitope. Interestingly, Parama et al. [8] reported the cysteine protease with similar size to our result in exceretion/secetion products and suggested the enzyme was released into culture medium as active form. The cysteine proteases of P. dicentrarchi participate in host immune response evasion, host tissue invasion, and nutritional degradation [8,25]. Therefore, synthetic PdCtL might facilitate studies of its potential as a candidate for developing novel immunoprophylactic or chemotherapeutic modalities. Acknowledgment This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (2013R1A1A2006794). Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.parint.2013.12.007. References [1] Dragesco A, Dragesco J, Coste F, Gasc C, Romestand B, Raymond J-C, et al. Philasterides dicentrarchi, n. sp., (ciliophora, scuticociliatida), a histophagous opportunistic parasite of Dicentrarchus labrax (Linnaeus, 1758), a reared marine fish. Eur J Protistol 1995;31:327–40. [2] Yoshinaga T, Nakazoe J-i. Isolation and in vitro cultivation of an unidentified ciliate causing scuticociliatosis in Japanese flounder (Paralichthys olivaceus). Fish Pathol 1993;28:131–4. [3] Jee BY, Kim YC, Park MS. Morphology and biology of parasite responsible for scuticociliatosis of cultured olive flounder Paralichthys olivaceus. Dis Aquat Organ 2001;47:49–55. [4] Shin SP, Han JE, Gomez DK, Kim JH, Choresca CHJ, Jun JW, et al. Identification of scuticociliate Philasterides dicentrarchi from indo-pacific seahorses Hippocampus kuda. Afr J Microbiol Res 2011;5:738–41. [5] Kim SM, Cho JB, Kim SK, Nam YK, Kim KH. Occurrence of scuticociliatosis in olive flounder Paralichthys olivaceus by Philasterides dicentrarchi (Ciliophora: scuticociliatida). Dis Aquat Organ 2004;62:233–8. [6] Kim SM, Cho JB, Lee EH, Kwon SR, Kim SK, Nam YK, et al. Pseudocohnilembus persalinus (Ciliophora: Scuticociitida) is an additional species causing scuticociliatosis in olive flounder Paralichthys olivaceus. Dis Aquat Organ 2004;62:239–44. [7] Jin CN, Harikrishnan R, Moon YG, Kim MC, Kim JS, Balasundaram C, et al. Histopathological changes of Korea cultured olive flounder, Paralichthys olivaceus due to

[8]

[9] [10] [11] [12]

[13]

[14]

[15]

[16] [17]

[18]

[19]

[20] [21]

[22]

[23]

[24] [25]

365

scuticociliatosis caused by histophagous scuticociliate, Philasterides dicentrarachi. Vet Parasitol 2009;161:292–301. Parama A, Iglesias R, Alvarez MF, Leiro J, Ubeira FM, Sanmartin ML. Cysteine proteinase activities in the fish pathogen Philasterides dicentrarchi (Ciliophora: Scuticociliatida). Parasitology 2004;128:541–8. Piazzon C, Lamas J, Leiro JM. Role of scuticociliate proteinases in infection success in turbot, Psetta maxima (L.). Parasite Immunol 2011;33:535–44. Lozupone CA, Knight RD, Landweber LF. The molecular basis of nuclear genetic code change in ciliates. Curr Biol 2001;11:65–74. Lin Y, Cheng G, Wang X, Clark TG. The use of synthetic genes for the expression of ciliate proteins in heterologous systems. Gene 2002;288:85–94. Dalton JP, Neill SO, Stack C, Collins P, Walshe A, Sekiya M, et al. Fasciola hepatica cathepsin L-like proteases: biology, function, and potential in the development of first generation liver fluke vaccines. Int J Parasitol 2003;33:1173–81. Mottram JC, Souza AE, Hutchison JE, Carter R, Frame MJ, Coombs GH. Evidence from disruption of the lmcpb gene array of Leishmania mexicana that cysteine proteinases are virulence factors. Proc Natl Acad Sci U S A 1996;93:6008–13. Ahn SJ, Seo JS, Kim MS, Jeon SJ, Kim NY, Jang JH, et al. Cloning, site-directed mutagenesis and expression of cathepsin L-like cysteine protease from Uronema marinum (Ciliophora: Scuticociliatida). Mol Biochem Parasitol 2007;156:191–8. Lim SU, Seo JS, Kim MS, Ahn SJ, Jeong HD, Kim KH, et al. Molecular cloning and characterization of Cathepsin B from a scuticociliate, Uronema marinum. Comp Biochem Physiol B Biochem Mol Biol 2005;142:283–92. Karrer KM, Peiffer SL, DiTomas ME. Two distinct gene subfamilies within the family of cysteine protease genes. Proc Natl Acad Sci U S A 1993;90:3063–7. Vernet T, Berti PJ, de Montigny C, Musil R, Tessier DC, Menard R, et al. Processing of the papain precursor. The ionization state of a conserved amino acid motif within the Pro region participates in the regulation of intramolecular processing. J Biol Chem 1995;270:10838–46. Iglesias R, Parama A, Alvarez MF, Leiro J, Sanmartin ML. Antiprotozoals effective in vitro against the scuticociliate fish pathogen Philasterides dicentrarchi. Dis Aquat Organ 2002;49:191–7. Quintela JM, Peinador C, Gonzalez L, Iglesias R, Parama A, Alvarez F, et al. Piperazine N-substituted naphthyridines, pyridothienopyrimidines and pyridothienotriazines: new antiprotozoals active against Philasterides dicentrarchi. Eur J Med Chem 2003;38:265–75. Dai JR, Wang W, Liang YS, Li HJ, Guan XH, Zhu YC. A novel molluscicidal formulation of niclosamide. Parasitol Res 2008;103:405–12. Cogliano VJ, Grosse Y, Baan RA, Straif K, Secretan MB, Ghissassi FE. Meeting report: summary of IARC monographs on formaldehyde, 2-butoxyethanol, and 1-tertbutoxy-2-propanol. Environ Health Perspect 2005;113:1205–8. Iglesias R, Parama A, Alvarez MF, Leiro J, Ubeira FM, Sanmartin ML. Philasterides dicentrarchi (Ciliophora:Scuticociliatida) expresses surface immobilization antigens that probably induce protective immune responses in turbot. Parasitology 2003;126:125–34. Ruszczyk A, Forlenza M, Savelkoul HF, Wiegertjes GF. Molecular cloning and functional characterisation of a cathepsin L-like proteinase from the fish kinetoplastid parasite Trypanosoma carassii. Fish Shellfish Immunol 2008;24:205–14. Verity CK, McManus DP, Brindley PJ. Vaccine efficacy of recombinant cathepsin D aspartic protease from Schistosoma japonicum. Parasite Immunol 2001;23:153–62. Parama A, Castro R, Lamas J, Sanmartin ML, Santamarina MT, Leiro J. Scuticociliate proteinases may modulate turbot immune response by inducing apoptosis in pronephric leucocytes. Int J Parasitol 2007;37:87–95.