A novel murrel Channa striatus mitochondrial manganese superoxide dismutase: gene silencing, SOD activity, superoxide anion production and expression.

Fish Physiol Biochem (2014) 40:1937–1955 DOI 10.1007/s10695-014-9981-0

A novel murrel Channa striatus mitochondrial manganese superoxide dismutase: gene silencing, SOD activity, superoxide anion production and expression Jesu Arockiaraj • Rajesh Palanisamy • Prasanth Bhatt Venkatesh Kumaresan • Annie J. Gnanam • Mukesh Pasupuleti • Marimuthu Kasi

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Received: 2 January 2014 / Accepted: 26 August 2014 / Published online: 3 September 2014 Ó Springer Science+Business Media Dordrecht 2014

Abstract We have reported the molecular characterization including gene silencing, superoxide activity, superoxide anion production, gene expression and molecular characterization of a mitochondrial manganese superoxide dismutase (mMnSOD) from striped murrel Channa striatus (named as CsmMnSOD). The CsmMnSOD polypeptide contains 225 amino acids with a molecular weight of 25 kDa and a theoretical isoelectric point of 8.3. In the N-terminal region,

J. Arockiaraj (&) R. Palanisamy P. Bhatt V. Kumaresan Division of Fisheries Biotechnology and Molecular Biology, Department of Biotechnology, Faculty of Science and Humanities, SRM University, Kattankulathur, Chennai 603 203, Tamil Nadu, India e-mail: [email protected] A. J. Gnanam Institute for Cellular and Molecular Biology, The University of Texas at Austin, 1 University Station A4800, Austin, TX 78712, USA M. Pasupuleti Lab PCN 206, Microbiology Division, CSIR-Central Drug Research Institute, B.S. 10/1, Sector 10, Jankipuram Extension, Sitapur Road, Lucknow 226031, Uttar Pradesh, India M. Kasi Department of Biotechnology, Faculty of Applied Sciences, AIMST University, Semeling Bedong, 08100 Bedong, Kedah, Malaysia

CsmMnSOD carries a mitochondrial targeting sequence and a superoxide dismutases (SOD) Fe domain (28–109), and in C-terminal region, it carries another SOD Fe domain (114–220). The CsmMnSOD protein sequence shared significant similarity with its homolog of MnSOD from rock bream Oplegnathus fasciatus (96 %). The phylogenetic analysis showed that the CsmMnSOD fell in the clade of fish mMnSOD group. The monomeric structure of CsmMnSOD possesses 9 a-helices (52.4 %), 3 b-sheets (8.8 %) and 38.8 % random coils. The highest gene expression was noticed in liver, and its expression was inducted with fungal (Aphanomyces invadans) and bacterial (Aeromonas hydrophila) infections. The gene silencing results show that the fish that received dsRNA exhibited significant (P \ 0.05) changes in expression when compared to their non-injected and fish physiological saline-injected controls. The SOD activity shows that the activity increases with the spread of infection and decreases once the molecule controls the pathogen. The capacity of superoxide anion production was determined by calculating the granular blood cell count during infection in murrel. It shows that the infection influenced the superoxide radical production which plays a major role in killing the pathogens. Overall, this study indicated the defense potentiality of CsmMnSOD; however, further research is necessary to explore its capability at protein level. Keywords Channa striatus MnSOD Gene silencing SOD activity Superoxide production

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Abbreviations CsmMnSOD Channa striatus mitochondrial manganese superoxide dismutase ROS Reactive oxygen species –OH Hydroxyl radical H2O2 Hydrogen peroxide O 2Superoxide radical Cu/ZnSOD Copper/zinc SOD FeSOD Iron SOD NiSOD Nickel SOD MTS Mitochondrial translocation sequence EUS Epizootic ulcerative syndrome MTP Mitochondrial targeting peptide FPS Fish physiological saline

Introduction The reactive oxygen species (ROS) such as hydroxyl radical (–OH), hydrogen peroxide (H2O2) and superoxide radical (O2-) are generated as by-products of aerobic metabolism which is necessary for energy production. ROS generation is induced by either endogenous or exogenous sources (Winston and Di Giulio 1991). They are shown to be cellular beneficial by acting as secondary messengers in signaling pathway, elimination of invading viruses, bacteria, fungi and protozoa through the activation of the respiratory burst and in regulation of a variety of cellular activities (Schreck et al. 1991; Anderson 1994; Roch 1999; Hensley et al. 2000). When ROS level goes beyond the threshold level of antioxidant system, it tends to cause oxidative damage to many cellular components such as lipids, proteins and nucleic acids due to alteration of the cellular redox homeostasis (Lesser 2006). To protect their cellular components from such oxidative damages, oxygenrespiring organisms are furnished with various enzymatic and non-enzymatic antioxidant systems to compensate and detoxify the elevated oxy-radicals (Mu et al. 2012). The cells contain various antioxidant systems that include superoxide dismutase, catalase and glutathione peroxidases (Apel and Hirt 2004). The superoxide dismutase (SOD) is one of the vital antioxidant enzymes which is the first-line defense mechanism for protecting all the aerobic life from the detrimental oxidative damage of ROS by means of catalytically converting the O2- into O2 and H2O2 (Fridovich 1995). SODs are widely present in both

