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Antimicrobial peptides (AMPs) are considered a novel approach to stimulate fish antiviral mechanisms for defense against a broad range of viral infections by enhancing immunomodulatory activities. Octominin is an AMP derived from the defense proteins of Octopus minor. In this study, preliminary screening of octominin against viral hemorrhagic septicemia virus (VHSV), infectious hematopoietic necrosis virus (IHNV), and infectious pancreatic necrosis virus (IPNV) was carried out. Moreover, immune responses upon octominin treatment and IHNV challenge were investigated using fathead minnow (FHM) cells. The CC 50 s of octominin for FHM and Chinook salmon embryo-214 (CHSE-214) cells were 2146.2 and 1865.2 μg/mL, respectively. With octominin treatment, EC 50 resulted in 732.8, 435.1, and 925.9 μg/mL for VHSV, IHNV, and IPNV, respectively. The selectivity indices were 2.9, 4.9, and 2.0, respectively. The transcriptional analysis results demonstrated the induced transcription factors (Irf3; 143-fold, Irf7; 105-fold, and NF-κB; 8-fold), stress response gene (HspB8; 2-fold), and apoptosis functional gene (p53; 3-fold) in octominin treated (500 μg/mL) FHM cells for 48 h. Moreover, IHNV viral copy number was slightly decreased with the octominin treatment (500 μg/mL) in FHM cells. Overall results suggest that octominin could be a potential antiviral agent, although further studies are necessary to understand its mode of action and the mechanism of its antiviral activity., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

The emergence of carbapenem-resistant Acinetobacter baumannii has increased the risk of nosocomial infections, which pose a huge health threat. There is an urgent need to develop alternative therapies, including broad-spectrum antimicrobial peptides. In this study, we designed, characterized, and studied the antibacterial, antibiofilm effects and possible mode of actions of a novel synthetic peptide Octopromycin, derived from the proline-rich protein 5 of Octopus minor. Octopromycin consists of 38 amino acids, (+5) net positive charge, high hydrophobic residue ratio (36%), and two α-helix secondary structures. The minimum inhibitory concentration and minimum bactericidal concentration against A. baumannii were 50 and 200 μg/mL, respectively. Time-kill kinetics and bacterial viability assays confirmed the concentration-dependent antibacterial activity of Octopromycin. Field emission scanning electron microscopy images clearly showed ultrastructural alterations in Octopromycin-treated A. baumannii cells. Propidium iodide penetrated into Octopromycin-treated A. baumannii cells, demonstrating the loss of cell membrane integrity. Octopromycin treatment increased the production of reactive oxygen species in a concentration-dependent manner, and it inhibited the biofilm formation and showed biofilm eradication activity against A. baumannii. In vitro and in vivo safety evaluation revealed that Octopromycin was nontoxic to HEK293T and Raw 264.7 cells (<400 μg/mL), as well as mice red blood cells (<300 μg/mL), and zebrafish embryos (<4 μg/mL). An in vivo study results revealed that the A. baumannii-infected fish treated with Octopromycin exhibited a significantly higher relative percent survival (37.5%) than the infected mock-treated fish with PBS (16.6%). Furthermore, a decreased bacterial load and fewer alterations in histological analysis confirmed the successful control of A. baumannii by Octopromycin in vivo. Collectively, the results indicate that the antibacterial peptide Octopromycin may achieve rapid control of A. baumannii through multi-target interactions; it presents a desirable therapeutic option for the prevention and control of the infections., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Streptococcus parauberis is a pathogenic gram-positive bacterium that causes streptococcosis infection in fish. Since S. parauberis is becoming resistant to multiple antibiotics, the development of alternatives, such as antimicrobial peptides, has gained great attention. Octominin, derived from the defense protein of Octopus minor, showed a significant antimicrobial activity against multidrug resistance S. parauberis, with a minimum inhibitory concentration (MIC) and a minimum bactericidal concentration (MBC) of 50 and 100 μg/mL, respectively. Furthermore, time-kill kinetics, agar diffusion, and bacterial viability assays confirmed the concentration-dependent antibacterial activity of Octominin against S. parauberis. Field emission scanning electron microscopy analysis showed morphological and ultra-structural changes in S. parauberis upon Octominin treatment. Moreover, Octominin treatment demonstrated changes in membrane permeability, induced reactive oxygen species (ROS), and its binding ability to genomic DNA, suggesting its strong bactericidal activity with multiple modes of action. We confirmed the inhibition of biofilm formation and the eradication of existing biofilms in a concentration-dependent manner. Additionally, Octominin on S. parauberis at transcriptional level exhibited downregulation of membrane formation (pgsA and cds1), DNA repairing (recF), biofilm formation (pgaB and epsF) genes, while upregulation of ROS detoxification (sodA) and DNA protecting (ahpF) related genes. An in vivo study confirmed a significantly (P < 0.05) higher relative percentage survival in Octominin-treated larval zebrafish exposed to S. parauberis (93.3%) compared to the control group (20.0%). Collectively, our results confirm that Octominin could be a potential antibacterial and anti-biofilm agent against S. parauberis., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Paramyxean parasites in the genus Marteilia deteriorate digestive tissues of the host organisms, resulting in mortality of oysters, cockles, and mussels. Most reports of infection by Marteilia spp. are from Europe, while a new species of Marteilia was identified recently in Japan. Here, we report a previously unidentified species in the genus Marteilia from digestive diverticula of Manila clam Ruditapes philippinarum from the south coast of Korea. Prevalence of the parasite was low, 0.5-3.3% in the study sites. We characterized this species using light and transmission electron microscopy (TEM), and analyzed the 18S rDNA sequence. Light microscopy revealed the sporulation process from uninucleated stage to spore in the epithelial tissues of the digestive gland. TEM revealed that the parasites produced four secondary cells containing four tri-cellular spores. An electron-dense haplosporosome-like structure and striated inclusions were evident in the spore and the primary cells, respectively, while refringent granules were rarely observed in the secondary cells. Phylogenetic analyses of the 18S rDNA sequence placed this isolate in the genus Marteilia, although it is not identical to other known species in the genus. Based on morphological and molecular characters, we describe this species as Marteilia tapetis sp. nov., the second Marteilia species reported parasitizing Manila clams in Asian waters., (Copyright © 2019. Published by Elsevier Inc.)

