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Experimental Parasitology 130 (2012) 189–194

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Experimental Parasitology

journal homepage: www.elsevier.com/locate/yexpr

Cloning and characterization of a surface antigen CiSA-32.6 from Cryptocaryon irritans ⇑ Xiaohong Huang , Zhiyu Sun, Guowei Guo, Changfeng Zheng, Yang Xu, Liping Yuan, Cheng Liu

Fujian Key Laboratory of Developmental and Neuro Biology, College of Life Science, Fujian Normal University, Fuzhou 350108, Fujian, PR China article info abstract

Article history: Cryptocaryon irritans is a ciliated parasite causing cryptocaryosis in marine fish. To isolate functional Received 17 September 2011 genes, a cDNA library of C. irritans trophonts was constructed and a gene designated CiSA-32.6 (GenBank Received in revised form 17 January 2012 ID: JF812643) was cloned and characterized. The full-length cDNA (1158 bp) encoded a deduced polypep- Accepted 18 January 2012 tide of 330 amino-acid (aa) with a signal peptide of 22 aa. To express the ciliate gene, a truncated open Available online 1 February 2012 reading frame (CiSA-32.6t) was synthesized to remove fragments encoding the signal peptide and hydrophobic C-terminal and to modify non-universal genetic codes. CiSA-32.6t was subcloned into Escherichia Keywords: coli DH5a strain using the pGEX-4T-1 vector and then expressed as a glutathione S transferase fusion pro- Cryptocaryon irritans tein (rCiSA-32.6t). Western blotting analysis showed that sera from mice immunized with rCiSA-32.6t Surface protein Molecular characterization reacted specifically with a native protein (32.6 kDa) in parasite lysates. Moreover, rCiSA-32.6t reacted Prokaryotic expression specifically with sera from mice immunized with a C. irritans trophont lysate. Expression of the Antigenicity CiSA-32.6 gene in C. irritans was detected at all developmental stages by reverse transcriptase PCR and Western blotting analysis. This study provides the basis of further investigations into the pathogenic biology of C. irritans and the control of cryptocaryosis. Ó 2012 Elsevier Inc. All rights reserved.

1. Introduction 2007; Misumi et al., 2011, 2012). This has promoted the development of vaccines capable of inducing a protective response in the Cryptocaryon irritans is a parasitic ciliate, belonging to Class host. In previous studies, live or killed parasites have been used Prostomatea, Order Prorodontida, Family Cryptocaryonidae, Genus for immunization (Luo et al., 2007; Misumi et al., 2011, 2012). Cryptocaryon (Wrighta and Colorni, 2002). Parasites penetrate the However, techniques for in vitro propagation of C. irritans are skin, gills and eyes of marine ﬁshes, causing cryptocaryosis, more insufﬁciently developed rendering collection of the parasites for commonly known as white spot disease, which is a devastating vaccine production extremely laborious. Production of genetically problem in commercial marine aquaculture in tropical and sub- engineered vaccines has therefore become a focus for research. A tropical regions (Colorni and Burgess, 1997). The increased inci- full-length cDNA library of C. irritans was constructed and screened dence of cryptocaryosis outbreaks in southeast of China has led for vaccine candidates, from which a large number of novel func- to severe economic losses in aquaculture. For example, hundreds tional genes were isolated. In this study, one of these genes encod- of millions of dollars were lost during one cryptocaryosis outbreak ing a C. irritans surface antigen (GenBank ID: JF812643) was cloned in Xiapu county, Fujian province in 2009. To date, no effective and expressed in bacteria for molecular characterization. methods have been developed to control this disease although re- cently, immunoprophylaxis has been considered as a potential 2. Materials and methods control measure (Colorni, 1985; Burgess and Matthews, 1995; Yoshinaga and Nakazoe, 1997; Bryant et al., 1999; Luo et al., 2.1. Parasites and experimental animals