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Fish Physiol Biochem (2014) 40:1937–1955

prokaryotes and eukaryotes. It is classified into four major types on the basis of their metal–ligand pattern such as copper/zinc SOD (Cu/ZnSOD), manganese SOD (MnSOD), iron SOD (FeSOD) and nickel SOD (NiSOD). Though the basic role of all SODs is same, they significantly varied in their genomic and proteomic structural features and subcellular distribution (Bannister et al. 1987; Zelko et al. 2002). MnSOD was initially identified, isolated and characterized from both prokaryotic and eukaryotic mitochondria (Zelko et al. 2002; Ken et al. 2005). It is a homodimeric or tetrameric enzyme bound to Mn through its active site (Holley et al. 2011). So far, two types of MnSOD have been reported from eukaryotic cells; they are mitochondrial MnSOD (mMnSOD) that harbors a mitochondrial translocation sequence (MTS) and cytosolic MnSOD (cMnSOD) that lacks the MTS (Brouwer et al. 1997). Both MnSODs are synthesized as precursor protein in eukaryotic cytoplasm, and cMnSOD is retained in cytosol, but the mMnSOD transported into the mitochondrial matrix (MM) with the help of MTS. The mMnSOD is entailed in scavenging superoxide radicals which is synthesized locally in mitochondria (Bannister et al. 1987). The early investigations indicated that the overexpressed recombinant mMnSOD protein enhances the life span of adult Drosophila melanogaster (Sun et al. 2002) and diminishes oxidative stress in cells (Greenberger et al. 2003; Greenberger and Epperly 2004). Kim et al. (2010) and Wang et al. (2010) reported that the MnSOD induced the immune response against heat, coldness, starvation and heavy metals. Under the normal physiological circumstances, the basal expression of MnSOD tackles the required level of ROS in cells by holding the balance between synthesis and scavenging. The variation in transcription of MnSOD against endotoxic shock (Abe et al. 1995) and pathogens (Zhang et al. 2007; Bao et al. 2008; Cho et al. 2009) indicates that the MnSOD protects the host cells from ROS induced by pathogens or stress during phagocytosis. Channa striatus, an air-breathing freshwater fish, is a favorite food fish in many parts of India due to its delicious flesh. Moreover, it fetches good market due to its medicinal values. This species is also widely cultured in China and Southeast Asian Countries. The improper management of murrel farming activities has lead to various kinds of diseases, especially epizootic ulcerative syndrome (EUS), a fungus Aphanomyces


invadans (primary causative agent) and bacterial Aeromonas hydrophila (secondary causative agent) causing disease. Thus, research into murrel immune system is imperative to develop disease control strategies. So far, a number of MnSOD orthologs have been characterized from mammals (Ho et al. 1991; Wan et al. 1994; Meyrick and Magnuson 1994; Jones et al. 1995), fish (Lin et al. 2009; Zhang et al. 2011) and invertebrates (Gomez-Anduro et al. 2006; Bao et al. 2008; Lin et al. 2010; Yu et al. 2011). However, reports on the mechanisms of transcriptional regulation of MnSOD from fish are fewer than from mammals (Yeh et al. 1998; Xu et al. 2002; Clair et al. 2002), and from striped murrel Channa striatus, the details are nil. Hence, it is necessary to study the characterization of mMnSOD from C. striatus at molecular level to understand its immune system. In this study, we reported the CsmMnSOD gene silencing for better understanding of its functions. To obtain insights into the redox status in MnSODregulated stress tolerance, double-stranded RNA (dsRNA)-mediated RNA interference (RNAi) was used to determine whether CsmMnSOD is involved in the antioxidant system. Further, we examined the changes in O2- accumulation and transcripts of stress responses in C. striatus after injection of the dsRNA. Moreover, we focused to evaluate the SOD activity, superoxide anion production, gene expression and molecular characterization of mMnSOD from C. striatus (named as CsmMnSOD).

Materials and method Striped murrel Healthy fish (70 ± 6 g) were obtained from a commercial fish farm, Tirunelveli, Tamil Nadu, India. The fishes were transported to the laboratory in oxygenated polythene bags. They were maintained in 15 flatbottomed plastic containers (150 L) with aerated and filtered dechlorinated freshwater. The water quality was maintained as reported in our earlier reports (Arockiaraj et al. 2003; Dhanaraj et al. 2008). The fishes were acclimatized for a week before being injected with immune stimulants. A maximum of 10 fish per tank were maintained during the experiment. During acclimatization period, the fishes were fed ad libitum two times daily at 0900 and 1600 hours with

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a commercially available fish feed (Cargill Animal Nutrition, Andhra Pradesh, India). RNA extraction and cDNA synthesis Total RNA was extracted using Tri ReagentTM (Life Technologies, India) as suggested by the manufacturer with slight modifications (Arockiaraj et al. 2011 and 2012). Using 2.5 lg of RNA, the cDNA was synthesized using a SuperScriptÒ VILOTM cDNA Synthesis Kit (Life technologies) as suggested by the manufacturer with slight modifications (Abirami et al. 2013; Arockiaraj et al. 2013a). The isolated cDNA was stored at -20 °C for further analysis. Construction of normalized striped murrel cDNA library and identification of CsmMnSOD The striped murrel cDNA library was established using total RNA isolated from spleen, liver, kidney, muscle and gills of striped murrel. In our earlier studies (Abirami et al. 2013; Arockiaraj et al. 2013b), we reported the construction of striped murrel cDNA library. With the constructed library, the sequences were screened and a cDNA sequence encoding CsmMnSOD was identified. The sequence was obtained from the library by internal sequencing using ABI Prism-Bigdye Terminator Cycle Sequencing Ready Reaction kit. The following primers were used for the internal sequencing: CsmMnSOD F1, ATG CTG TGC AGA GTT GGT CAA ATA CGC AGG and CsmMnSOD R2, GAG CGA GCG TCT CCA GAG TGC CAA AAA G. The obtained sequence and its coding region were confirmed using ABI 3730 sequencer (Sambrook et al. 1989; Arockiaraj et al. 2012). Moreover, the obtained CsmMnSOD cDNA sequence including its 50 and 30 untranslated region (UTR), coding region and polypeptide sequences were analyzed on DNAssist 2.2 (Patterton and Graves 2000). Sequence analysis The CsmMnSOD sequence was analyzed on BLAST program (http://blast.ncbi.nlm.nih.gov/Blast) to find out its homologous sequences. Domains and motifs of CsmMnSOD were determined using Pfam database (http://pfam.sanger.ac.uk/). The presence of the N-terminal pre-sequences such as mitochondrial

