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Two classes of glutathione S-transferase genes with different response profiles to bacterial challenge in Venerupis philippinarum
Fish & Shellfish Immunology 32 (2012) 219e222
Contents lists available at SciVerse ScienceDirect
Fish & Shellfish Immunology journal homepage: www.elsevier.com/locate/fsi
Short communication
Two classes of glutathione S-transferase genes with different response profiles to bacterial challenge in Venerupis philippinarum Chenghua Li a, *, Xiurong Su a, *, Ye Li a, Taiwu Li a, b, Chongjie Sun a, Tingting Zhou a, Haipeng Liu c a
Faculty of Life Science and Biotechnology, Ningbo University, 818 Fenghua Road, Ningbo, Zhejiang Province 315211, PR China Ningbo City College of Vocational Technology, Ningbo 315100, PR China c State Key Laboratory of Marine Environmental Science, Xiamen University, Xiamen 361005, PR China b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 4 August 2011 Received in revised form 20 October 2011 Accepted 21 October 2011 Available online 18 November 2011
Glutathione S-transferase (GST) is major cytosolic detoxification enzymes involved in many pathological and physiological processes. In the present study, two classes of GSTs (VpGST-1 and VpGST-2) were cloned from Venerupis philippinarum haemocytes by cDNA library and RACE approaches. Sequence alignments and phylogenetic analysis together supported that they belonged to a new member of sigma and pi classes GSTs protein family, respectively. The expression profiles of these two genes under Vibrio anguillarum challenge were investigated by quantitative RT-PCR. The bacterial challenge could significantly up-regulate the mRNA expression of both VpGST-1 and VpGST-2 with larger amplitude in VpGST2, and the feedback speed for VpGST-2 was more rapid than that of VpGST-1. The differences in the response to bacterial challenge indicated that they were functional diversity and probably played cooperative roles in mediating the Vibrio challenge in clam. Ó 2011 Elsevier Ltd. All rights reserved.
Keywords: Venerupis philippinarum Glutathione S-transferase Expression profile Vibrio anguillarum
1. Introduction Glutathione S-transferases (GST, EC 2.5.1.18) are composed of many cytosolic, mitochondrial, and microsomal proteins participating in the detoxification of reactive electrophilic compounds by catalyzing their conjugation to glutathione [1,2]. The vital functions of GSTs have been demonstrated to be promising indicators for environmental pollutants [3e5], and involved into elimination of intracellular reactive oxidative species (ROS) in marine organisms [6]. There are at least eight major classes of GSTs, designated alpha, kappa, mu, pi, sigma, omega, theta and zeta based on their substrate specificity, immunological properties and protein sequence identity in mammalian [1,7e9]. Some classes of GSTs have been identified and characterized in aquatic invertebrates include bivalve mollusks [3,10e13], gastropods [14e16] and shrimp [17]. China, with a thousand year history of Manila clam Venerupis philippinarum farming, produces about 1.8 million tonne of Manila clams annually, accounting for about 90% the total global production [18]. However, with the development of intensive culture and environmental deterioration, various diseases caused by bacteria, protozoa occurred in cultured V. philippinarum populations, resulted in enormous losses to the clam aquaculture. As a major
* Corresponding authors. Tel./fax: þ86 574 87608368. E-mail addresses: [email protected] (C. Li), [email protected] (X. Su). 1050-4648/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.fsi.2011.10.032
pathogen in global fishery, Vibrio anguillarum had been widely isolated from ocean environment and demonstrated to be pathogenicity on oysters and other bivalves [19e21]. Manila clams like other bivalves filtered large amounts of seawater to cope with nutritional and respiratory needs, leading to its continuously exposure to the microorganism. However, the pathogenicity of V. anguillarum on Manila clam has not been investigated to our knowledge. The purposes of this study were: 1) to clone full-length cDNAs of two classes of clam GSTs; 2) to investigate their mRNA expression under Vibrio challenge and provide basic data for the host response against the bacterial challenge.