C. irritans trophonts were collected from gills of infected Pseu- Abbreviations: CiSA-32.6 or CiSA-32.6, the gene or protein of surface antigen from Cryptocaryon irritans with a molecular mass of 32.6 kDa; rCiSA-32.6t, dosciaena crocea cultured in netcages in the coastal area of Xiapu recombinant truncated CiSA-32.6; ESTs, expression sequence tags; ORF, open county, Fujian province, China on July 2nd, 2009, during a crypto- reading frame; IPTG, isopropylthio-b-D-galactoside; GST, glutathione S transferase; caryosis outbreak in the area. The collection was carried out by RT-PCR, reverse transcriptase PCR; aa, amino-acid. soaking gills with visible white spots in sterile sea water for 2 h be- ⇑ Corresponding author. Address: Fujian Key Laboratory of Developmental and Neuro Biology, College of Life Science, Fujian Normal University, Qishan Campus, fore the gills were discarded. Trophonts were collected in the sed- Fuzhou 350108, Fujian, PR China. iment and allowed to develop into tomonts, which were washed E-mail address: [email protected] (X. Huang). three times with sterile sea water. Tomonts were sampled for

190 X. Huang et al. / Experimental Parasitology 130 (2012) 189–194 extraction of genomic DNA and the remainder was incubated at and used as primers for RT-PCR detection of CiSA-32.6 expression 27 °C for development of theronts. The newly hatched theronts were at the transcriptional level. Expression of a C. irritans actin gene used for the infection and passage of Sebastiscus marmoratus main- (GenBank ID: JN399999) was used as a control and amplified using tained in an aquarium. The parasite was then defined as the same the following of primer pair: 50-ATGGCCGAAGACTAACAAGCAG-30 strain as PYH4.12 or Chiayi strain by molecular phylogeny based and 50-TCAGAAGCATTTTCTGTGTACA-30. on ribosomal DNA internal transcribed spacer sequences (Diggles and Adlard, 1997; H.Y. Sun et al., 2006; Z. Sun et al., 2011). The 2.5. Modification of the CiSA-32.6 gene sequence by artificial synthesis C. irritans life cycle was established in vitro and parasites were collected at every stage for further investigation. Theronts were har- The CiSA-32.6 gene open reading frame (ORF) was modified for vested by centrifugation at 1000g for 5 min at 4 °C. Trophonts, the the expression in bacteria by removal of the signal peptide, muta- parasitic stage of the ciliate, were collected from S. marmoratus 3d tion of TAA into CAA and also removal of the hydrophobic C-termi- post-infection by scraping fish body surfaces in a dish of sterile sea nal to improve the solubility of the recombinant protein. The signal water with a glass slide. Motile trophonts were harvested individu- peptide was predicted by using the online Signal P 3.0 Server (Cen- ally and immediately using a pipette. Protomonts and tomonts were ters for biological sequence analysis, Technical University of Den- collected 3 d post-infection of fish by placing dishes on the bottom of mark). The hydrophobicity of the polypeptide was predicted by the aquarium for 2 and 24 h, respectively, to allow mature trophonts DNAMAN (Lynnon Biosoft, Quebec, Canada). The truncated form lost from fish to sediment into the dishes automatically. Parasites at of CiSA-32.6 without genetic coding sequences for the signal pep- each stage were washed in sterile sea water and stored immediately tide and a 25 amino acid fragment at the C-terminal was synthe- at 80 °C for further use. All experiments were conducted in accor- sized artificially and inserted into plasmid pUC57 (Genscript Ltd. dance with the Guiding Principles for the Care and Use of Research Company in Nanjing, China). Animals outlined by Fujian Normal University. 2.6. Subcloning of the truncated ORF from recombinant pUC57 into 2.2. Construction of cDNA library pGEX-4T-1