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targeting peptide (MTP) or secretory pathway signal peptide (SP) was predicted using Target P 1.1 (http:// www.cbs.dtu.dk/services/TargetP/) program, a prediction program to study the subcellular location of eukaryotic proteins. Further, we also used MITOPROT server (http://ihg.gsf.de/ihg/mitoprot.html) to predict the mitochondrial targeting sequence (MTS) of CsmMnSOD. Multiple sequence alignment was constructed on ClustalW 2.0 program (http://www.ebi.ac. uk/Tools/msa/clustalw2/), and then, the constructed alignment was edited using Bioedit 7.1.3.0 software. To know the evolutionary relationship of CsmMnSOD with its homologs, a phylogenetic tree was constructed using neighbor-joining method on MEGA 5.05. The genetic distance was calculated using the Poisson correction method (Uinuk-Ool et al. 2003). The CsmMnSOD protein secondary structure was predicted using SOPMA program, and the structure was analyzed through PolyView program (http://polyview. cchmc.org). The homodimeric and monomeric structures of CsmMnSOD were established using SWISSMODEL protein modeling server (http://swissmodel. expasy.org/) and I-TASSER program (http://zhanglab. ccmb.med.umich.edu/I-TASSER), respectively. Disease challenge and tissue collection The relative expression of CsmMnSOD gene was quantified in healthy and disease-challenged striped murrel tissues using real-time PCR. To quantify the relative expression of CsmMnSOD gene during diseased state, the striped murrel were infected with fungus and bacteria through intraperitoneal injection. For fungal infection, 70 g fish were injected with 150 lL of A. invandans at a concentration of 103 spores. In our earlier findings (Abirami et al. 2013; Arockiaraj et al. 2013a), we described the isolation of A. invadans from EUS-infected muscle, culture in the laboratory and injection to the fishes. For bacterial challenge, the fish was injected with A. hydrophila (6 9 106 CFU/mL) suspended in 19 fish physiological saline (FPS preparation: 8 g NaCl, 0.2 g KCl, 1.44 g Na2HPO4 and 0.24 g KH2PO4 in 800 mL distilled H2O, adjust the pH to 7.4 by adding HCl, make up the volume to 1 L by adding distilled H2O and then sterilize the mixture by autoclaving before using) (150 lL/fish). A. hydrophila was also isolated from the muscle of C. striatus infected with EUS. The

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isolated bacteria were cultured in the laboratory and injected to the fishes as explained by Dhanaraj et al. (2008). Equal volume (150 lL/fish) of 19 FPS was injected to the control individuals. Tissue samples (blood, liver, spleen, kidney, muscle and gill) were collected before (0 h) and after injection (3, 6, 12, 24, 48 and 72 h), immediately snap-frozen in liquid nitrogen and stored at -80 °C until the total RNA was isolated. Using a sterilized syringe, blood (0.5–1.0 mL per fish) was collected from the caudal fin region of the fish and immediately centrifuged at 4,0009g for 10 min at 4 °C to allow blood cell collection for total RNA extraction. Five fishes were used for each experiment at various time points. Quantitative real-time PCR analysis The relative expression of CsmMnSOD gene in different tissues was quantified using qRT-PCR. The quantification was carried out using a ABI 7500 Realtime Detection System (Applied Biosystems, India) in 20 lL reaction volume containing 5 lL of cDNA from each tissue, 8 lL of Fast SYBRÒ Green Master Mix, 1 lL of each primer (20 pmol/lL) (CsmMnSOD F3, CCA GCC TCA GCC AAA CTA TAA and CsmMnSOD R4, TCC AGG GCA CCA TAA TCA TAA G) and 5 lL dH2O. The qRT-PCR cycle profile was 1 cycle of 95 °C for 10 s, followed by 35 cycles of 95 °C for 5 s, 58 °C for 10 s and 72 °C for 20 s and finally 1 cycle of 95 °C for 15 s, 60 °C for 30 s and 95 °C for 15 s. The same qRT-PCR cycle profile was used for the housekeeping gene, b-actin. b-actins of C. striatus primers (b-actin F5, TCT TCC AGC CTT CCT TCC TTG GTA and b-actin R6, GAC GTC GCA CTT CAT GAT GCT GTT) were designed from the sequence obtained from NCBI database (GenBank accession ID. EU570219). The amplification sizes of both CsmMnSOD gene and b-actin are 87 bp and 80 bp, respectively. After the PCR program, data were analyzed with ABI 7500 SDS software (Applied Biosystems). To maintain the consistency, the baseline was set automatically by the software. The comparative CT method (2-DDCT method) was used to analyze the expression level of CsmMnSOD (Livak and Schmittgenm 2001). All samples were analyzed in three duplications, and the results are expressed as relative fold of one sample as mean ± standard deviation.