2. Materials and methods 2.1. Clams and bacterial challenge The clams V. philippinarum (7.5e11 g in weight) were collected from Qingdao, Shandong Province, China. The clams were acclimated for a week before commencement of the experiment. The temperature was held at 20e22 C throughout the whole experiment. The salinity for the supplied seawater was kept at 30&. For V. anguillarum challenge experiment, the clams were cultured in seawater with high density of V. anguillarum (107 CFU mL1), and a group of unchallenged clams were used as control. The challenged clams were randomly sampled at 6 h, 12 h, 24 h, 48 h, 72 h and 96 h
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respectively. The haemolymphs from the control and the challenged groups were collected using a syringe and centrifuged at 2000 g, 4 C for 10 min to harvest the haemocytes. There were three replicates for each time point. 2.2. Cloning of the full-length cDNA of VpGST-1 and VpGST-2 by 50 RACE cDNA library construction, EST analysis, RNA isolation and cDNA synthesis were consistent with our published works [22,23]. Two sets of gene specific primers, P1 50 -CCATTTCCGAGAACATTTCCATC30 and P2 50 -ACACGGGACTGA TGCTCTTCGAG-30 for VpGST-1 and P3 50 -AAACCGGAAGCAGTGTATCGTCT- 30 and P4 50 -TTGAGGGTCGGTAATGCGTCTAT-30 for VpGST-2, were designed based on the two ESTs from cDNA library to clone the full-sequence cDNA of VpGST-1 and VpGST-2, respectively. Semi-nested PCR approaches were introduced to get the 50 end of the two genes with 1:100 dilutions of the first round PCR products as templates and oligodG as anchored primer. The PCR programs and PCR product sequencing were performed according to reference [23]. 2.3. Sequence analysis of VpGST-1 and VpGST-2 The VpGST-1 and VpGST-2 gene sequences were analyzed using the BLAST algorithm at NCBI web site (http://www.ncbi.nlm.nih. gov/blast), and the deduced amino acid sequence was predicted with the Expert Protein Analysis System (http://www.expasy.org/). For construction of phylogenetic tree, the deduced amino acid sequences of VpGST-1 and VpGST-2 were aligned with the corresponding sequences from various animals using the ClustalX software [24]. Based on this alignment, a NJ tree was constructed by Mega3.1 software package (http://www.megasoftware.net/). To derive the confidence value for the phylogeny analysis, bootstrap trials were replicated 1000 times. 2.4. Temporal expression profile of VpGST-1 and VpGST-2 mRNA in haemocytes post V. anguillarum challenge The expression profiles of VpGST-1 and VpGST-2 transcript in haemocytes after Vibrio challenge were measured by quantitative real-time PCR in Applied Biosystem 7500 fast Real-time PCR System. Two sets of gene specific primers, P5 50 -CATTGCCCGTGCTTACTATTGAC-30 and P6 50 -TGTTCCTTTCTTCCTCGTTAT CC-30 for VpGST-1 and P7 50 -GTATCCGAGCGATTTACAAGAGG-30 and P8 50 -CTTTGAGGGTCGGTAATGCGTCT-30 for VpGST-2, were designed to amplify products of 207 bp and 290 bp, respectively. Two clam b-actin primers, P9 50 CTCCCTTGAGAAGAGCTACGA-30 and P10 50 -GATACCAGCAGATTCCATA CCC-30 were used to amplify an 121 bp fragment as internal control to verify the successful reverse transcription and to calibrate the cDNA template. The reaction was performed in a total volume of 20 ml containing 10 ml of 2 SYBR Green Master Mix (Applied Biosystems), 4 ml of the diluted cDNA mix, 0.25 ml of each primer (10 mmol L1), 5.5 ml of DEPC-treated water. The thermal profile for real-time PCR was 50 C for 2 min and 95 C for 10 min followed by 45 cycles of 95 C for 15 s and 60 C for 1 min. Dissociation curve analysis of amplification products was performed at the end of each PCR reaction to confirm that only one PCR product was amplified and detected [22]. The 2DDCT method was used to analyze the expression level of VpGSTs. The Ct for the target amplified VpGSTs and the Ct for the internal control b-actin were determined for each sample. Difference in the Ct for the target and the internal control, called DCT, was calculated to normalize the differences in the amount of template and the efficiency of the RT-PCR. The DCT value for untreated sample at 0 h was used as the reference sample, called the calibrator. The DCT for each sample (including controls from other time points) was subtracted from the DCT of the
calibrator; the difference was called DDCT. The expression level of VpGSTs could be calculated by 2DDCT, and the value stood for an n-fold difference relative to the calibrator. All data were given in terms of relative mRNA expression as means S.D. The control data obtained from real-time PCR analysis were subjected to One-way Analysis of Variance (ANOVA) followed by multiple Duncan test to determine differences in the mean values among the controls. Then, nonparametric test was employed to compare challenged and control groups of each sampling time. Significant differences across control from each time point were indicated with an asterisk at P < 0.05 and two asterisks at P < 0.01. 3. Results and discussion 3.1. cDNA cloning and analysis of the VpGST-1 and VpGST-2 genes Blastx analysis indicated the two ESTs of 635 bp and 587 bp from clam cDNA library were similar to the sigma classes of GSTs from Haliotis discus discus (ABO26602) and pi classes of GSTs from Chlamys farreri (ACL80138), respectively. Based on these fragments, four gene specific primers (P1, P2, P3, and P4) were designed to clone the full-length cDNA of VpGST-1 and VpGST-2. A 330 bp fragment was produced by 50 RACE with primer P2 and oligodG for VpGST-1. Regarding to VpGST-2, the corresponding product was of 526 bp amplified with primer P4 and oligodG. By overlapping the fragments with respective EST, two sequences of 928 bp and 939 bp nucleotide sequence representing the full-length cDNA of VpGST-1 and VpGST-2 were assembled and deposited in GenBank under accession no. GQ384392 and GQ384393, respectively. The complete sequence of VpGST-1 cDNA contained a 50 untranslated region (UTR) of 32 bp, a 30 UTR of 263 bp with a polyA tail. Canonical polyadenylation signal sequence AATAAA was instead of ATTAAA. An open reading frame (ORF) of 633 bp encoded a polypeptide of 210 amino acids with the predicted molecular weight of 23.78 kDa and the theoretical isoelectric point of 5.32. The typical GST-N domain (G-site) was located from 4aa to 74aa, and GST-C (H-site) domain from 96aa to 204aa. Blastp search indicated the deduced amino acid of VpGST-1 showed weak similarity to other sigma classes of GSTs. The highest similarity was detected between VpGST-1 and its isoforms (ADI44317) in the same species with 39% identities. The similar phenomenon was consistent with other reports [4,16]. The lower similarity was attributed to its highest diversity of C-terminus. In Drosophila, the sigma GST exhibited a distinct N-terminal terminus with a proline/alaninerich extension [4], while the domain was absent from mollusk counterparts. To be mentioned, the central residue of Tyr (Y) for GSH stabilization in the sigma class GSTs was totally conserved in the deduced amino acid of VpGST-1. The full-length cDNA of VpGST-2 consisted of a 50 UTR of 8 bp, 0 a 3 UTR of 313 bp, and an ORF of 618 bp encoding a polypeptide of 205 amino acids with the predicted molecular weight of 23.93 kDa and the theoretical isoelectric point of 6.84. The typical GST-N and GST-C domain was located from 4aa to 73aa and from 94aa to 183aa, respectively. Search by Blastp indicated it had highest similarity to pi classes GSTs from other species, such as 91% to C. farreri (ACL80138), 74% to Mercenaria mercenaria (ABV29188), and 70% to Corbicula fluminea (AAX20374). Moreover, multiple alignment analysis revealed 11 highly conserved amino acid residues (F8, R14,W39,K45, Q52, L53, P54, Q65, S66,E98, D99) for G-site of GST [25] was almost totally conserved in the deduce amino acid of VpGST-2 with the exception of Ser, which was substituted for Leu53. This substitution has also been reported in Antarctic bivalve [13]. The conserved amino acid residues for H-site (F7, P8, V10, R13, V104, F108, N204, and G205) were also partially identified in VpGST-2 except absent from V10.