In this study, we describe the identification and characterization of a proto type galectin, galectin-1, from rock bream Oplegnathus fasciatus (OfGal-1). Galectins are evolutionarily conserved carbohydrate binding lectins that show a wide range of functions related to development and immune physiology. They have been identified as pattern recognition receptors of innate immune system that recognize a broad range of microbes. OfGal-1 cDNA comprised of 993 bp with an open reading frame of 408 bp that encodes 135 amino acids. A single carbohydrate recognition domain was present in the OfGal-1 amino acid sequence. The sequence comparison by multiple and pairwise alignments and the phylogenetic tree emphasized the strong evolutionary conservation of Gal-1. The typical β-sandwich structure was identified from the predicted tertiary structure. The constitutive expression of mRNA transcripts was detected in a wide range of tissues examined, with the highest expression in the heart. Immune challenges with live bacteria (Edwardsiella tarda and Streptococcus iniae), rock bream irido virus, and mitogens (lipopolysaccharide and poly I:C) modulated the expression of OfGal-1 mRNAs in the gills, head kidney, and liver. The recombinant OfGal-1 (rOfGal-1) strongly agglutinatinated the human erythrocytes, and this hemagglutination was inhibited by lactose and D-galactose. A wide range of bacteria (S. iniae, S. parauberis, Escherichia coli, Edwardsiella tarda, Vibrio anguillarum, Vibrio harveyi, and Vibrio tapetis) and a ciliate (Miamiensis avidus) were also effectively recognized by rOfGal-1. Significant antiviral activity against rock bream irido virus was also demonstrated by rOfGal-1. Collectively, results from the present study indicate that OfGal-1 can recognize a wide range of microbes and is a vital pattern recognition receptor in the innate immune system of rock bream., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Apoptosis inducing factor (AIF) is a flavoprotein that is involved in oxidative phosphorylation and induces apoptosis in eukaryotic cells. There are three isozymes of AIF that have been identified to date, designated as AIF1, AIF2, and AIF3; the human AIF3 is also known as an AIF-like protein (AIFL). This study aimed to identify and characterize a homologue of AIF3 from disk abalone (AbAIF3) that belongs to the phylum Mollusca. The open reading frame (ORF) of AbAIF3 is 1749 base pairs (bp) in length and encodes a protein of 583 amino acids, with a predicted molecular mass of 63.14 kDa. Based on our in-silico analysis, the AbAIF3 protein harbored the typical domain architecture as that of the known AIF family proteins, consisting of N-terminal Rieske and pyridine nucleotide-disulphide oxidoreductase domain. Comparative protein sequence analysis confirmed that AbAIF3 is a homolog of AIF3. Moreover, our phylogenetic analysis revealed that AbAIF3 had a close evolutionary relationship with the molluscan counterparts. Interestingly, AbAIF3 was shown to induce apoptosis in HEK293T cells using transfection assays followed by flow cytometric analysis. In addition, we found that AbAIF3 mRNA expression was ubiquitous in physiologically important tissues, and significantly modulated upon experimental immune stimulations in hemocytes. Collectively, our study illustrates the indispensable role of AbAIF3 in inducing apoptosis in disk abalones, which in turn might be involved in hosts' immune defense mechanisms against microbial infections., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Serine proteases and their inhibitors play vital roles in diverse biological processes. In this study, we identified and characterized cDNA coding for a Kunitz-type serine protease inhibitor (SPI), which we designated as RbKSPI, in a commercially important species, rock bream. The full-length cDNA sequence of RbKSPI consisted of 2452 bp with an open reading frame (ORF) of 1521 bp encoding a polypeptide of 507 amino acid (aa) residues. In the RbKSPI protein, MANEC, PKD, LDLa, and two Kunitz domains responsible for various functions were identified as characteristic features. Homology analysis revealed that RbKSPI shared the highest identity with the Kunitz homolog in Takifugu rubripes (77.6%). Phylogenetic analysis indicated that RbKSPI clusters with other teleostean KSPIs. In tissue-specific expression analysis, RbKSPI transcripts were detected in all the tested tissues, with the highest expression in gill tissue, followed by kidney and intestine. The mRNA expression of RbKSPI significantly increased in blood cells upon stimulation with two strains of bacteria (Edwardsiella tarda and Streptococcus iniae) and two pathogen-associated molecular patterns (PAMPs; LPS and poly I:C). Meanwhile, down-regulated expression of RbKSPI was observed in response to tissue injury. Collectively, these results suggest that the RbKSPI may be involved in essential immune defense against microbial pathogens and in the wound-healing process., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Haptoglobin (Hp) and serum amyloid A (SAA) are two vital proteins involved in inflammatory reactions and are classified as acute-phase proteins. They are released from hepatocytes under inflammatory conditions to protect healthy cells from being damaged by pathogens or from self-destructive mechanisms. In this study, a previously constructed black rockfish (Sebastes schlegeli) cDNA library was used to identify the full-length cDNA sequences of Hp and SAA homologs (RfHp and RfSAA, respectively) and characterize them at the molecular level. As expected, in silico analysis of these homologs showed the typical domain architectures of their known counterparts. Open reading frames of RfHp and RfSAA consisted of 942-bp and 313-bp DNA sequences, respectively. The derived polypeptide sequence of RfHp was composed of 313 amino acids (aa) with a predicted molecular weight of 34 kD, whereas RfSAA had a 121-amino acid sequence with a molecular weight of 13 kD. Phylogenetic analysis as well as pairwise sequence alignment results showed that RfHp was more closely related to Oreochromis mossambicus from an evolutionary perspective while RfSAA was closely related to the Epinephelus coioides ortholog. Although both genes were expressed ubiquitously in the tissues analyzed, they were particularly expressed in liver tissue, suggesting their origin in hepatocytes. Quantitative real-time PCR analysis indicated that both RfHp and RfSAA were significantly up-regulated by both bacterial and viral stimulation in liver tissue, affirming their putative importance in the acute phase of first-line host immune defenses., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Serine protease inhibitors (SERPINs) control cellular protease activity in order to maintain cellular homeostasis. The immune and inflammatory responses of invertebrate clade B SERPINs have not been widely reported. In the present study, three proteins with high similarity to clade B SERPINs, referred to as AbSERPIN-1, AbSERPIN-2 and AbSERPIN-3, were identified from disk abalone (Haliotis discus discus). While AbSERPIN-1 (399 aa) was of a typical size for this protein class, AbSERPIN-2 (506 aa) and AbSERPIN-3 (532 aa) were relatively larger. Bioinformatic analysis revealed the characteristic SERPIN domain in each AbSERPIN. In addition, the N-terminal region of both AbSERPIN-2 and AbSERPIN-3 contained a predicted low complexity region (LCR) and a signal peptide, suggesting that these proteins are secretory proteins and are, thus, novel peptides. Tertiary structural models of the AbSERPINs highlighted their structural and functional conservation. Ubiquitous expression of AbSERPIN transcripts was evaluated by quantitative real time PCR (qPCR) analysis in seven tissue types. AbSERPIN-1, AbSERPIN-2, and AbSERPIN-3 transcript levels were highest in mantle, hemocytes, and muscles, respectively. Temporal expression analysis revealed that AbSERPINs were significantly (P < 0.05) elevated in hemocytes during the early/middle stages following the injection of a bacterial pathogen (Vibrio parahaemolyticus or Listeria monocytogenes) or an immuno-stimulant (lipopolysaccharide). Moreover, mantle tissue injury led to significant changes in the temporal expression of AbSERPIN mRNA. Specifically, transcription of AbSERPIN-1 and AbSERPIN-3 was