Total RNA was extracted from trophonts using TRIzol™ reagent The truncated ORF (CiSA-32.6t) was amplified using a set of oli- (Invitrogen, Carlsbad, CA). Poly A+ RNA was then purified using oligonucleotide primers, 50-CGGGATCCGAAAAAACTGCAGTTGCA-30 go-dT resin (Wako, Tokyo, Japan). First strand cDNA was reverse and 50-CGGAATTCTCAAGTAGAGGTAACTTC-30 (underlining in each transcribed from poly A+ RNA using SMARTScribe™ MMLV Reverse primer indicates BamHI and EcoRI recognition sites, respectively), Transcriptase, SMART IV Oligonucleotide and CDS III/30 Primer using pUC57/CiSA-32.6 as template DNA. The PCR product was (TaKaRa, Otsu, Japan). Second strand cDNA was amplified by double-digested with BamHI and EcoRI (TaKaRa, Otsu, Japan), li- long-distance PCR using the first strand as a template. Following gated to the BamHI–EcoRI ends of pGEX-4T-1 and transformed into restriction enzyme digestion, double-stranded cDNA was size-frac- E. coli DH5a. tionated using a CHROMA SPIN-400 column. All cDNAs larger than 0.4 kbp were inserted into the k phage vector kTriplE 2 and pack- 2.7. Expression, extraction and purification of recombinant CiSA-32.6t aged using Gigapack III Gold Packaging Extract (Stratagene, La Jolla, protein CA). The titer of the primary cDNA library was determined and amplification was carried out. Individual phage plaques from the Bacteria containing plasmid pGEX-4T/CiSA-32.6t were cultured primary cDNA library were picked at random and subjected to at 37 °CtoOD600nm between 0.3 and 0.5. Expression of the foreign PCR to amplify the DNA insert. The recombination rate and the gene was induced by the addition of isopropylthio-b-D-galactoside length of DNA inserts were estimated. (IPTG) and cultured for a further 6 h at 25 °C. Recombinant rCiSA- 32.6t protein was extracted and purified using glutathione Sephar- 2.3. Gene isolation from the cDNA library ose 4B (GE Healthcare Life Sciences, Uppsala, Sweden) according to the instructions provided by the manufacturer. For sequencing the expression sequence tags (ESTs), recombinant kTriplE 2 was converted into recombinant plasmid 2.8. Preparation of antisera to rCiSA-32.6t and to trophont cell lysates kTriplE 2 by transduction of the recombinant phage into Esche- richia coli BM25.8 strain expressing Cre recombinase. Sequence Specific pathogen free Kunming mice from Shanghai Laboratory alignments of ESTs with reported genes in GenBank predicted the Animal Co. Ltd., China were injected intraperitoneally with rCiSA- identity of one gene to be a surface antigen. This gene was isolated 32.6t (100 lg/mouse) emulsified with equal volume (200 ll/ for characterization and was shown to encode a deduced mature mouse) of Freund’s complete adjuvant (Sigma–Aldrich, St. Louis, protein of 32.6 kDa and therefore was designated CiSA-32.6. MO, USA). Animals were boosted twice at 14 d intervals using the same dose of antigen emulsified with an equal volume of Fre- 2.4. Reverse transcriptase PCR detection of CiSA-32.6 gene expression und’s incomplete adjuvant (Sigma–Aldrich). Ten days after the last at different stages in the life cycle of the parasite immunization, blood was obtained by cardiac puncture and antisera against rCiSA-32.6t were separated. Antisera against trophont Total mRNA was extracted from each developmental stage of C. cell lysates were prepared using similar procedures. irritans using QIAGEN RNeasy Mini kit (QIAGEN) and used as template DNAs to synthesize first strand cDNAs by reverse transcriptase 2.9. Western blotting analysis PCR (RT-PCR). RT-PCR was carried by using TaKaRa RNA PCR kit (TaKaRa) according to the instructions provided by the manufac- Cell lysates of the trophonts/protomonts, tomonts and theronts turer. Several sequences predicted to encode surface antigens, were prepared and subjected to sodium dodecyl sulfate polyacryl- which had or had not been deposited in GenBank, were compared amide gel electrophoresis as described previously (Huang et al., using CLUSTAL X1.83 software. As a result, the specific fragments 2003). Proteins were then transferred to polyvinylidene fluoride in the CiSA-32.6 gene were identified. According to the specific membranes (Millipore, Billerica, MA, USA). Murine sera against CiSA-32.6 gene sequence, a pair of oligonucleotides, 50-GTATTTGCAA rCiSA-32.6t were used as the primary detection reagent and horse- CTGGTATG-30 and 50-ACCTGAATCAGTCCATGT-30, were synthesized radish peroxidase conjugated goat anti-murine IgG antibody (ICN Author's personal copy