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3.2. Phylogenetic analysis To detect evolutionary relationships between different classes of GSTs and further elucidate the identities of VpGST-1 and VpGST-2, a NJ phylogenetic tree was re-constructed based on 18 amino acid sequences of four classes of GSTs including VpGST-1 and VpGST-2 (Fig. 1). Four classes of GSTs with different animal origins were clustered into four major branches in the tree according to their classification criterion. Pi classes of GSTs were firstly clustered with sigma ones, subsequently with mu and omega counterparts in turn. VpGST-1 was placed together into sigma classes of GSTs, indicating it belonged to a new member of this protein family. VpGST-2 was clustered with pi classes GSTs. From this tree, we could draw the conclusion that VpGST-1 and VpGST-2 belonged to a distinctive category in GSTs protein family. 3.3. The expression profile of VpGST-1 and VpGST-2 after Vibrio challenge As antioxidant proteins, GSTs are expressed in response not only to a wide range of environmental pollutants, but also to biological stressors such as infectious pathogens. In bacterial-challenged abalone, the expression of sigma GST was increases in all time points with the peak expression at 48 h, and 45-fold increase was detected at this time point compared to control group [16]. In Chinese mitten crab, GST transcription was significantly induced in haemocytes at 6 h post-bacterial challenge and dropped to basal levels at 12 h [26]. To know the different function of different classes of GSTs in the microbial challenge of the clam, expression profiles of VpGST-1 and VpGST-2 in haemocytes after microorganism challenge were recorded by qT-PCR. In order to elinimate the effection of environmental disturbance on VpGST expression, the statistical difference among the control at different time points was firstly analyzed by one-way ANOVA and multiple Duncan test. No difference was detected in all time point of control samples (Fig. 2 and Fig. 3). Based on the analysis, non-parametric test was employed to compare challenged and control groups of each sampling time, and
Fig. 2. Time-course expression level of VpGST-1 transcript in haemocytes after Vibrio anguillarum challenge measured by quantitative real-time PCR at 0 h, 6 h, 12 h, 24 h, 48 h, 72 h and 96 h. Each symbol and vertical bar represented the mean S.D (n ¼ 3).
the results were also shown in Fig. 2 (for VpGST-1) and Fig. 3 (for VpGST-2), respectively. The expression level of VpGST-1 mRNA remained at control level from 6 h to 12 h. After that, the expression level was increased sharply from 12 h to 48 h with 31.5-fold increase at 72 h compared with their control group. Control and challenged groups compared analysis showed statistically significant difference in VpGST-1 gene expression at 6 h (P ¼ 0.0115), 24 h (P ¼ 0.0213), 48 h (P ¼ 0.0095) and 72 h (P ¼ 0.0087) postchallenge. However, no significant difference was observed in other time points of the challenge group. The expression profile of VpGST-2 was different from that of VpGST-1. The expression level of VpGST-2 was increased continuously during the first 48 h post-challenge and reached the maximum expression at 48 h with 164.2-fold increase compared with that in the control group. Then, the expression level of VpGST2 was sharply decreased from 72 h to 96 h. At 72 h, VpGST-2 mRNA level dropped greatly and only was 5.0-fold compared to its control. Statistical analysis with control and challenged groups indicated
Fig. 1. Consensus neighbour-joining tree based on the sequences of different classes of GSTs. The numbers at the forks indicated the bootstrap. The detail information of alignment sequences were as follows: 1) omega classes of GSTs: Crassostrea gigas (CAD89618), Drosophila melanogaster (ACZ02428), Haliotis discus-1 (ABO26600), Haliotis discus-2 (ABO26601), Takifugu rubripes (AAL08414); 2) pi classes of GSTs: Ruditapes philippinarum (ACM16805), Danio rerio (BAD98445), Mercenaria mercenaria (ABV29188), Laternula elliptica (ABV44413); 3) sigma classes of GSTs: Nototodarus sloanii (P46088), Drosophila melanogaster-S (NP_725653), Haliotis discus-S-1 (ABF67507), Haliotis discus-S-2 (ABO26602); 4) mu classes of GSTs: Cyphoma gibbosum (ABS32298), Crassostrea gigas-Mu (AJ558252), Thais clavigera (ACD13785).