considerably up-regulated, while expression of AbSERPIN-2 was suppressed. These results suggest a potential role of AbSERPINs in response to pathogen invasion and tissue injury in disk abalone., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Chemokines are small, structurally related chemotactic cytokines characterized by the presence of conserved cysteine residues. In the present study, we identified the cDNA of a CXC chemokine from Oplegnathus fasciatus, designated as OfCXCL12. An open reading frame of 297 bp encoded a 98 amino acid peptide with a putative signal peptide of 23 amino acids. The CXC family-specific small cytokine domain (SCY), which is highly conserved among vertebrates, was located between residues 29 and 87. The characteristic conserved cysteine residues in the CXC motif of OfCXCL12 were separated by tyrosine (Y). Similar to other vertebrate CXCL12 proteins, OfCXCL12 also lacked the ELR motif and hence belongs to ELR(-) subfamily. Phylogenetic analysis revealed two distinct clades, consisting of fish and tetrapod CXCL12 homologs. Constitutive expression with significantly higher levels of OfCXCL12 mRNA transcription was detected in immune-related organs, including the head kidney, spleen, and kidney. Infection with bacterial and viral agents led to significant upregulation of mRNA expression in both the head kidney and spleen, in a stimulant-specific manner. Stimulation of peripheral blood leukocytes by the mitogen concanavalin-A significantly induced OfCXCL12 transcription. Results from the present study suggest an important role for OfCXCL12 in immune defense against bacterial and viral infection in rock bream., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Glutathione peroxidase (GPx) is an essential member of the antioxidant systems of living organisms and may be involved in immune defense against pathogenic invasion. In the current study, two selenium-dependent glutathione peroxidases (AbSeGPxs) that shared 54.3% identity were identified from the disk abalone Haliotis discus discus. The open reading frames (ORFs) of AbSeGPx-a and AbSeGPx-b coded for 222 and 220 amino acids, respectively, with a characteristic selenocysteine residue encoded by an opal stop codon (TGA). The conserved selenocysteine insertion sequence (SECIS) element was predicted in the 3' untranslated region (UTR) of both isoforms, and they were found to form two stem-loop structures. Amino acid comparison and phylogenetic studies revealed that the AbSeGPxs were closely related to those in other mollusk species and were evolutionarily distinct from those of other taxonomic groups. The SYBR Green qPCR was employed in investigating the transcripts of AbSeGPxs. The expression of AbSeGPxs mRNA was examined in different embryonic developmental stages and differential expression patterns for AbSeGPx-a and AbSeGPx-b were noted. Meanwhile, the highest expression of AbSeGPxs was detected in the hepatopancreas of healthy adult animals. Next, transcriptional levels were profiled in hemocytes of adults to determine the immune responses of AbSeGPxs to microbial infections. The results revealed the significant up-regulation of AbSeGPx-a in a time-dependent manner after bacterial (Listeria monocytogenes and Vibrio parahaemolyticus) and viral (viral hemorrhagic septicemia virus) infections. Consequently, these findings indicate that AbSeGPx-a and AbSeGPx-b might be involved in the embryonic development of disk abalone and the regulation of immune defense system of adult animals., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Myeloperoxidases (MPOs) are heme-linked oxidative stress-generating enzymes found abundantly in azurophilic granules of polymorphonuclear neutrophils. Mature MPOs act as potent antimicrobial agents by producing hypohalous acids using hydrogen peroxide and halide ions as substrates. These acids can readily oxidize reactive groups of biomolecules on invading microbes. In this study, we identified and characterized a homolog of MPO from rock bream (Oplegnathus fasciatus), designated as RbMPO. We analyzed the RbMPO gene for its basal expression level in physiologically important tissues and for transcriptional changes under different pathogenic stress conditions. The complete coding sequence of RbMPO consisted of 2652 nucleotides encoding an 884 amino acid sequence with a predicted molecular mass of 99.7 kDa. Our in silico analysis confirmed the typical MPO domain arrangement in RbMPO, including the propeptide, large chain and heavy chain, along with the heme peroxidase signature. Intriguingly, a C1q domain was also identified in the C-terminal region of the derived amino acid sequence. Most of the known functionally important residues of MPOs are found to be well conserved in RbMPO, showing a close evolutionary relationship with other teleostan MPOs, particularly with that of mandarin fish. RbMPO exhibited a ubiquitous basal expression in physiologically relevant tissues, with particularly high expression levels in blood cells. Basal transcript levels of RbMPO in gill and spleen tissues were found to change upon different pathogen or pathogen-derived mitogen stimulation, with detectable inductive responses. Together, these data suggest the potential involvement of RbMPO in the innate immune response in rock bream., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

The CXC chemokine receptor 4 (CXCR4) is the cognate receptor of the CXC chemokine ligand 12 (CXCL12) and plays a pivotal role under immune-pathophysiological conditions. In the current study, the CXCR4 homolog of Oplegnathus fasciatus (OfCXCR4) was sequenced and the mRNA expression levels were characterized. The genomic structure of the cloned OfCXCR4 coding region (2094 bp) revealed a bi-exonic element, where the open reading frame (ORF) appears split by a single intron. Analysis of the ORF (1134 bp) of OfCXCR4 revealed a predicted protein of 42.1 kDa with typical seven transmembrane (TM) domain architecture and several conserved structural features, including two cysteine residues forming a predicted disulfide bond, a characteristic CXC motif (containing CYC) and a G-protein-coupled receptor (GPCR) family 1 signature. Furthermore, based on comparative analysis, the structure OfCXCR4 appears well conserved at both the genomic DNA and the amino acid levels. Phylogenic analysis of OfCXCR4 revealed that the greatest homology was with its teleostean relatives. Expression studies showed ubiquitous OfCXCR4 transcription, mainly in immune organs, with the highest levels in the head kidney. Examination of OfCXCR4 transcriptional regulation post injection to different stimuli or pathogens revealed a significant modulation of mRNA expression as detected by reverse transcription-quantitative real-time PCR. Evidence of various transcription factor binding sites present in the 5'-flanking region of OfCXCR4 coupled with its observed regulated mRNA expression suggest that it may have an important role in immune surveillance in rock bream., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Antioxidative defense renders a significant protection against environmental stress in organisms and maintains the correct redox balance in cells, thereby supporting proper immune function. Catalase is an indispensable antioxidant in organisms that detoxifies hydrogen peroxides produced in cellular environments. In this study, we sought to molecularly characterize a homolog of catalase (RfCat), identified from black rockfish (Sebastes schlegelii). RfCat consists of a 1581 bp coding region for a protein of 527 amino acids, with a predicted molecular weight of 60 kD. The protein sequence of RfCat harbored similar domain architecture to known catalases, containing a proximal active site signature and proximal heme ligand signature, and further sharing prominent homology with its teleostan counterparts. As affirmed by multiple sequence alignments, most of the functionally important residues were well conserved in RfCat. Furthermore, our phylogenetic analysis indicates its common vertebrate ancestral origin and a close evolutionary relationship with teleostan catalases. Recombinantly expressed RfCat demonstrated prominent peroxidase activity that varied with different substrate and protein concentrations, and protected against DNA damage. RfCat mRNA was ubiquitously expressed among different tissues examined, as detected by qPCR. In addition, RfCat mRNA expression was modulated in response to pathogenic stress elicited by Streptococcus iniae and poly I:C in blood and spleen tissues. Collectively, our findings indicate that RfCat may play an indispensable role in host response to oxidative stress and maintain a correct redox balance after a pathogen invasion., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