X. Huang et al. / Experimental Parasitology 130 (2012) 189–194 191

Biochemicals, Aurora, Ohio) was used as the secondary detection 3.4. Expression of recombinant CiSA-32.6t protein in E. coli reagent (1:4000). Color was developed using 3,30-diaminobenzi- dine substrate and H2O2. Western blotting analysis of rCiSA-32.6t rCiSA-32.6t was expressed as a GST fusion protein. The molecu- was performed using trophont cell lysates as a control to detect lar mass of rCiSA-32.6t (55.9 kDa) was shown to be equivalent to the reaction of murine sera against trophont cell lysates containing the calculated size of GST (26 kDa) fused with truncated CiSA- native protein CiSA-32.6. 32.6 (29.9 kDa) (Fig. 3). rCiSA-32.6t was mainly expressed in the insoluble fraction (Lane 1, Fig. 3). However, the fusion protein was also partially present in the soluble fraction (Lane 2, Fig. 3), 2.10. Localization of native CiSA-32.6 by immune fluorescent antibody suggesting that the solubility of the hydrophobic C-terminal trun- testing (IFAT) cated rCiSA-32.6t was improved compared with that of rCiSA-32.6 (data not shown). Theronts and trophonts were collected and divided into two groups, respectively. Parasites in the first group were incubated 3.5. Western blotting analysis of native parasite protein with murine with murine sera against rCiSA-32.6t (1:100 in PBS containing 3% anti-rCiSA-32.6t protein fetal bovine serum) for 30 min, while those in the second group were incubated with murine sera against glutathione S transferase Murine sera against rCiSA-32.6t were produced by immuniza- (GST) as a control. After five washes with PBS, both groups were tion of mice with rCiSA-32.6t for use as the primary detection re- incubated with Alexa 488 conjugated goat anti-mouse IgG anti- agent in Western blotting analysis of native CiSA-32.6 in lysates body (1:400 in PBS containing 3% fetal bovine serum) (Sigma) for of trophonts/protomonts, tomonts and theronts. Murine serum 30 min. After a further five washes, samples were observed using against GST was used as a control. Results revealed that murine fluorescence microscopy and photographed. sera against rCiSA-32.6t specifically reacted with a molecule of the expected size in all stages of C. irritans life-cycle (Fig. 4B). No reaction with murine anti-GST serum was observed (Fig. 4C). The 3. Results molecular mass of native CiSA-32.6 protein corresponded with the calculated size (32.6 kDa). 3.1. Evaluation of the cDNA library 3.6. Western blotting analysis of recombinant CiSA-32.6t protein with 6 The titer of the primary library was 3.25 10 pfu/ml, with a sera from mice immunized with native proteins of C. irritans recombination rate of 98%. The majority of DNA inserts were between 0.4 and 2 kbp in length. The ESTs of 463 clones detected The reactogenicity of rCiSA-32.6t was detected by Western blot- were aligned with known gene sequences by NCBI-blastx. No hits ting analysis using sera from mice immunized with C. irritans tro- were detected for ESTs of 46 clones, while those of the other 417 phont cell lysates. Sera from immunized mice not only reacted clones were clustered into 306 putative unique transcripts, with strongly to multiple native molecules from the parasite 222 singletons and 84 contigs. The 306 putative unique transcripts (Fig. 5B1), but also reacted specifically with rCiSA-32.6t were homologous to genes encoding five agglutination/immobili- (Fig. 5B2). No reaction with GST was observed. In contrast, sera zation antigens, 70 enzymes, 28 ribosomal proteins, 103 proteins from SPF mice did not react with any of these proteins (Fig. 5C). with other functions, 64 chromosome undeterminated scaffold proteins and 36 unknown putative proteins. These results demon- 3.7. Localization of the native CiSA-32.6 protein strated successful construction of the cDNA library. Further analy- ses of more ESTs are being undertaken. Localization of CiSA-32.6 in living theronts and trophonts was determined using IFAT and the results were observed by fluorescence microscopy. The surfaces of the parasites reacted with mur- 3.2. Characterization of the CiSA-32.6 gene ine sera against rCiSA-32.6t (Fig. 6A and C), but did not react with murine sera against GST (Fig. 6B and D), suggesting that the native The full-length CiSA-32.6 cDNA (1158 bp with an ORF of 993 bp) CiSA-32.6 protein was expressed on the surface of the parasites. encoded a polypeptide of 330 aa containing a signal peptide, the cleavage site of which was predicted to be located between resi- dues 22 and 23 (Fig. 1). This polypeptide also contained hydropho- 4. Discussions bic transmembrane domains. The ratio of AT to GC in the nucleotide sequence was 63:37. After cleavage of the signal pep- In this study a cDNA library was constructed using SMART tech- tide, the molecular weight of the deduced mature CiSA protein niques to obtain full-length cDNAs of functional genes. Sixty per- was 32.6 kDa. Therefore, this gene was designated CiSA-32.6. This cent of the cDNAs in this library were full-length, which gene exhibited 99% identity with the full-length cDNA of an agglu- facilitated cloning of functional genes. Genes coding for surface tination/immobilization antigen precursor deposited in GenBank antigens were then analyzed in detail. So-called surface antigens (GenBank ID: FJ167512.1). The ORF of CiSA-32.6 contained 6 TAAs, here are the surface membrane proteins of cells, each of which is codons for glutamine in the ciliate (Hatanaka et al., 2007; Salim characterized by a signal peptide at N-terminal, a highly hydropho- et al., 2008). bic C-terminal and transmembrane domains. Due to their direct interactions with hosts, surface antigens of protozoan parasites are usually considered as not only receptors of signals which reg- 3.3. CiSA-32.6 mRNA expression ulate cell activities, but also major immunodorminant molecules. They are therefore potential candidates for vaccine targets. RT-PCR was used to determine CiSA-32.6 gene expression at dif- CiSA-32.6 was first confirmed as a surface antigen in this study ferent stages in the life cycle of the parasite. CiSA-32.6 gene tran- by sequence analysis, since it was shown to contain a signal pep- scription was detected at all stages (trophonts/protomonts, tide, highly hydrophobic C-terminal and transmembrane domains, tomonts and theronts), at levels similar to the house-keeping gene and it was highly homologous to the reported surface proteins of actin (Fig. 2). the parasites (Hatanaka et al., 2007, 2008). Secondly, the surface Author's personal copy