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Fig. 3. Time-course expression level of VpGST-2 transcript in haemocytes after Vibrio anguillarum challenge measured by quantitative real-time PCR at 0 h, 6 h, 12 h, 24 h, 48 h, 72 h and 96 h. Each symbol and vertical bar represented the mean S.D (n ¼ 3).
that statistically significant differences in VpGST-2 gene expression were detected at 6 h, 12 h, 24 h, 48 h and 72 h with the P value of 0.0267, 0.0064, 0.0064, 0.0025, 0.0321, respectively. It had been reported that generation of reactive oxygen species (ROS) was one of the earliest cellular responses in aerobic organisms, following successful pathogen recognition [27]. As a major member of antioxidant defence system, GSTs should be involved into this anti-infection process. In the mosquito and Drosophila, the expression of GSTs was increased in response to oxidative stress, further suggesting its protective role against oxidative stress [28,29]. The up-regulation of VpGST-1 and VpGST-2 in the study was speculated to their protecting roles against oxidative stress caused by Vibrio stimulation. On the other hand, take into account of multiple physiological roles of GSTs, the conclusion should be further validated by analyzing whether ROS was produced in challenged clams, or clams haemocytes phagocytosed activity. Concerning to each type of VpGST, The different expression profiles like two isoforms of small HSP in our previous work [30] might be indicated that they were involved in bacterial challenge in different manners. To cope with microorganism entrance, VpGST-2 might be acted as the acute response protein and VpGST-1 as the late-stage one. The different expression profile between VpGST-1 and VpGST-2 from clam further elucidated different classes of GST family diversified their major role in evolution process. Acknowledgements This work was financially supported by Start Research Fund projects, K.C.Wong Magna Fund at Ningbo University, MEL Visiting Fellowship Program and partially supported by NSFC grant (31101919). References [1] Sheehan D, Meade G, Foley VM, Dowd CA. Structure, function and evolution of glutathione transferases: implications for classification of non-mammalian members of an ancient enzyme superfamily. Biochem J 2001;360:1e16. [2] Fan C, Zhang S, Liu Z, Li L, Luan J, Saren G. Identification and expression of a novel class of glutathione-S-transferase from amphioxus Branchiostoma belcheri with implications to the origin of vertebrate liver. Int J Biochem Cell Biol 2007;39:450e61. [3] Yang H, Zeng Q, Li E, Zhu S, Zhou X. Molecular cloning, expression and characterization of glutathione S-transferase from Mytilus edulis. Comp Biochem Physiol B 2004;139:175e82. [4] Wan Q, Whang I, Lee J. Molecular cloning and characterization of three sigma glutathione S-transferases from disk abalone (Haliotis discus discus). Comp Biochem Physiol B 2008;151:257e67.
[5] Wan Q, Whang I, Lee J, Lee J. Novel omega glutathione S-transferases in disk abalone: characterization and protective roles against environmental stress. Comp Biochem Physiol C 2009;150:558e68. [6] Dixon DP, Cummins L, Cole DJ, Edwards R. Glutathione-mediated detoxification systems in plants. Curr Opin Plant Biol 1998;1:258e66. [7] Pemble SE, Wardle AF, Taylor JB. Glutathione S-transferase class kappa: characterization by the cloning of rat mitochondrial GST and identification of a human homologue. Biochem J 1996;319:749e54. [8] Hayes JD, Flanagan JU, Jowsey IR. Glutathione transferases. Annu Rev Pharmacol Toxicol 2005;45:51e88. [9] Mannervik B, Alin P, Guthenberg C, Jensson H, Tahir MK, Warholm M, et al. Identification of three classes of cytosolic glutathione transferase common to several mammalian species: correlation between structural data and enzymatic properties. Proc Nat Acad Sci USA 1985;82:7202e6. [10] Boutet I, Tanguy A, Moraga D. Characterisation and expression of four mRNA sequences encoding glutathione S-transferases pi, mu, omega and sigma classes in the Pacific oyster Crassostrea gigas exposed to hydrocarbons and pesticides. Mar Biol 2004;146:53e64. [11] Doyen P, Bigot A, Vasseur P, Rodius F. Molecular cloning and expression study of pi-class glutathione S-transferase (pi-GST) and selenium-dependent glutathione peroxidase (Se-GPx) transcripts in the freshwater bivalve Dreissena polymorpha. Comp Biochem Physiol C 2008;147:69e77. [12] Feng X, Singh BR. Molecular identification of glutathione S-transferase gene and cDNAs of two isotypes from northern quahog (Mercenaria mercenaria). Comp Biochem Physiol B 2009;154:25e36. [13] Kim M, Ahn I, Cheon J, Park H. Molecular cloning and thermal stress-induced expression of a pi-class glutathione S-transferase (GST) in the Antarctic bivalve Laternula elliptica. Comp Biochem Physiol A 2009;152:207e13. [14] Rhee JS, Raisuddin S, Hwang DS, Horiguchi T, Cho H, Lee J. A mu-class glutathione S-transferase (GSTM) from the rock shell Thais clavigera. Comp Biochem Physiol C 2008;148:195e203. [15] Whalen KE, Morin D, Lin C, Tjeerdema RS, Goldstone JV, Hahn ME. Proteomic