As a pivotal signaling mediator of toll-like receptor (TLR) and interleukin (IL)-1 receptor (IL-1R) signaling cascades, the IL-1R-associated kinase 4 (IRAK4) is engaged in the activation of host immunity. This study investigates the molecular and expressional profiles of an IRAK4-like homolog from Oplegnathus fasciatus (OfIRAK4). The OfIRAK4 gene (8.2 kb) was structured with eleven exons and ten introns. A putative coding sequence (1395bp) was translated to the OfIRAK protein of 464 amino acids. The deduced OfIRAK4 protein featured a bipartite domain structure composed of a death domain (DD) and a kinase domain (PKc). Teleost IRAK4 appears to be distinct and divergent from that of tetrapods in terms of its exon-intron structure and evolutionary relatedness. Analysis of the sequence upstream of translation initiation site revealed the presence of putative regulatory elements, including NF-κB-binding sites, which are possibly involved in transcriptional control of OfIRAK4. Quantitative real-time PCR (qPCR) was employed to assess the transcriptional expression of OfIRAK4 in different juvenile tissues and post-injection of different immunogens and pathogens. Ubiquitous basal mRNA expression was widely detected with highest level in liver. In vivo flagellin (FLA) challenge significantly intensified its mRNA levels in intestine, liver and head kidney indicating its role in FLA-induced signaling. Meanwhile, up-regulated expression was also determined in liver and head kidney of animals challenged with potent immunogens (LPS and poly I:C) and pathogens (Edwardsiella tarda and Streptococcus iniae and rock bream iridovirus (RBIV)). Taken together, these data implicate that OfIRAK4 might be engaged in antibacterial and antiviral immunity in rock bream., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

IkBa is a member of IkB family, which sequesters NF-kB in an inactivate form in the cytoplasm and blocks the translocation of NF-kB to nucleus. The IkBa paralogs of rock bream (OfIkBa-A and OfIkBa-B) encoded IkBa proteins with typical features including, highly conserved IkB degradation motif, six ankyrin repeats and a PEST sequence. However, their amino acid identity and similarity were only 55.6 and 69.7%, respectively suggesting that these two genes could be the two different isoforms of IkBa. The number and size of the exons of OfIkBa-A and OfIkBa-B were conserved well with all the compared vertebrate species, although they have significantly different genomic sizes. Phylogenetic analysis revealed that OfIkBa-A and OfIkBa-B proteins cluster with IkBa family members; however, they were grouped with different subclades in IkBa family. Tissue specific expression of OfIkBa mRNA was constitutively detected in all the tested tissues, and they showed the higher transcription level in heart, liver, gill and peripheral blood cells, respectively. The injection of flagellin stimulated the mRNA expression of OfIkBa paralogs in head kidney and intestine. Moreover, the OfIkBa mRNA expression in gill and liver was significantly upregulated by LPS, poly I:C and Edwardsiella tarda challenges. The transcription of OfIkBa was up-regulated in early-phase of injection and then rapidly restored. These results suggest that the OfIkBa paralogs might be involved in rapid immune responsive reactions in rock bream against bacterial and viral pathogens.

Antimicrobial immune defense is evolutionarily preserved in all organisms. Mammals have developed robust, protein-based antiviral defenses, which are under constant investigation. Studies have provided evidences for the various fish immune factors sharing similarity with those of mammals. In this study, we have identified an ortholog of mitochondrial antiviral signaling protein from rock bream, Oplegnathus fasciatus. RbMAVS cDNA possesses an open reading frame (ORF) of 1758 bp coding for a protein of 586 amino acids with molecular mass of approximately 62 kDa and isoelectric point of 4.6. In silico analysis of RbMAVS protein revealed a caspase recruitment domain (CARD), a proline rich domain and a transmembrane domain. RbMAVS protein also contains a putative TRAF2 binding motif, (319)PVQDT(323). Primary sequence comparison of RbMAVS with other orthologues revealed heterogeneity towards the C-terminus after the CARD region. RbMAVS transcripts were evident in all the examined tissues. RbMAVS expression was induced in vivo after poly I:C challenge in peripheral blood cells, liver, head kidney and spleen tissues. Over-expression of RbMAVS potently inhibited marine birnavirus (MABV) infection in rock bream heart cells and induced various cytokines and signaling molecules in vitro. Thus, RbMAVS is an antiviral protein and potentially involved in the recognition and signaling of antiviral defense mechanism in rock bream., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

The CXCR1 and CXCR2 are the prototypical receptors and are the only known receptors for mammalian ELR+ (Glu-Leu-Arg) CXC chemokines, including CXCL8 (interleukin 8). These receptors transduce the ELR+ chemokine signals and operate the downstream signaling pathways in inflammation and innate immunity. In this study, we report the identification and characterization of CXCR1 and CXCR2 genes from rock bream fish (OfCXCR1 and OfCXCR2) at the molecular level. The cDNA and genomic DNA sequences of the OfCXCR1 and OfCXCR2 were identified from a transcriptome library and a custom-constructed BAC library, respectively. Both OfCXCR genes consisted of two exons, separated by an intron. The 5'-flanking regions of OfCXCR genes possessed multiple putative transcription factor binding sites related to immune response. The coding sequences of OfCXCR1 and OfCXCR2 encoded putative peptides of 355 and 360 amino acids (aa), respectively. The deduced aa sequences of OfCXCR1 and OfCXCR2 comprised of a G-protein coupled receptors (GPCR) family 1 profile with a GPCR signature and a DRY motif. In addition, seven conserved transmembrane regions were predicted in both OfCXCRs. While our multiple alignment study revealed the functionally significant conserved elements of the OfCXCR1 and OfCXCR2, phylogeny analyses further confirmed their position in teleost sub clade, in which they manifested an evolutionary relatedness with other fish counterparts. Based on comparative analyses, teleost CXC chemokine receptors appear to be distinct from their non-fish orthologs in terms of evolution (both CXCR1 and CXCR2) and genomic organization (CXCR2). Quantitative real-time PCR (qPCR) detected the transcripts of OfCXCR1 and OfCXCR2 in eleven examined tissues, with higher levels in head kidney, kidney and spleen highlighting their crucial importance in immunity. In vitro stimulation of peripheral blood leukocytes (PBLs) with concanavalin A (Con A) resulted in modulation of OfCXCR2 transcription, but not that of OfCXCR1. In addition, the magnitude of the OfCXCR1 and OfCXCR2 transcripts in head kidney and spleen was differentially increased after the in vivo administration of immune stimulants, LPS and poly I:C and in the infection models injected with rock bream irido virus, Edwardsiella tarda and Streptococcus iniae. These lines of evidence suggest that these receptors may play an important role(s) in immune responsive signaling during pathogenesis of rock bream., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Immune signaling cascades have an indispensable role in the host defense of almost all the organisms. Tumor necrosis factor (TNF) signaling is considered as a prominent signaling pathway in vertebrate as well as invertebrate species. Within the signaling cascade, TNF receptor-associated factor (TRAF) and TNF receptor-associated protein (TTRAP) has been shown to have a crucial role in the modulation of immune signaling in animals. Here, we attempted to characterize a novel molluskan ortholog of TTRAP (AbTTRAP) from