Double-stranded RNAs (dsRNAs) synthesis The dsRNAs were synthesized as instructed by Amparyup et al. (2009) with slight modifications. In brief, to synthesis the dsRNAs of CsmMnSOD, DNA fragments of CsmMnSOD (258 nucleotides) were amplified by PCR using the following primers: CsmMnSOD F7, GGG CAC ATT AAC CAC ACT ATC T and CsmMnSOD R8, GAG AGG AAT GAG ACC TGT TGT G. T7 RNA polymerase was used to produce sense and antisense RNA strands by in vitro transcription. The sense and antisense DNA templates containing the T7 promoter sequence at the 50 end on each strand were produced by PCR using oligonucleotide primers containing the T7 promoter RNA polymerase recognition sequence at the 50 end as follows: CsmMnSODT7 F9, GGA TCC TAA TAC GAC TCA CTA TAG GGG GCA CAT TAA CCA CAC TAT CT and CsmMnSODT7 R10, GGA TCC TAA TAC GAC TCA CTA TAG GGA GAG GAA TGA GAC CTG TTG TG. For the negative control, DNA template amplification was performed on the green fluorescent protein (GFP) gene of pEGFP-1 vector (Clontech, India) using the following primers: GFPT7 F11, TAA TAC GAC TCA CTA TAG GAT GGT GAG CAA GGG CGA GGA and GFP R12, TTA CTT GTA CAG CTC GTC CA for the sense strand template and GFP F13, ATG GTG AGC AAG GGC GAG GA and GFPT7 R14, TAA TAC GAC TCA CTA TAG GTT ACT TGT ACA GCT CGT CCA for the antisense strand template. According to the methodology of Amparyup et al. (2009), we used T7 RiboMAXTM Express Large Scale RNA Production Systems (Promega, India) to synthesize RNA by in vitro transcription. The obtained dsRNAs were determined using a UV spectrophotometer (Nano View). Injection of dsRNAs to striped murrel The prepared dsRNAs were injected to the fishes using a 500-lL insulin syringe (22-gauge needle). In brief, various concentrations (10, 25 and 50 lg) of CsmMnSOD dsRNA or 10 lg GFP (as negative control) dsRNAs dissolved in 100 lL of 19 FPS were injected intramuscularly. The control individuals also received similar quantity of 19 FPS. Tissues were collected from the fishes after 24 h post-injection (p.i) of CsmMnSOD dsRNA, immediately frozen in liquid

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nitrogen and stored at -80 °C until the total RNA and cDNA were isolated. The synthesized cDNA was used as a template and the expression level analyzed using real-time PCR. Three replicates were used for each treatment. SOD activity assay The assay was performed as described by GarcıáTriana et al. (2010) with slight modifications. The activity was measured by utilizing xanthine and xanthine oxidase (XO) to produce superoxide radicals which react with 2-(4-iodophenyl)-3-(4-nitrophenol)5-phenyltetrazolium chloride (INT) to form a red formazan dye. The activity was determined by the level of inhibition of the reaction. SOD assay kit (Sigma, India) was used to determine the activity. Briefly, the tissue samples (50 mg, n = 3) were homogenized in 100 lL of 19 FPS. Then, the aqueous solution was separated by centrifugation at 12,0009g for 12 min in 4 °C. In a 96-wells microplate, 5 lL of the aqueous solution from the tissue samples were mixed with 85 lL of xanthine substrate (pH = 10.2). The substrate contained 0.05 mmol/L xanthine, 0.025 mmol/L INT, 40 mmol/L 3-(cyclohexylamino)-1-propanesulfonic acid and 0.94 mmol/ L ethylenediaminetetraacetic acid. To this mixture, a 12.5 lL of XO (80 U/L) was added. This reaction mixture was gently mixed and incubated for 30 s at room temperature. After incubation, the optical density (OD) was measured at 490 nm using a microplate reader (Thermo scientific). The SOD activity was presented as the difference in OD after 3 min of reaction per mg of protein. Protein concentration was measured using a Bradford protein assay kit (Bio Rad, India) as suggested by the manufacturer. The unit of SOD activity was presented as the amount of enzyme that causes an absorbance change of 0.001/min/mg protein. The activity was performed in three replications. Strength of CsmMnSOD superoxide anion generation The study was performed using a nitroblue tetrazolium (NBT) reduction assay as explained by Munoz et al. (2000) with slight modifications. In brief, blood (0.5 mL, n = 3) was collected from the caudal fin region of the fish using a 1-mL disposable syringe.

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Before collection of blood, the syringe was rinsed with isotonic anticoagulant solution (Alsever’s solution). In a microplate well, 100 lL of blood was taken and an equal volume of Hank’s solution was added into the well. Then, the reaction was induced by adding 100 lL of laminarin (2 mg/mL) followed by 100 lL of NBT (0.3 %) into the mixture. A non-stimulated treatment was also experimented. The reaction mixture was incubated for 30 min at room temperature. After incubation, the mixture was washed with 100 % methanol (400 lL), followed by 70 % methanol (400 lL) and dried in the room temperature for 2 min. Then finally, 240 lL of potassium hydroxide (2 M) and 280 lL of dimethyl sulfoxide were also added into the mixture, and the OD was read at 650 nm using a microplate reader (Thermo scientific, India). The superoxide radical production capacity was determined by calculating the difference between the stimulated and non-stimulated samples. The activity was performed on healthy as well as fungaland bacterial-infected individuals as reported elsewhere. Statistics For comparison of gene silencing, SOD activity, superoxide anion production and gene expression, statistical analysis was performed using one-way ANOVA and the mean comparisons were performed by Tukey’s multiple range test using SPSS 11.5 at the 5 % significance level.

Results Bioinformatics characterization A cDNA encoding mitochondrial MnSOD was identified during screening from the constructed striped murrel cDNA library using GS-FLXTM technique. The sequence was obtained by internal sequencing and deposited in EMBL GenBank database under the accession number HF674398. The nucleotide is 678 base pair (bp) in length that comprises a 675-bp open reading frame (ORF). The ORF encodes a polypeptide of 225 amino acids which is 25 kDa in molecular weight and has a theoretical isoelectric point of 8.3. The TargetP (version 1.1) analysis showed the presence of MTS at N-terminal of CsmMnSOD