identification, cDNA cloning and enzymatic activity of glutathione S-transferases from the generalist marine gastropod, Cyphoma gibbosum. Arch Biochem Biophys 2008;478:7e17. [16] Ren H, Xu D, Gopalakrishnan S, Qiao K, Huang W, Wang K. Gene cloning of a sigma class glutathione S-transferase from abalone (Haliotis diversicolor) and expression analysis upon bacterial challenge. Dev Comp Immunol 2009;33: 980e90. [17] Contreras-Vergara CA, Harris-Valle C, Sotelo-Mundo RR, Yepiz-Plascencia G. A mu-class glutathione s-transferase from the marine shrimp Litopenaeus vannamei: molecular cloning and active-site structural modeling. J Biochem 2004;18(5):245e52. [18] Zhang G, Yan X. A new three-phase culture method for Manila clam, Ruditapes philippinarum, farming in northern China. Aquaculture 2006;258:452e61. [19] DiSalvo LH, Blecka J, Zebal R. Vibrio anguillarum and larval mortality in a California coastal shellfish hatchery. Appl Environ Microbiol 1978;35:219e21. [20] Olafsen JA, Mikkelsen HV, Giaever HM, Høvik Hansen G. Indigenous bacteria in hemolymph and tissues of marine bivalves at low temperatures. Appl Environ Microbiol 1993;59:1848e54. [21] Zhang H, Wang H, Wang L, Song L, Song X, Zhao J, et al. Cflec-4, a multidomain C-type lectin involved in immune defense of Zhikong scallop Chlamys farreri. Dev Comp Immunol 2009;33:780e8. [22] Zhao J, Li C, Chen A, Li L, Su X, Li T. Molecular characterization of a novel big defensin from clam Venerupis philippinarum. PLoS ONE 2010;5(10):e13480. doi:10.1371/journal.pone.0013480. [23] Zhang L, Zhao J, Li C, Su X, Chen A, Li T, et al. Cloning and characterization of allograft inflammatory factor-1 (AIF-1) from manila clam Venerupis philippinarum. Fish Shellfish Immunol 2011;30:148e53. [24] Thompson JD, Higgins DG, Gibson TJ. CLUSTALW: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 1994;22:4673e80. [25] Dirr H, Reinemer P, Huber R. X-ray crystal structures of cytosolic glutathione S-transferases. Implications for protein architecture, substrate recognition and catalytic function. Eur J Biochem 1994;220:645e61. [26] Zhao D, Chen L, Qin C, Zhang H, Wu P, Zhang F. A delta-class glutathione transferase from the Chinese mitten crab Eriocheir sinensis: cDNA cloning, characterization and mRNA expression. Fish Shellfish Immunol 2010;29: 698e703. [27] Chiu C, Guu Y, Liu C, Pan T, Cheng W. Immune responses and gene expression in white shrimp, Litopenaeus vannamei, induced by Lactobacillus plantarum. Fish Shellfish Immunol 2007;23:364e77. [28] Singh SP, Coronella JA, Benes H, Cochrane BJ, Zimniak P. Catalytic function of Drosophila melanogaster glutathione S-transferase DmGSTS1-1 (GST-2) in conjugation of lipid peroxidation end products. Eur J Biochem 2001;268: 2912e23. [29] Ding Y, Hawkes N, Meredith J, Eggleston P, Hemingway J, Ranson H. Characterization of the promoters of epsilon glutathione transferases in the mosquito Anopheles gambiae and their response to oxidative stress. Biochem J 2005;387:879e88. [30] Li C, Wang Li, Ning X, Chen A, Zhang L, Qin S, et al. Identification of two small heat shock proteins with different response profile to cadmium and pathogen stresses in Venerupis philippinarum. Cell Stress Chaperones 2010;15: 897e904.
Contents lists available at SciVerse ScienceDirect
Fish & Shellfish Immunology journal homepage: www.elsevier.com/locate/fsi
Short communication
Two classes of glutathione S-transferase genes with different response profiles to bacterial challenge in Venerupis philippinarum Chenghua Li a, *, Xiurong Su a, *, Ye Li a, Taiwu Li a, b, Chongjie Sun a, Tingting Zhou a, Haipeng Liu c a
Faculty of Life Science and Biotechnology, Ningbo University, 818 Fenghua Road, Ningbo, Zhejiang Province 315211, PR China Ningbo City College of Vocational Technology, Ningbo 315100, PR China c State Key Laboratory of Marine Environmental Science, Xiamen University, Xiamen 361005, PR China b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 4 August 2011 Received in revised form 20 October 2011 Accepted 21 October 2011 Available online 18 November 2011
Glutathione S-transferase (GST) is major cytosolic detoxification enzymes involved in many pathological and physiological processes. In the present study, two classes of GSTs (VpGST-1 and VpGST-2) were cloned from Venerupis philippinarum haemocytes by cDNA library and RACE approaches. Sequence alignments and phylogenetic analysis together supported that they belonged to a new member of sigma and pi classes GSTs protein family, respectively. The expression profiles of these two genes under Vibrio anguillarum challenge were investigated by quantitative RT-PCR. The bacterial challenge could significantly up-regulate the mRNA expression of both VpGST-1 and VpGST-2 with larger amplitude in VpGST2, and the feedback speed for VpGST-2 was more rapid than that of VpGST-1. The differences in the response to bacterial challenge indicated that they were functional diversity and probably played cooperative roles in mediating the Vibrio challenge in clam. Ó 2011 Elsevier Ltd. All rights reserved.