disk abalone (Haliotis discus discus) and analyzed its expression levels under pathogenic stress. The complete coding sequence of AbTTRAP consisted of 1071 nucleotides, coding for a 357 amino acid peptide, with a predicted molecular mass of 40 kDa. According to our in-silico analysis, AbTTRAP resembled the typical TTRAP domain architecture, including a 5'-tyrosyl DNA phosphodiesterase domain. Moreover, phylogenetic analysis revealed its common ancestral invertebrate origin, where AbTTRAP was clustered with molluskan counterparts. Quantitative real time PCR showed universally distributed expression of AbTTRAP in selected tissues of abalone, from which more prominent expression was detected in hemocytes. Upon stimulation with two pathogen-derived mitogens, lipopolysaccharide (LPS) and polyinosinic:polycytidylic acid (poly I:C), transcript levels of AbTTRAP in hemocytes and gill tissues were differentially modulated with time. In addition, the recombinant protein of AbTTRAP exhibited prominent endonuclease activity against abalone genomic DNA, which was enhanced by the presence of Mg(2+) in the medium. Collectively, these results reinforce the existence of the TNF signaling cascade in mollusks like disk abalone, further implicating the putative regulatory behavior of TTRAP in invertebrate host pathology., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

The membrane-attack complex/perforin (MACPF) domain-containing proteins play an important role in the innate immune response against invading microbial pathogens. In the current study, a member of the MACPF domain-containing proteins, macrophage expressed gene-1 (MPEG1) encoding 730 amino acids with the theoretical molecular mass of 79.6 kDa and an isoelectric point (pI) of 6.49 was characterized from disk abalone Haliotis discus discus (AbMPEG1). We found that the characteristic MACPF domain (Val(131)-Tyr(348)) and transmembrane segment (Ala(669)-Ile(691)) of AbMPEG1 are located in the N- and C-terminal ends of the protein, respectively. Ortholog comparison revealed that AbMPEG1 has the highest sequence identity with its pink abalone counterpart, while sequences identities of greater than 90% were observed with MPEG1 members from other abalone species. Likewise, the furin cleavage site KRRRK was highly conserved in all abalone species, but not in other species investigated. We identified an intron-less genomic sequence within disk abalone AbMPEG1, which was similar to other mammalian, avian, and reptilian counterparts. Transcription factor binding sites, which are important for immune responses, were identified in the 5'-flanking region of AbMPEG1. qPCR revealed AbMPEG1 transcripts are present in every tissues examined, with the highest expression level occurring in mantle tissue. Significant up-regulation of AbMPEG1 transcript levels was observed in hemocytes and gill tissues following challenges with pathogens (Vibrio parahemolyticus, Listeria monocytogenes and viral hemorrhagic septicemia virus) as well as pathogen-associated molecular patterns (PAMPs: lipopolysaccharides and poly I:C immunostimulant). Finally, the antibacterial activity of the MACPF domain was characterized against Gram-negative and -positive bacteria using a recombinant peptide. Taken together, these results indicate that the biological significance of the AbMPEG1 gene includes a role in protecting disk abalone through the ability of AbMPEG1 to initiate an innate immune response upon pathogen invasion., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Peptidoglycan recognition proteins (PGRPs) are a widely studied group of pattern recognition receptors found in invertebrate as well as vertebrate lineages, and are involved in bacterial pathogen sensing. However, in addition to this principal role, they can also function in multiple host defense processes, including cell phagocytosis and hydrolysis of peptidoglycans (PGNs). In this study, a novel invertebrate short-type PGRP was identified in disk abalone (Haliotis discus discus) designated as AbPGRP. The complete coding sequence of AbPGRP was 534 bp, encoding a 178-amino acid protein with a predicted molecular mass of 20 kDa. The AbPGRP gene had a bipartite arrangement consisting of two exons separated by a single intron. Homology analysis revealed that AbPGRP shares conserved features, including amino acid residues critical for substrate and ion binding as well as for its amidase activity, with homologs of other species. Phylogenetic analysis of AbPGRP revealed that it likely evolved from a common ancestor of invertebrates, having significant homology with other molluscan PGRPs. Recombinant AbPGRP exhibited detectable, dose-dependent PGN-hydrolyzing activity with the presence of Zn(2+), and strong antibacterial activity against Vibrio tapetis, consistent with the functional properties previously reported for PGRPs in other mollusks. Moreover, AbPGRP transcription was induced upon treatment of healthy abalones with bacterial peptidoglycan and lipopolysaccharide, although the expression profiles differed with treatment, suggesting a capacity for discriminating between bacterial pathogens through molecular pattern recognition. Collectively, the findings of this study indicate that AbPGRP is a true homolog of invertebrate PGRPs and likely plays an indispensable role in host immunity., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

The interferon regulatory factor 5 (IRF5) is a key mediator of the Toll-like receptor (TLR)7 and TLR8 signaling pathways. In this study, we describe the identification of IRF5 (Rb-IRF5) from rock bream fish (Oplegnathus fasciatus) and its characteristics features at the genomic and expression levels. The full-length Rb-IRF5 sequence was identified from a cDNA library and its genomic sequence was obtained by screening and sequencing of a bacterial artificial chromosome (BAC) genomic DNA library of rock bream. The genomic sequence is comprised of 8 exons interrupted by 7 introns. The complete coding sequence of Rb-IRF5 is 1497 bp in length and encodes for 498 amino acids. The putative Rb-IRF5 protein consists of 3 important conserved domains: a DNA-binding domain (DBD) at the N-terminus, an IRF-associated domain (IAD), and a virus-activated domain (VAD) at the C-terminus. Based on pairwise sequence analysis, the highest sequence similarity/identity for Rb-IRF5 was observed with the IRF5 gene from turbot fish (>87%) and Japanese flounder (83%). Several important putative transcription factor-binding sites shared by the IRF gene family, including the NF-κB, Ap-1, IRF-1, and ICSBP/ISRE sites, were found in the 5' flanking region of Rb-IRF5. The predicted tertiary structure of the dimerized IAD and VAD of the Rb-IRF5 protein resembled that of its orthologs from humans. In healthy rock bream, the highest constitutive expression of Rb-IRF5 was detected in the liver. After iridovirus and polyinosinic-polycytidylic acid (poly(I:C)) challenge, Rb-IRF5 expression was significantly induced in the head kidney. Furthermore, rock bream recombinant type I interferon (Rb-IFN1) was also found to be an efficient inducer of Rb-IRF5 in a head kidney primary cell culture model. Upon IRF5 transfection, rock bream Mx (Rb-Mx), interferon I (Rb-IFN1) and tumor-necrosis factor α (Rb-TNFα) genes get significantly upregulated in rock bream heart cells. The findings of the present study explain the involvement of Rb-IRF5 in the induction of interferons and pro-inflammatory cytokines and thereby provide a model for how IRF5 modulates immune responses against viral infections in rock bream., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