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Fish Physiol Biochem (2014) 40:1937–1955 Fig. 1 Nucleotide and deduced amino acid of C. striatus c mitochondrial MnSOD. a Domain architecture of CsmMnSOD. The CsmMnSOD harbors a mitochondrial translocation sequence (MTS) domain, consists of 27 residues which is highlighted in yellow color and two superoxide dismutase (SOD) Fe domains (highlighted in blue shade) which contains three MnSOD signature motifs (highlighted in violet shade). Four putative amino acid residues (His53, His101, Asp186 and His190) which are responsible for manganese binding (Mn-BS) are given. b Nucleotide and their corresponding amino acid residues of CsmMnSOD. The CsmMnSOD consists of a putative MTS region which is shown in brown shade and three MnSOD signature motifs indicated in blue shade. The residues responsible for Mn binding are circled. The start codon (ATG) and the stop codon (TAG) are boxed and shown in red color. Potential N-glycosylation sites are depicted by italic and double underline. The secondary structure elements (a-helices: a1, a2, a3, a4, a5, a6, a7, a8 and a9 and b-sheets: b1, b2 and b3) that acquired from the tertiary structure of human MnSOD (PDB ID: 2adqB) are shown. (Color figure online)

protein between 1 and 27. Moreover, the MITOPROT program analysis also indicated that the CsmMnSOD protein was localized in mitochondria. Hence, it is confirmed that the obtained sequence CsmMnSOD is a mitochondrial MnSOD. The difference between mitochondrial MnSOD and cytosolic MnSODs (cMnSOD) is that cMnSOD contains a conserved N-terminal extension that is not present in mMnSOD, instead it contains 27 residues of MTS region. The Pfam domain search program showed the presence of two conserved SOD Fe domain at N (28–109) and C (114–220) terminals (Fig. 1a). Further analysis indicated that these two SOD Fe domains carry three potential MnSOD family signatures at 93 Phe-Asn-Gly-Gly-Gly-His-Leu-Asn-His101, 145ValGln-Gly-Ser-Gly-Trp-Gly-Trp-Leu-Gly154 and 186Aspval-Trp-Glu-His-Ala-Tyr-Tyr193. Moreover, these conserved SOD Fe domains contain four putative Mn2?binding residues at N (H53 and H101) and C (D186 and H190) terminals (Fig. 1b). There are four potential N-glycosylation sites observed in the CsmMnSOD at 66–69, 100–103, 107–110 and 215–218 which was predicted using NetNGlyc (version 1.0) server program. Homology analysis The homology analysis of CsmMnSOD was conducted using BLASTP program. The CsmMnSOD protein sequence shared significant homology with its homologs of MnSOD from O. fasciatus (96 %) followed by Sparus aurata (95 %), Rachycentran


canadum (95 %), Gallus gallus (82 %), Xenopus laevis (78 %), Mus musculus (78 %) and Macrobrachium rosenbergii (74 %) (data not shown).

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Multiple sequence alignment showed that the CsmMnSOD signature motifs and the amino acid residues which are responsible for Mn2?-binding ions are

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Fig. 2 Multiple sequence alignment of CsmMnSOD along with its orthologs which are as follows: Of-MnSOD, rock bream Oplegnathus fasciatus (AFO64916), Sa-MnSOD, gilthead sea bream Sparus aurata (AFV39807), Rc-MnSOD, cobia Rachycentran canadum (ABC71306), Mr-MnSOD, freshwater prawn Machrobrachium rosenbergii (ABB05539). Xl-MnSOD, African clawed frog (Xenopus laevis (NP_001083968), Gg-MnSOD, red jungle fowl Gallus gallus (NP_989542), and Mm-MnSOD, house mouse Mus musculus (AAB60902). The CsmMnSOD-

containing MTS region is boxed. The CsmMnSOD signatures motifs (DVWEHAYY, FNGGGHINH and VQGSGWGWLGY) are highlighted in pink color. The conserved sequences are highlighted in black shade. The amino acid residues responsible for Mn2?-binding ions are indicated in asterisk. The secondshell residues (His57, Tyr61, Trp150, Gln170 and Trp188) that are responsible for stabilizing the active site are shown in down arrow mark. (Color figure online)

highly conserved among the sequences taken for analysis. Moreover, the second-shell residues (His57, Tyr61, Trp150, Gln170 and Trp188) that are responsible for stabilizing the active site are also highly conserved among the sequences considered for analysis (Fig. 2).

Phylogenetic tree

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A phylogenetic tree of CsmMnSOD was constructed along with its homologs using MEGA 5.05. The tree exactly clustered into two separate groups including


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Fig. 3 Consensus neighbor-joining phylogenetic tree of CsmMnSOD along with other homologous sequences. The tree was constructed using Mega 5.05. The bootstrap confidence values given at the forks of the tree are based on 5,000 bootstrap replications

mMnSOD and cMnSOD. Further, the mMnSOD formed two groups of vertebrates and invertebrates, wherein CsmMnSOD located within fish monophyletic clade of vertebrate mMnSOD group. The CsmMnSOD was closely related to its ortholog from rock bream O. fasciatus (Fig. 3). MnSOD of Triticum aestivum and zea mays was set as out-group. Based on the bioinformatics and phylogenetic analysis, it is confirmed that the characterized sequence from C. striatus is a mitochondrial MnSOD.

template. The identity between the template and CsmMnSOD is 86.4 % (Fig. 4a). A monomeric model was also constructed using I-TASSER server (Fig. 4b). The analysis indicated that the CsmMnSOD contains 9 a-helices and 3 b-sheets. The Mn ionbinding active residues including H53, H101 and H190 are located in the a-helical region, whereas D186 is located in the random coil (Fig. 4c). Overall, the structure of CsmMnSOD contains 52.4 % of a-helices, 8.8 % of b-sheets and 38.6 % of random coils.