Keywords: Venerupis philippinarum Glutathione S-transferase Expression profile Vibrio anguillarum
1. Introduction Glutathione S-transferases (GST, EC 2.5.1.18) are composed of many cytosolic, mitochondrial, and microsomal proteins participating in the detoxification of reactive electrophilic compounds by catalyzing their conjugation to glutathione [1,2]. The vital functions of GSTs have been demonstrated to be promising indicators for environmental pollutants [3e5], and involved into elimination of intracellular reactive oxidative species (ROS) in marine organisms [6]. There are at least eight major classes of GSTs, designated alpha, kappa, mu, pi, sigma, omega, theta and zeta based on their substrate specificity, immunological properties and protein sequence identity in mammalian [1,7e9]. Some classes of GSTs have been identified and characterized in aquatic invertebrates include bivalve mollusks [3,10e13], gastropods [14e16] and shrimp [17]. China, with a thousand year history of Manila clam Venerupis philippinarum farming, produces about 1.8 million tonne of Manila clams annually, accounting for about 90% the total global production [18]. However, with the development of intensive culture and environmental deterioration, various diseases caused by bacteria, protozoa occurred in cultured V. philippinarum populations, resulted in enormous losses to the clam aquaculture. As a major
* Corresponding authors. Tel./fax: þ86 574 87608368. E-mail addresses: [email protected] (C. Li), [email protected] (X. Su). 1050-4648/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.fsi.2011.10.032
pathogen in global fishery, Vibrio anguillarum had been widely isolated from ocean environment and demonstrated to be pathogenicity on oysters and other bivalves [19e21]. Manila clams like other bivalves filtered large amounts of seawater to cope with nutritional and respiratory needs, leading to its continuously exposure to the microorganism. However, the pathogenicity of V. anguillarum on Manila clam has not been investigated to our knowledge. The purposes of this study were: 1) to clone full-length cDNAs of two classes of clam GSTs; 2) to investigate their mRNA expression under Vibrio challenge and provide basic data for the host response against the bacterial challenge.
2. Materials and methods 2.1. Clams and bacterial challenge The clams V. philippinarum (7.5e11 g in weight) were collected from Qingdao, Shandong Province, China. The clams were acclimated for a week before commencement of the experiment. The temperature was held at 20e22 C throughout the whole experiment. The salinity for the supplied seawater was kept at 30&. For V. anguillarum challenge experiment, the clams were cultured in seawater with high density of V. anguillarum (107 CFU mL1), and a group of unchallenged clams were used as control. The challenged clams were randomly sampled at 6 h, 12 h, 24 h, 48 h, 72 h and 96 h
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respectively. The haemolymphs from the control and the challenged groups were collected using a syringe and centrifuged at 2000 g, 4 C for 10 min to harvest the haemocytes. There were three replicates for each time point. 2.2. Cloning of the full-length cDNA of VpGST-1 and VpGST-2 by 50 RACE cDNA library construction, EST analysis, RNA isolation and cDNA synthesis were consistent with our published works [22,23]. Two sets of gene specific primers, P1 50 -CCATTTCCGAGAACATTTCCATC30 and P2 50 -ACACGGGACTGA TGCTCTTCGAG-30 for VpGST-1 and P3 50 -AAACCGGAAGCAGTGTATCGTCT- 30 and P4 50 -TTGAGGGTCGGTAATGCGTCTAT-30 for VpGST-2, were designed based on the two ESTs from cDNA library to clone the full-sequence cDNA of VpGST-1 and VpGST-2, respectively. Semi-nested PCR approaches were introduced to get the 50 end of the two genes with 1:100 dilutions of the first round PCR products as templates and oligodG as anchored primer. The PCR programs and PCR product sequencing were performed according to reference [23]. 2.3. Sequence analysis of VpGST-1 and VpGST-2 The VpGST-1 and VpGST-2 gene sequences were analyzed using the BLAST algorithm at NCBI web site (http://www.ncbi.nlm.nih. gov/blast), and the deduced amino acid sequence was predicted with the Expert Protein Analysis System (http://www.expasy.org/). For construction of phylogenetic tree, the deduced amino acid sequences of VpGST-1 and VpGST-2 were aligned with the corresponding sequences from various animals using the ClustalX software [24]. Based on this alignment, a NJ tree was constructed by Mega3.1 software package (http://www.megasoftware.net/). To derive the confidence value for the phylogeny analysis, bootstrap trials were replicated 1000 times. 2.4. Temporal expression profile of VpGST-1 and VpGST-2 mRNA in haemocytes post V. anguillarum challenge The expression profiles of VpGST-1 and VpGST-2 transcript in haemocytes after Vibrio challenge were measured by quantitative real-time PCR in Applied Biosystem 7500 fast Real-time PCR System. Two sets of gene specific primers, P5 50 -CATTGCCCGTGCTTACTATTGAC-30 and P6 50 -TGTTCCTTTCTTCCTCGTTAT CC-30 for VpGST-1 and P7 50 -GTATCCGAGCGATTTACAAGAGG-30 and P8 50 -CTTTGAGGGTCGGTAATGCGTCT-30 for VpGST-2, were designed to amplify products of 207 bp and 290 bp, respectively. Two clam b-actin primers, P9 50 CTCCCTTGAGAAGAGCTACGA-30 and P10 50 -GATACCAGCAGATTCCATA CCC-30 were used to amplify an 121 bp fragment as internal control to verify the successful reverse transcription and to calibrate the cDNA template. The reaction was performed in a total volume of 20 ml containing 10 ml of 2 SYBR Green Master Mix (Applied Biosystems), 4 ml of the diluted cDNA mix, 0.25 ml of each primer (10 mmol L1), 5.5 ml of DEPC-treated water. The thermal profile for real-time PCR was 50 C for 2 min and 95 C for 10 min followed by 45 cycles of 95 C for 15 s and 60 C for 1 min. Dissociation curve analysis of amplification products was performed at the end of each PCR reaction to confirm that only one PCR product was amplified and detected [22]. The 2DDCT method was used to analyze the expression level of VpGSTs. The Ct for the target amplified VpGSTs and the Ct for the internal control b-actin were determined for each sample. Difference in the Ct for the target and the internal control, called DCT, was calculated to normalize the differences in the amount of template and the efficiency of the RT-PCR. The DCT value for untreated sample at 0 h was used as the reference sample, called the calibrator. The DCT for each sample (including controls from other time points) was subtracted from the DCT of the