192 X. Huang et al. / Experimental Parasitology 130 (2012) 189–194

Fig. 1. Molecular characterization of the CiSA-32.6 gene from C. irritans trophont/protomonts. Panel A, full-length cDNA and deduced amino acid sequence. Genetic codes to be reassigned are marked by underlining; Panel B, transmembrane helical regions of CiSA-32.6 predicted using TMHMM and the truncated CiSA-32.6.

Fig. 3. Expression of recombinant CiSA-32.6t protein in E. coli induced by IPTG at 25 °C. Lane M, proteins with standard molecular weight; Lanes 1 and 2, the Fig. 2. Transcriptional expression of CiSA-32.6 gene detected by RT-PCR. Lane M, insoluble and soluble fractions of the bacterial lysate, respectively; Lane 3, puriﬁed DNA ladder; Lanes 1–3, RT-PCR products of the truncated CiSA-32.6 gene from rCiSA-32.6t; Lane 4, GST. trophonts/protomonts, tomonts and theronts, respectively, using an actin gene as a control. CiSA-32.6 protein was expressed at all stages in the life cycle of the parasitic ciliate at both transcriptional and translational levels. expression of the native CiSA-32.6 protein in the parasites was val- Therefore, this protein was deemed to be suitable for evaluation of idated by immune ﬂuorescent antibody test. Furthermore, native its potential to induce protective immunity in hosts. Author's personal copy

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well as in previous reports (Hatanaka et al., 2007, 2008). Six TAAs were identified in the CiSA-32.6 gene ORF sequence, making protein expression using conventional expression systems, such as bacteria, yeast, insect cells and mammal cells expression systems problematic. A tetrahymena expression system has been constructed for the expression of some foreign genes although, it is neither as sophisticated as the conventional systems nor commer- cially available (Gaertig et al., 1999). Therefore, the CiSA-32.6 ORF sequence was modified for expression in a more sophisticated expression system. Results showed that the modified foreign gene was expressed effectively in E. coli. Antibodies against recombinant Fig. 4. Western blotting analysis demonstrating that sera from mice immunized CiSA-32.6t also recognized native CiSA-32.6 in the ciliate. Further- with rCiSA-32.6t protein reacted specifically with native CiSA-32.6 protein in more, antibodies against C. irritans trophont lysates were shown to parasites cell lysates. (A) Amido black 10B protein staining; (B) primary detection reagent, mouse antisera against rCiSA-32.6t; (C) primary detection reagent, mouse react specifically with recombinant CiSA-32.6t, thus demonstrating antisera against GST. Lane M, standard molecular weight markers; Lanes 1–3, cell both the immunogenicity and the reactogenicity of the bacteria- lysates of trophonts/protomonts, theronts and tomonts, respectively; Lane 4, GST. expressed CiSA-32.6t protein. However, induction of protective immunity by the CiSA-32.6t protein against parasites in host fish remains to be evaluated.

Acknowledgments

This work was supported by Grants from the National Natural Science Foundation of China (No. 31040084), the Natural Science Foundation of Fujian Province, China (No. 2008J004) and the Scien- tiﬁc Research Foundation for the Returned Overseas Chinese Schol- ars, Fujian Province, China.

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