CsmMnSOD structural prediction

Tissue distribution and mRNA transcription

A putative homodimeric structure of CsmMnSOD protein was established using the SWISS-MODEL protein modeling server. The structure was predicted based on human MnSOD (PDB ID: 2adqB) as a

The relative gene expression of CsmMnSOD was quantified using real-time PCR. The analysis showed that the CsmMnSOD gene expression was noticed in all the tissues including muscle, blood, kidney, liver,

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Fig. 4 The tertiary structure of C. striatus mitochondrial MnSOD predicted using I-TASSER server. a The predicted homodimeric structure of CsmMnSOD based on human MnSOD (PDB ID: 2adqB, 86.4 % identity). b Cartoon representation of CsmMnSOD monomer with Mn ion-binding active residues. c Closer view of the active site, Mn ion ligand along with its binding residues, His53, His101, Asp186 and His190. C0 . The a-helices, random coil and b-strands are shown in red, blue and green color, respectively. (Color figure online)

spleen and gill taken for analysis (Fig. 5a). But significantly (P \ 0.05) highest expression was noticed in liver tissue. Based on the tissue distribution analysis, liver tissue was selected to study the mRNA transcription upon fungal (A. invadans) and bacterial (A. hydrophila) infections. In fungal-injected liver tissue, a steady increase was noticed in gene expression until 48 h post-injection (p.i) time; thereafter, the expression fell down (Fig. 5b). The statistical analysis showed that the expressions at 12, 24, 48 and 72 h p.i were significantly different (P \ 0.05) when compared to their corresponding controls. In bacteriainfected samples, a significant (P \ 0.05) expression was observed at 12, 24 and 48 h p.i time when compared to their respective controls. At 72 h p.i time, the expression nearly reached the basal level (Fig. 5c).

and FPS-injected individuals, no significant difference in gene expression was observed, whereas the individuals treated as negative controls showed significant (P \ 0.05) differences in expression. The individuals that received various concentrations of dsRNA showed significant (P \ 0.05) changes in expression when compared to their non-injected and FPS-injected controls (Fig. 6). Moreover, the results showed that knockdown of CsmMnSOD transcript impaired the total O2- level. The impairment of total O2- level is based on the concentration of dsRNA injected. Overall, the results indicated that the treatment is concentration dependent, i.e., as dsRNA dose increased, a decrease in CsmMnSOD transcription was observed.

CsmMnSOD gene knockdown

SOD activity was observed in the gene-silenced liver samples. The results showed the highest activity in non-injected individuals and the lowest activity in the negative control-injected fishes. Moreover, the results

Based on the tissue distribution analysis, we selected liver tissue for the gene silencing study. In non-injected

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SOD activity


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Fig. 5 Relative quantification of CsmMnSOD gene expression by real-time PCR. a Results of tissue distribution analysis of CsmMnSOD from various organs of C. striatus. Data are given as a ratio to CsmMnSOD mRNA expression in muscle. b, c The time course of CsmMnSOD mRNA expression in liver at 0, 3, 6, 12, 24, 48 and 72 h postinjection with A. invadans and A. hydrophila, respectively

showed that the activity is dose dependent (Fig. 7a). The up-regulated CsmMnSOD expression in liver tissues upon fungal and bacterial infections has attracted us to examine whether the activity of the enzyme is present or lost during the infection by interaction between the host and pathogen. The study indicated that the fungus-injected fish steadily

decreased in SOD activity from 3 h p.i to 48 h p.i (Fig. 7b); and the bacteria-injected fish also showed a significantly (P \ 0.05) lowest activity at 48 h p.i time (Fig. 7c). Overall, the results showed that the SOD activity decreases with the spread of infection and increases once the molecule controls the toxic substances released by the pathogens.

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Fig. 6 Quantitative real-time PCR analysis of CsmMnSOD relative expression in liver tissue collected from C. striatus injected with various concentrations of dsRNA. Liver tissue was

selected to study the gene knockdown parameter on the basis of tissue distribution results which is given in Fig. 5a

Strength of superoxide anion production

saving cells from oxidative stresses due to the presence of mMnSOD in mitochondria (Boveris and Chance 1973; Cadenas and Davies 2000). MnSOD has been reported from many vertebrate and invertebrate species. However, comparatively the information on MnSOD from fish is limited compared with mammals; especially there is no information on mitochondrial MnSOD from C. striatus. In this study, a cDNA sequence encoding mMnSOD was identified from the constructed striped murrel cDNA library and the sequence obtained from the library by internal sequencing. Most of the mMnSODs possessed a short MTP at the N-terminal region, which is also known as secretory pathway SP. In this study too, we observed a short peptide as MTP at the N-terminal region of CsmMnSOD which consists of 27 amino acid residues. This short peptide is necessary for the translocation into the mitochondria (Fukuhara et al. 2002). Wispe et al. (1989) reported that the mMnSOD is encoded by the nuclear gene and it is synthesized and translocated into the MM with mature enzyme activity. Interestingly, this putative CsmMnSOD contains four N-glycosylation sites, but most of the mitochondrial MnSOD contains only two (Duttaroy et al. 1994; Cheng et al. 2006; Zhang et al. 2007; Bao et al. 2008; Zhang et al. 2013). Hence, it is possible to suggest that the CsmMnSOD may be a new glycoprotein. Moreover, the Mn2?-binding residues at the N (H53 and H101) and C (D186 and H190) terminals of the conserved SOD Fe domains of CsmMnSOD are necessary for the catalytic activity as observed by Bao et al. (2008). They (Bao et al. 2008) also reported that these conserved sequences are likely to be involved in

The strength of superoxide anion production was determined by calculating the granular blood cell count during infection. In both fungus- and bacteriainfected individuals, significantly (P \ 0.05) lowest count was observed at 48 h p.i (Fig. 8a, b). The superoxide radical production capacity related to the granular blood cells was 11.71 9 10-3 and 10.6 9 10-3 in fungus- and bacteria-infected individuals at 48 h p.i, respectively. The results indicated that the infection influenced the superoxide radical production.