calibrator; the difference was called DDCT. The expression level of VpGSTs could be calculated by 2DDCT, and the value stood for an n-fold difference relative to the calibrator. All data were given in terms of relative mRNA expression as means S.D. The control data obtained from real-time PCR analysis were subjected to One-way Analysis of Variance (ANOVA) followed by multiple Duncan test to determine differences in the mean values among the controls. Then, nonparametric test was employed to compare challenged and control groups of each sampling time. Significant differences across control from each time point were indicated with an asterisk at P < 0.05 and two asterisks at P < 0.01. 3. Results and discussion 3.1. cDNA cloning and analysis of the VpGST-1 and VpGST-2 genes Blastx analysis indicated the two ESTs of 635 bp and 587 bp from clam cDNA library were similar to the sigma classes of GSTs from Haliotis discus discus (ABO26602) and pi classes of GSTs from Chlamys farreri (ACL80138), respectively. Based on these fragments, four gene specific primers (P1, P2, P3, and P4) were designed to clone the full-length cDNA of VpGST-1 and VpGST-2. A 330 bp fragment was produced by 50 RACE with primer P2 and oligodG for VpGST-1. Regarding to VpGST-2, the corresponding product was of 526 bp amplified with primer P4 and oligodG. By overlapping the fragments with respective EST, two sequences of 928 bp and 939 bp nucleotide sequence representing the full-length cDNA of VpGST-1 and VpGST-2 were assembled and deposited in GenBank under accession no. GQ384392 and GQ384393, respectively. The complete sequence of VpGST-1 cDNA contained a 50 untranslated region (UTR) of 32 bp, a 30 UTR of 263 bp with a polyA tail. Canonical polyadenylation signal sequence AATAAA was instead of ATTAAA. An open reading frame (ORF) of 633 bp encoded a polypeptide of 210 amino acids with the predicted molecular weight of 23.78 kDa and the theoretical isoelectric point of 5.32. The typical GST-N domain (G-site) was located from 4aa to 74aa, and GST-C (H-site) domain from 96aa to 204aa. Blastp search indicated the deduced amino acid of VpGST-1 showed weak similarity to other sigma classes of GSTs. The highest similarity was detected between VpGST-1 and its isoforms (ADI44317) in the same species with 39% identities. The similar phenomenon was consistent with other reports [4,16]. The lower similarity was attributed to its highest diversity of C-terminus. In Drosophila, the sigma GST exhibited a distinct N-terminal terminus with a proline/alaninerich extension [4], while the domain was absent from mollusk counterparts. To be mentioned, the central residue of Tyr (Y) for GSH stabilization in the sigma class GSTs was totally conserved in the deduced amino acid of VpGST-1. The full-length cDNA of VpGST-2 consisted of a 50 UTR of 8 bp, 0 a 3 UTR of 313 bp, and an ORF of 618 bp encoding a polypeptide of 205 amino acids with the predicted molecular weight of 23.93 kDa and the theoretical isoelectric point of 6.84. The typical GST-N and GST-C domain was located from 4aa to 73aa and from 94aa to 183aa, respectively. Search by Blastp indicated it had highest similarity to pi classes GSTs from other species, such as 91% to C. farreri (ACL80138), 74% to Mercenaria mercenaria (ABV29188), and 70% to Corbicula fluminea (AAX20374). Moreover, multiple alignment analysis revealed 11 highly conserved amino acid residues (F8, R14,W39,K45, Q52, L53, P54, Q65, S66,E98, D99) for G-site of GST [25] was almost totally conserved in the deduce amino acid of VpGST-2 with the exception of Ser, which was substituted for Leu53. This substitution has also been reported in Antarctic bivalve [13]. The conserved amino acid residues for H-site (F7, P8, V10, R13, V104, F108, N204, and G205) were also partially identified in VpGST-2 except absent from V10.
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3.2. Phylogenetic analysis To detect evolutionary relationships between different classes of GSTs and further elucidate the identities of VpGST-1 and VpGST-2, a NJ phylogenetic tree was re-constructed based on 18 amino acid sequences of four classes of GSTs including VpGST-1 and VpGST-2 (Fig. 1). Four classes of GSTs with different animal origins were clustered into four major branches in the tree according to their classification criterion. Pi classes of GSTs were firstly clustered with sigma ones, subsequently with mu and omega counterparts in turn. VpGST-1 was placed together into sigma classes of GSTs, indicating it belonged to a new member of this protein family. VpGST-2 was clustered with pi classes GSTs. From this tree, we could draw the conclusion that VpGST-1 and VpGST-2 belonged to a distinctive category in GSTs protein family. 3.3. The expression profile of VpGST-1 and VpGST-2 after Vibrio challenge As antioxidant proteins, GSTs are expressed in response not only to a wide range of environmental pollutants, but also to biological stressors such as infectious pathogens. In bacterial-challenged abalone, the expression of sigma GST was increases in all time points with the peak expression at 48 h, and 45-fold increase was detected at this time point compared to control group [16]. In Chinese mitten crab, GST transcription was significantly induced in haemocytes at 6 h post-bacterial challenge and dropped to basal levels at 12 h [26]. To know the different function of different classes of GSTs in the microbial challenge of the clam, expression profiles of VpGST-1 and VpGST-2 in haemocytes after microorganism challenge were recorded by qT-PCR. In order to elinimate the effection of environmental disturbance on VpGST expression, the statistical difference among the control at different time points was firstly analyzed by one-way ANOVA and multiple Duncan test. No difference was detected in all time point of control samples (Fig. 2 and Fig. 3). Based on the analysis, non-parametric test was employed to compare challenged and control groups of each sampling time, and
Fig. 2. Time-course expression level of VpGST-1 transcript in haemocytes after Vibrio anguillarum challenge measured by quantitative real-time PCR at 0 h, 6 h, 12 h, 24 h, 48 h, 72 h and 96 h. Each symbol and vertical bar represented the mean S.D (n ¼ 3).