The Janus tyrosine kinase (JAK)/signal transducer and activator of transcription (STAT) signaling pathway plays a critical role in host defense against viral and bacterial infections. STAT proteins are a group of transcription factors that translocate into the nucleus and are critical for the induction of many genes crucial for the allergic cascade and immune defense. In the present study, a member of the STAT4 family was identified from rock bream (RbSTAT4) at the genomic level, and its transcriptional regulation in response to different pathological stimuli under in vivo conditions was investigated. The genomic sequence of RbSTAT4 is approximately 15.6 kb in length, including a putative core promoter region and 24 exons interrupted by 23 introns. Bioinformatics analysis of RbSTAT4 identified the presence of typical and conserved features of the STAT4 family, including the STAT&#95;int domain, STAT alpha domain, STAT bind domain, linker domain, SH2 domain, and transcriptional activation domain. According to the phylogenetic analysis, RbSTAT4 exhibited the closest evolutionary proximity with the STAT4 member from mandarin fish (Siniperca chuatsi). The RbSTAT4 transcript in healthy rock breams was detected to have ubiquitous expression in 11 different tissues examined, where liver and spleen tissues showed moderate expressions compared with the highest expression level detected in gill tissue. The time-course in vivo immune stimulation of rock bream with lipopolysaccharide, poly I:C, live Edwardsiella tarda, and rock bream iridovirus caused significant transcriptional regulation of the RbSTAT4 expression in gill, head kidney, and spleen tissues, suggesting that RbSTAT4 is involved in immune regulation mechanisms and/or signaling cascades, orchestrating against both bacterial and viral pathogens., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Ferritins are biological iron chelators that can sequestrate excess iron to maintain iron homeostasis in the body. Ferritins basically consist of 2 types of subunits, designated as H and L. However, another new subunit, ferritin "M" which possesses characteristic features of both the H and L subunits, was recently identified in lower vertebrates, mostly in fish. In this study, a ferritin M-like subunit from rock bream (Oplegnathus fasciatus) (RbFerM) was characterized at the molecular level, and its transcriptional profile was analyzed in healthy fish, as well as in pathogen- and mitogen-stimulated fish. Furthermore, its functional properties were evaluated using the recombinant protein. The complete coding sequence of RbFerM was 528 bp in length, encoding a 176-amino acid peptide with a calculated molecular mass of 20 kDa. In silico analysis of RbFerM revealed that it has features similar to both the mammalian ferritin subunits, H and L. Phylogenetic analysis depicted the higher evolutionary proximity of RbFerM with its fish counterparts. Quantitative real time polymerase chain reaction (PCR) analysis detected a ubiquitous transcriptional profile of RbFerM in selected tissues of rock bream, in which more pronounced expression was observed in blood and liver tissues. Significant transcriptional inductions of RbFerM were detected in liver tissues upon lipopolysaccharides (LPS), Edwardsiella tarda, Streptococcus iniae, and rock bream irido virus (RBIV) exposures in time-course immune-challenge experiments. The purified recombinant protein of RbFerM demonstrated detectable iron chelating activity that varied with the temperature. Moreover, the recombinant RbFerM rendered a detectable protection effect against iron (II) and H2O2-mediated DNA damage., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Complement component 1q (C1q) is a subcomponent of the C1 complex and the key protein that recognizes and binds to a broad range of immune and non-immune ligands to initiate the classical complement pathway. In the present study, we identified and characterized three novel C1q family members from rock bream, Oplegnathus fasciatus. The full-length cDNAs of C1q A-like (RbC1qAL), C1q B-like (RbC1qBL), and C1q C-like (RbC1qCL) consist of 780, 720 and 726 bp of nucleotide sequence encoding polypeptides of 260, 240 and 242 amino acids, respectively. All three RbC1qs possess a leading signal peptide and collagen-like region(s) (CLRs) in the N-terminus, and a C1q domain at the C-terminus. The C1q characteristic Gly-X-Y repeats are present in all three RbC1qs, while the CLR-associated sequence that enhances phagocytic activity is present in RbC1qAL ((49)GEKGEP(54)) and RbC1qCL ((70)GEKGEP(75)). Moreover, the coding region was distributed across six exons in RbCqAL and RbC1qCL, but only five exons in RbC1qBL. Phylogenetic analysis revealed that the three RbC1qs tightly cluster with the fish clade. All three RbC1qs are most highly expressed in the spleen and liver, as indicated by qPCR tissue profiling. In addition, all three are transcriptionally responsive to immune challenge, with liver expression being significantly up-regulated in the early phase of infection with intact, live bacteria (Edwardsiella tarda and Streptococcus iniae) and virus (rock bream iridovirus) and in the late phase of exposure to purified endotoxin (lipopolysaccharide). These data collectively suggest that the RbC1qs may play defense roles as an innate immune response to protect the rock bream from bacterial and viral infections., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Lysozymes are ubiquitously distributed enzymes with hydrolytic activity against bacterial peptidoglycan and function to protect organisms from microbial pathogens. In this study, an invertebrate goose-type lysozyme, designated as abLysG, was identified in the disk abalone, Haliotis discus discus. The full-length cDNA of abLysG was 894 bp in length with an open reading frame of 789 bp encoding a polypeptide of 263 amino acids containing a signal peptide and a characteristic soluble lytic transglycosylase domain. Six cysteine residues and two catalytic residues (Glu(142) and Asp(168)) conserved among molluscs were also identified. The 3D homology structural models of abLysG and hen egg white lysozyme had similar conformations of the active sites involved in the binding of substrate. BAC sequence data revealed that the genomic structure of disk abalone g-type lysozyme comprises 7 exons with 6 intervening introns. The deduced amino acid sequence of abLysG shared 45.2-61.6% similarity with those of other molluscs and vertebrates. The TFSEARCH server predicted a variety of transcription factor-binding sites in the 5'-flanking region of the abLysG gene, some of which are involved in transcriptional regulation of the lysozyme gene. abLysG expression was detected in multiple tissues with the highest expression in mantle. Moreover, qPCR analysis of abLysG mRNA expression demonstrated significant up-regulation in gill in response to infection by live bacteria (Vibrio parahaemolyticus and Listeria monocytogenes), virus (viral hemorrhagic septicemia) and bacterial mimics (LPS and PGN). Expression of the recombinant disk abalone g-type lysozyme in Escherichia coli BL21, demonstrated its bacteriolytic activity against several Gram-negative and Gram-positive bacterial species. Collectively these data suggest that abLysG is an antimicrobial enzyme with a potential role in the disk abalone innate immune system to protect it from bacterial and viral infections., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Antimicrobial peptides are important innate effector molecules, playing a vital role in antimicrobial immunity in all species. Glyrichin is a transmembrane protein and an antibacterial peptide, exerting its functions against a wide range of pathogenic bacteria. In this study, cDNA and a BAC clone harboring the glyrichin gene were identified from rock bream and characterized. Genomic characterization showed that the OfGlyrichin gene exhibited a 3 exon-2 intron structure. OfGlyrichin is a 79-amino-acid protein with a transmembrane domain at (22)GFMMGFAVGMAAGAMFGTFSCLR(44). Pairwise and multiple sequence alignments showed high identity and conservation with mammalian orthologues. Phylogenetic analysis showed a close relationship with fish species. Higher levels of OfGlyrichin transcripts were detected in the liver from healthy rock bream which were induced by immunogens like lipopolysaccharide, poly I:C, rock bream irido virus, Edwardsiella tarda and Streptococcus iniae. The synthetic peptide (pOf19) showed antibacterial activity against Escherichia coli, E. tarda, and S. iniae. Analysis of the bacterial morphological features after pOf19 peptide treatment showed breakage of the cell membrane, affirming that antibacterial function is accomplished through membrane lysis. The pOf19 peptide also showed antiviral activity against RBIV infection. The high conservation of the genomic structure and protein, together with the antimicrobial roles of OfGlyrichin, provide evidence for the evolutionary existence of this protein playing a vital role in