Discussion In aerobic life, the generation of ROS is one of the important protective defense mechanism. At low concentration, ROS have been shown to be beneficial in many biological processes including immune response against pathogenic infections, whereas the high level of ROS and reactive oxygen intermediates (ROI) lead to cause oxidative stress which result in cell damage (Winston and Di Giulio 1991; Schreck et al. 1991; Anderson 1994; Roch 1999; Hensley et al. 2000; Lesser 2006). The MM is the main source for the generation of ROS. MnSOD is shown to be a major antioxidant enzyme in the MM, which can catalytically convert the radicals into O2 and H2O2 and prevent the damage of mitochondrial membrane, proteins and nucleic acids. So, it can be concluded that the mMnSOD likely plays a more critical role in

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Fig. 7 SOD activity in C. striatus liver. a SOD activity in liver tissue collected from C. striatus injected with various concentrations of dsRNA. b, c SOD activity in liver infected with fungus and bacteria, respectively

stabilizing the structure and function of MnSOD in evolutionary terms. Based on the pair-wise comparison analysis, CsmMnSOD shared a maximum identity (96 %) with MnSOD from O. fasciatus. The analysis showed that

the MnSOD locus has been strongly conserved among various taxa; this is in accordance with the earlier hypothesis of Cho et al. (2009). Moreover, the higher similarity ([80 %) of CsmMnSOD with its homologs indicates the evolutionary conservation of MnSOD

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Fig. 8 Strength of superoxide anion production in non-infected and fungus (a) and bacteria (b)-infected C. striatus granular blood cell count

locus among the entire vertebrate lineage. The identity and similarity of CsmMnSOD was notably higher with the vertebrate orthologs than the invertebrate orthologs. Furthermore, the multiple polypeptide sequence alignment of CsmMnSOD with its orthologs from vertebrate and invertebrate organisms showed that the four metal-binding residues and MnSOD signature motifs are strongly conserved. Hence, it is possible to suggest that the CsmMnSOD may have the same function as that in other organisms due to the conserved sequences and motifs. Though the function of CsmMnSOD may be similar to other vertebrate and invertebrate mMnSOD due to their conserved domains and motifs, the variations in the number of N-glycosylation sites in CsmMnSOD may lead it to a different activity, which needs to be explored. In the phylogenetic analysis, two major groups such as mMnSOD and cMnSOD were observed. These two groups have the same origin but differ in subcellular localization (Zelko et al. 2002). CsmMnSOD placed within the vertebrate clade and clustered with the fish mMnSOD group. Based on the taxonomical classification as well as the cluster formation, it is proposed

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that our sequence is a mitochondrial MnSOD. In this study, the higher bootstrap value of the phylogenetic tree is in accordance with the earlier findings of Zelko et al. (2002) who observed a constant rate of evolution for MnSOD. The homodimeric structure of CsmMnSOD was constructed based on a template, human MnSOD, which shared 86.4 % of identity. Parker and Blake (1988) and Borgstahl et al. (1992) reported that the MnSOD structures in prokaryotes as homodimer, whereas the eukaryotic MnSODs are homotetramer (Borgstahl et al. 1992; Parker and Blake 1988). The monomeric structure of CsmMnSOD consisted of 9 ahelices and 3 antiparallel b-strands. It is reported that the human MnSOD also contains similar number of ahelices and b-sheets. Moreover, both human MnSOD and CsmMnSOD contain two SOD Fe domains at N and C terminals, respectively. In both cases, the N-terminal domain contains three a-helices, among them 2nd and 3rd helix are larger than the 1st helix. Hence, it is also known as helical domain. Wool et al. (2008) reported that the number and size of a-helix potentially influenced the anti-atherogenic properties.


However, the C-terminal domain contains 3 antiparallel b-sheets and six smaller a-helices; hence, it is otherwise known as a/b domain (Porta et al. 2010). Apart from the metal-binding active sites, the secondshell residues of CsmMnSOD including His57, Tyr61, Trp150, Gln170 and Trp188 which are responsible for stabilizing the active site topology were also evolutionary conserved (Porta et al. 2010). Abreu and Cabelli (2010) reported that the water molecule bound to Mn2? active site cavity and interacted with the side chain of the conserved second-shell residues forming a hydrogen-bonded network. The network mediates the proton transferring process when superoxide radicals are reduced to H2O2 (Abreu and Cabelli 2010). The structural analysis revealed that the active site of Mn ligand is coordinated with the catalytic residues of H53, H101, D186 and H190 as shown by Borgstahl et al. (1992). The results of tissue distribution demonstrated that the CsmMnSOD was distributed in all the tested tissues and it is highly distributed in liver. The variations in the expression of CsmMnSOD in all the tested tissues may be due to the tissue-dependent mitochondrial content and oxidative load (Cho et al. 2009), since this antioxidant enzyme is the primary scavenger of ROS produced during mitochondrial respiration (Benard et al. 2006). Cho et al. (2009) reported that the highest gene expression in liver showed its high energy demand. Moreover, the results showed that the liver is the main tissue which is involved in the defense process. Bell and Smith (1993) reported that the hemopoietic organs including liver as the important immune organ which release superoxide anions in response against the pathogenic invaders. The hemopoietic organs play a critical role in defense process of both vertebrates and invertebrates and exhibited significant expressions to the stress created by the microbial pathogens including bacteria and fungus. Cho et al. (2005) and Sokolova et al. (2005) stated that the tissue-specific gene expression of ionbinding proteins such as MnSOD is widely distributed in aquatic vertebrate and invertebrate organisms. It may be due to the different rates of ion uptake during respiration and osmoregulation and release of ions during excretion through the various organs which are involved in those physiological processes. The mRNA transcription in liver was inducted with fungal and bacterial infections, and the induction was varied at different time points. The decrease in