the results were also shown in Fig. 2 (for VpGST-1) and Fig. 3 (for VpGST-2), respectively. The expression level of VpGST-1 mRNA remained at control level from 6 h to 12 h. After that, the expression level was increased sharply from 12 h to 48 h with 31.5-fold increase at 72 h compared with their control group. Control and challenged groups compared analysis showed statistically significant difference in VpGST-1 gene expression at 6 h (P ¼ 0.0115), 24 h (P ¼ 0.0213), 48 h (P ¼ 0.0095) and 72 h (P ¼ 0.0087) postchallenge. However, no significant difference was observed in other time points of the challenge group. The expression profile of VpGST-2 was different from that of VpGST-1. The expression level of VpGST-2 was increased continuously during the first 48 h post-challenge and reached the maximum expression at 48 h with 164.2-fold increase compared with that in the control group. Then, the expression level of VpGST2 was sharply decreased from 72 h to 96 h. At 72 h, VpGST-2 mRNA level dropped greatly and only was 5.0-fold compared to its control. Statistical analysis with control and challenged groups indicated
Fig. 1. Consensus neighbour-joining tree based on the sequences of different classes of GSTs. The numbers at the forks indicated the bootstrap. The detail information of alignment sequences were as follows: 1) omega classes of GSTs: Crassostrea gigas (CAD89618), Drosophila melanogaster (ACZ02428), Haliotis discus-1 (ABO26600), Haliotis discus-2 (ABO26601), Takifugu rubripes (AAL08414); 2) pi classes of GSTs: Ruditapes philippinarum (ACM16805), Danio rerio (BAD98445), Mercenaria mercenaria (ABV29188), Laternula elliptica (ABV44413); 3) sigma classes of GSTs: Nototodarus sloanii (P46088), Drosophila melanogaster-S (NP_725653), Haliotis discus-S-1 (ABF67507), Haliotis discus-S-2 (ABO26602); 4) mu classes of GSTs: Cyphoma gibbosum (ABS32298), Crassostrea gigas-Mu (AJ558252), Thais clavigera (ACD13785).
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Fig. 3. Time-course expression level of VpGST-2 transcript in haemocytes after Vibrio anguillarum challenge measured by quantitative real-time PCR at 0 h, 6 h, 12 h, 24 h, 48 h, 72 h and 96 h. Each symbol and vertical bar represented the mean S.D (n ¼ 3).
that statistically significant differences in VpGST-2 gene expression were detected at 6 h, 12 h, 24 h, 48 h and 72 h with the P value of 0.0267, 0.0064, 0.0064, 0.0025, 0.0321, respectively. It had been reported that generation of reactive oxygen species (ROS) was one of the earliest cellular responses in aerobic organisms, following successful pathogen recognition [27]. As a major member of antioxidant defence system, GSTs should be involved into this anti-infection process. In the mosquito and Drosophila, the expression of GSTs was increased in response to oxidative stress, further suggesting its protective role against oxidative stress [28,29]. The up-regulation of VpGST-1 and VpGST-2 in the study was speculated to their protecting roles against oxidative stress caused by Vibrio stimulation. On the other hand, take into account of multiple physiological roles of GSTs, the conclusion should be further validated by analyzing whether ROS was produced in challenged clams, or clams haemocytes phagocytosed activity. Concerning to each type of VpGST, The different expression profiles like two isoforms of small HSP in our previous work [30] might be indicated that they were involved in bacterial challenge in different manners. To cope with microorganism entrance, VpGST-2 might be acted as the acute response protein and VpGST-1 as the late-stage one. The different expression profile between VpGST-1 and VpGST-2 from clam further elucidated different classes of GST family diversified their major role in evolution process. Acknowledgements This work was financially supported by Start Research Fund projects, K.C.Wong Magna Fund at Ningbo University, MEL Visiting Fellowship Program and partially supported by NSFC grant (31101919). References [1] Sheehan D, Meade G, Foley VM, Dowd CA. Structure, function and evolution of glutathione transferases: implications for classification of non-mammalian members of an ancient enzyme superfamily. Biochem J 2001;360:1e16. [2] Fan C, Zhang S, Liu Z, Li L, Luan J, Saren G. Identification and expression of a novel class of glutathione-S-transferase from amphioxus Branchiostoma belcheri with implications to the origin of vertebrate liver. Int J Biochem Cell Biol 2007;39:450e61. [3] Yang H, Zeng Q, Li E, Zhu S, Zhou X. Molecular cloning, expression and characterization of glutathione S-transferase from Mytilus edulis. Comp Biochem Physiol B 2004;139:175e82. [4] Wan Q, Whang I, Lee J. Molecular cloning and characterization of three sigma glutathione S-transferases from disk abalone (Haliotis discus discus). Comp Biochem Physiol B 2008;151:257e67.
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