innate immune defense in rock bream., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Proteasomes are multicatalytic subunit complexes involved in the degradation of cytosolic proteins and antigen presentation. In this study, we have characterized the alpha and beta subunits of proteasome activator complex from rock bream at the molecular level. RbPA28α and RbPA28β possessed the characteristic features of the subunits identified from mammals and teleosts. The RbPA28α and RbPA28β proteasome subunits contained a proline-rich motif (Region A), subunit-specific insert in the region corresponding to the KEKE motif of the known PA28α (Region B), conserved activation loop (Region C), a potential protein kinase C recognition site (Region D) and a highly homologous C-terminal region (Region E) among all three PA28 subunits. Multiple sequence alignment and pairwise alignment revealed that RbPA28α and RbPA28β proteins shared high homology with the teleosts and mammals. RbPA28α and RbPA28β genome possessed 11 exons interrupted by 10 introns. In silico promoter analysis of RbPA28α and RbPA28β revealed various transcription factor-binding sites displaying their regulation under various stress conditions. Tissue distribution profiling showed a higher expression in blood and gills. Transcriptional expression analysis of RbPA28α and RbPA28β showed up-regulation in the immune tissues following LPS and poly I:C challenges, providing further evidence for the immunological role of RbPA28α and RbPA28β., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Catalases are well known antioxidant enzymes that can mainly dismutate hydrogen peroxide into water and oxygen in order to prevent oxidative stress. The complete genomic DNA (gDNA) sequence of the catalase gene from rock bream (Oplegnathus fasciatus) was identified from our custom-constructed BAC genomic DNA library and designated as RbCat. RbCat consists of 13 exons, separated by 12 introns, within a 13,722-bp gDNA sequence. The complete cDNA sequence (3303 bp) of RbCat is comprised of a 1581-bp coding region, encoding a peptide of 527 amino acids (aa) in length, with a predicted molecular mass of 60 kDa and a theoretical isoelectric point of 8.34. The anticipated promoter region of RbCat contains several transcription factor-binding sites, including sites that bind with immune- and antioxidant-responsive signaling molecules, suggesting its substantial transcriptional regulation. RbCat resembles the typical catalase family signature, i.e., it is composed of the catalase proximal active site motif along with a catalase proximal heme-ligand signature motif and shares great homology with its fish counterparts. According to multiple sequence alignment, functionally important amino acids present in RbCat were thoroughly conserved among its vertebrate counterparts. Phylogenetic analysis revealed that RbCat evolved from a vertebrate origin, and further positioned it in the fish clade. Recombinant RbCat had noticeable peroxidase activity against its substrate, hydrogen peroxide, in a dose-dependent manner. However, it demonstrated substantial peroxidase activity within a broad range of temperatures and pH values. Constitutive RbCat mRNA expression of different magnitudes was detected in a tissue-specific manner, suggesting its diverse role in physiology with respect to the tissue type. Moreover, immune challenge experiments using Edwardsiella tarda and rock bream iridovirus (RBIV) as live pathogens and polyinosinic:polycytidylic acid and lipopolysaccharide as mitogens revealed that the transcription of RbCat can be modulated by immune stimulation. Collectively, the results obtained in this study suggest that RbCat can function as a potent antioxidant enzyme in rock bream and may play a role in post-immune responses with respect to its peroxidase activity., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Akirins are conserved nuclear resident NF-κB signaling pathway molecules. Isoforms of akirins found in various organisms are known to play diverse roles. In this study, we have characterized two akirin2 homologues from rock bream, OfAk2(1) and OfAk2(2). The proteins derived from OfAk2(1) and OfAk2(2) revealed the presence of nuclear localization signal. Multiple sequence alignment and pairwise alignment of OfAk2(1) and OfAk2(2) with the akirin homologues, revealed high conservation and identity. Phylogenetic tree analysis revealed that the distinct position of OfAk2(1) and OfAk2(2) was close to the fish homologues and separated from the mammals and invertebrates. Genomic structure characterization revealed two distinct structures. OfAk2(1) possessed 6 exons interrupted by 5 introns whereas OfAk2(2) possessed 5 exons interrupted by 4 introns. The promoter analysis revealed the presence of significant transcription factors, which suggests its regulation by diverse stimuli. In addition, transcript expression analysis using real time quantitative reverse-transcriptase polymerase chain reaction post immune challenges with lipopolysaccharide, Edwardsiella tarda and poly I:C revealed upregulation of both OfAk2(1) and OfAk2(2) in liver, spleen and head kidney., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Proteinases and proteinase inhibitors are involved in several biological and physiological processes in all multicellular organisms. Proteinase inhibitors play a key role in regulating the activity of the respective proteinases. Among serine proteinase inhibitors, kazal-type proteinase inhibitors (KPIs) are widely found in mammals, avians, and a variety of invertebrates. In this study, we describe the identification of a kazal-type serine proteinase inhibitor (Ab-KPI) from the disk abalone, Haliotis discus discus, which is presumably involved in innate immunity. The full-length cDNA of Ab-KPI includes 600 bp nucleotides with an open reading frame (ORF) encoding a polypeptide of 143 amino acids. The deduced amino acid sequence of Ab-KPI contains a putative 17-amino acid signal peptide and two tandem kazal domains with high similarity to other kazal-type SPIs. Each kazal domain consists of reactive site (P1) residue containing a leucine (L), and a threonine (T) located in the second amino acid position after the second conserved cysteine of each domain. Temporal expression of Ab-KPI was assessed by real time quantitative PCR in hemocytes and mantle tissue following bacterial and viral hemorrhagic septicemia virus (VHSV) challenge, and tissue injury. At 6 h post-bacterial and -VHSV challenge, Ab-KPI expression in hemocytes was increased 14-fold and 4-fold, respectively, compared to control samples. The highest up-regulations upon tissue injury were shown at 9 h and 12 h in hemocytes and mantle, respectively. The transcriptional modulation of Ab-KPI following bacterial and viral challenges and tissue injury indicates that it might be involved in immune defense as well as wound healing process in abalone., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Toll-like receptors (TLRs) are well-characterized pattern recognition receptors of innate immunity, known to induce immune responses against the pathogens by interacting with evolutionarily conserved pathogen-associated molecular patterns (PAMPs). In this study, a novel TLR homolog from disk abalone (Haliotis discus discus) was identified and characterized at molecular level. The open reading frame (ORF) of AbTLR is 3804 bp in length and encodes a 1268 amino acid peptide with a calculated molecular mass of 143.5 kDa. The deduced protein shows typical TLR domain architecture, with leucine rich repeats (LRR) and the toll-interleukin receptor (TIR) domain. Phylogenetic analysis revealed a close evolutionary relationship for AbTLR to its invertebrate counterparts, with close clustering to the molluscan homologs. Quantitative real-time PCR detected ubiquitous transcription of AbTLR in healthy tissues, but with highest levels in hemocytes. Differential transcriptional modulation of AbTLR was observed in abalone hemocytes and gills upon immune challenge, whereby Vibrio parahaemolyticus and purified lipopolysaccharide (LPS) enhanced the transcript level prominently. In addition, the viral hemorrhagic septicemia virus induced AbTLR transcription in hemocytes and gills, representing the first evidence of viral-induced immune response in mollusks