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transcription after infection showed the level of ROS production which involved in destroying the pathogen in liver. On the other hand, the increment in the expression showed that the ROS have extremely gathered and the microbial killing process took place slowly. In other words, the increment in transcription due to the accumulation of pathogenic microbes such as A. invadans and A. hydrophila thus produced too much toxic effect to the host organisms which results in either cell death or loss of cellular immunity (Lee and Lim 2007; Rodrıguez et al. 2008). Moreover, the invaded microbial pathogens inhibit the phagocytic process and host immunity, thus causing pathogenesis. Hansen et al. (2006) observed the activation of ROS by the positive transcription factors including cytokines, serine protease and nuclear factor kappa B in such a condition and induct the mMnSOD mRNA transcription to eradicate the excess ROS. Once the excess ROS was eradicated, the mMnSOD mRNA transcription gradually decreased and reached the basal level. Hence, the variations in the CsmMnSOD mRNA transcription due to the interaction between the molecule and microbial pathogens including fungus and bacteria are confirmed. Silencing of CsmMnSOD was conducted to study whether RNA interference (RNAi) conciliates the silencing of the CsmMnSOD expression using dsRNA. Hence, we targeted the CsmMnSOD gene to silence and evaluate the effect of RNAi in C. striatus. The gene expression was tested in C. striatus liver after 24 h of dsRNA injection. The results show that the CsmMnSOD dsRNA capacity is strong enough even after 24 h p.i. No harmful effect was visualized in the individuals injected with dsRNA. RNAi was not able to completely arrest the gene expression of CsmMnSOD. The results showed that the dsRNA function is dose dependent in C. striatus. So it is suggested that the RNAi was less likely to be lethal at lower dosage of dsRNA and more at higher dosage (Mouyna et al. 2004). The differences in the expression may be due to the variations in small interference RNA (siRNA) receptors in liver as observed by Yu et al. (2009). This is in accordance with the earlier findings of Garcıá-Triana et al. (2010) who examined the MnSOD silencing in gill and hepatopancreas of Litopenaeus vannamei. Similar results were also obtained by Maningas et al. (2008) in shrimp. The results also showed that the abundance expression in non-injected and FPS-injected individuals may due to

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the presence of higher amount of CsmMnSOD mRNA transcription. Moreover, the higher dose of dsRNAinjected individuals showed the lowest expression; it represents the appropriate CsmMnSOD silencing activation. The SOD activity was analyzed in CsmMnSOD gene-silenced C. striatus along with its controls. The results indicated a significant silencing effect in liver. Moreover, the results show that the dsRNA function is dose dependent in C. striatus. Therefore, the results indicated that the CsmMnSOD is a necessary parameter for the antioxidant activity in liver. Similar results were obtained by Garcıá-Triana et al. (2010) in cMnSOD gene-silenced gills and hepatopancreas of L. vanameii. In fungus- and bacteria-infected individuals, the SOD activity varied at different time points. The variation is in accordance with the results of CsmMnSOD mRNA transcription; whereas the mRNA transcription increases, the SOD activity decreases. It shows that when the CsmMnSOD is controlling the pathogenic infection, the mRNA transcription is low, during that time the SOD activity is high. Mouyna et al. (2004) reported that the differences in SOD activity of pathogenic microbesinfected organisms are due to the variations in the genome context of ectopic integration events. Moreover, Matityahu et al. (2008) stated that the reduced activity of MnSOD achieved by RNAi hinders the growth of the organism. Overall, this study indicated that the RNAi is an essential tool to down-regulate the gene expression as well as to reduce the enzyme activity in striped murrel. Since the SOD activity varied in accordance with the CsmMnSOD mRNA transcription in fungus- and bacteria-infected individuals, it was necessary to determine the level of superoxide radical production during infection. Hence, we measured the superoxide radical production in granular blood cells infected with fungus and bacteria. Similar to the SOD activity, the superoxide radical production also varied at different time points for fungal- and bacterial-infected fish. The radical production is in correspondence with the SOD activity, i.e., the lowest radical production represents the lowest SOD activity. Olker et al. (2004) reported that the superoxide anions are toxic substances which are released by the defense system of the organisms to destroy the invading pathogens. In this study also, we observed the variations in the radical production in the infected fish at different post-

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infection time points; it is due to the variations in the capacity of CsmMnSOD which produce the superoxide radicals to kill the microbes. During phagocytosis, the radicals were generated in large amount by NADPH oxidase, an oxygen-dependent killing mechanism of invading microbes, thus killing the microbes. Moreover, the radicals are also generated as a byproduct during mitochondrial respiration as well as several other enzymes including XO (Muller et al. 2007). Overall, this study indicates the capacity of CsmMnSOD superoxide radical production which plays a major role in the killing of microbial pathogens. Conclusively, a cDNA sequence encoding CsmMnSOD was obtained from the established striped murrel cDNA library by internal sequencing. The sequence was characterized using various bioinformatic tools. We also observed its relative gene expression in a healthy as well as fungal- and bacterial-infected C. striatus. Gene silencing was studied to evaluate whether RNAi mediates the silence of CsmMnSOD expression using dsRNA. The SOD activity was analyzed in gene-silenced and fungal- and bacterialinfected C. striatus. The analysis indicated that the RNAi is essential to regulate the gene expression and to influence the SOD activity in fish. The capacity of superoxide radical production was examined in fungal- and bacterial-infected granular blood cells of C. striatus. The results showed the strength of CsmMnSOD superoxide radical production which plays a major role in killing the microbial pathogens. Overall, this study showed the molecular importance of CsmMnSOD and its potential involvement in defense system of C. striatus. However, further study is necessary to evaluate its potentiality at proteomic level. Acknowledgments This research is supported by DBT’s Prestigious Ramalingaswami Re-entry Fellowship (D.O.NO. BT/HRD/35/02/2006) funded by Department of Biotechnology, Ministry of Science and Technology, Government of India, New Delhi.

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High-level expression of a manganese superoxide dismutase (PoMn-SOD) from Pleurotus ostreatus in Pichia pastoris.

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