to date. Collectively, our findings support a putative role for AbTLR in abalone antiviral and antibacterial defense., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Lysozymes are crucial antibacterial proteins that are associated with catalytic cleavage of peptidoglycan and subsequent bacteriolysis. The present study describes the identification of two lysozyme genes from disc abalone Haliotis discus discus and their characterization at sequence-, genomic-, transcriptional- and functional-levels. Two cDNAs and BAC clones bearing lysozyme genes were isolated from abalone transcriptome and BAC genomic libraries, respectively and sequences were determined. Corresponding deduced amino acid sequences harbored a chicken-type lysozyme (LysC) family profile and exhibited conserved characteristics of LysC family members including active residues (Glu and Asp) and GS(S/T)DYGIFQINS motif suggested that they are LysC counterparts in disc abalone and designated as abLysC1 and abLysC2. While abLysC1 represented the homolog recently reported in Ezo abalone [1], abLysC2 shared significant identity with LysC homologs. Unlike other vertebrate LysCs, coding sequence of abLysCs were distributed within five exons interrupted by four introns. Both abLysCs revealed a broader mRNA distribution with highest levels in mantle (abLysC1) and hepatopancreas (abLysC2) suggesting their likely main role in defense and digestion, respectively. Investigation of temporal transcriptional profiles post-LPS and -pathogen challenges revealed induced-responses of abLysCs in gills and hemocytes. The in vitro muramidase activity of purified recombinant (r) abLysCs proteins was evaluated, and findings indicated that they are active in acidic pH range (3.5-6.5) and over a broad temperature range (20-60 °C) and influenced by ionic strength. When the antibacterial spectra of (r)abLysCs were examined, they displayed differential activities against both Gram positive and Gram negative strains providing evidence for their involvement in bacteriolytic function in abalone physiology., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Macrophage migration inhibitory factor (MIF) is a pleiotropic molecule playing vital roles in various signaling cascades, including cell proliferation, and activation of immune responses against infections. It is well known as a pivotal regulator of innate immunity. In this study, we have rescued and characterized two members of the MIF family, macrophage migration inhibitory factor (OfMIF) and D-Dopachrome tautomerase (OfDDT) from rock bream, Oplegnathus fasciatus. The deduced OfMIF and OfDDT protein sequences revealed the presence of the catalytic oxidoreductase (CXXC), motif. They also possessed highly conserved proline (P(2)) and lysine residues (K(33)), responsible for their isomerase and tautomerase functions. Rock bream MIF and DDT homologues shared higher identity with fish homologues and also with mammals and occupied a distinct position in the phylogenetic tree, depicting their evolutionary conservation. The spatial expression analysis revealed the highest expression of both OfMIF and OfDDT in liver, while portraying constitutive expression in other tissues. The recombinant proteins purified using the Escherichia coli system revealed potent oxidoreductase activity against insulin with both dithiothreitol and glutathione as reducing agents. Stimulation of rock bream head kidney cells with recombinant OfMIF and OfDDT proteins induced the expression of proinflammatory cytokines like tumor necrosis factor alpha (TNF-α), interleukin-8 (IL-8) and interleukin-1β (IL-1β). These results together suggest their involvement in rock bream immune defense and this study on the novel MIF family member DDT from rock bream will pave the way for further studies of this homologue in other teleosts and delineate its multiple functions., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Apoptosis based immune responses are important component of host defense in mollusks. In this study, we have identified two novel molluscan BCL-2 cDNAs from Manila clam, Ruditapes philippinarum and named as RpBCL-2A and RpBCL-2B. There were four and three highly conserved BCL-2 homology (BH) regions in RpBCL-2A and RpBCL-2B, respectively suggesting these two genes could be different isoforms of anti-apoptotic BCL-2 family. Phylogenetic results revealed that Manila clam BCL-2 genes were clustered closely with invertebrate BCL-2 members. It gives evidence of their common origin and conserved features of invertebrate BCL-2 family. RpBCL-2A and 2B were expressed in tissue-specific manner showing the highest and lowest level of expression in gills and hemocytes, respectively. However there was no clear expression profile difference between two genes. After Vibrio tapetis challenge, transcriptional responses of RpBCL-2A and RpBCL-2B were induced in gills and hemocytes with high variation that could be due to effects of immune reactions of other host defense molecules., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Innate immune system relies on the recognition of pathogen associated molecular patterns present in the microbes by the pattern recognition receptors leading to the activation of signaling cascade and subsequent synthesis of cytokines. NF-κB is a major stimulus activated transcription factor, which regulates the expression of a diverse array of genes. IκB is an inhibitor of NF-κB, retaining NF-κB in an inactive state in the cytoplasm. In this study, we have reported the characterization of first abalone IκB (HdIκB). The cDNA possessed an ORF of 1200 bp coding for a protein of 400 amino acids with molecular mass of 45 kDa and isoelectric point of 4.7. HdIκB protein possessed a conserved phosphorylation site (58)DSGIFS(63) in the N-terminal region, six ankyrin repeats, and a PEST sequence in the C-terminal region. A casein kinase II phosphorylation site could also be observed in the PEST sequence. Constitutive expression of HdIκB revealed its physiological significance since NF-κB is known to be activated by various stimuli. Elevated expression of HdIκB transcripts could be observed in abalones challenged with various mitogens and live microbes. This novel characterization of abalone IκB would further be a positive approach in the affirmation of evolutionary conservation and significance of this protein as a repressor/inhibitor of a pleiotropic transcription factor like NF-κB., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Cystatins are a well-characterized group of cysteine protease inhibitors, which play crucial roles in physiology and immunity. In the present study, an invertebrate ortholog of cystatin B was identified in Manila clam (Ruditapes philippinarum) (RpCytB) and characterized at the molecular level, demonstrating its inhibitory activity against the well-known cysteine protease, papain. The complete coding sequence of RpCytB (297 bp in length) encodes a 99 amino acid peptide with a calculated molecular mass of 11 kDa and a theoretical isoelectric point of 5.9. The derived peptide was found to harbor typical features of cystatin proteins, including the 'Q-X-V-X-G' motif, which was identified as QLVAG in RpCytB. Phylogenetic analysis of RpCytB revealed close evolutionary relationships with its invertebrate counterparts, especially those from mollusks. Recombinant RpCytB (rRpCytB) was overexpressed as a fusion with maltose binding protein (MBP) in Escherichia coli BL21 (DE3) cells. Purified rRpCytB fusion protein exhibited a detectable inhibitory activity against papain, while the control MBP showed an almost constant negligible activity. While quantitative RT-PCR detected ubiquitous RpCytB expression in all tissues examined, the expressions in hemocytes and gills were relatively higher. Upon in vivo immune challenge with lipopolysaccharide (LPS), the expression of RpCytB in gills and hemocytes was down-regulated. Similar challenges with poly I:C and intact Vibrio tapetis bacteria revealed a complicated transcriptional regulation, wherein mRNA expression levels fluctuated over time of exposure. Moreover, a precise induction of RpCytB expression after bacterial infection was detected in gills by in situ hybridization. Collectively, our findings in this study indicate that RpCytB expression is sensitive to host pathological conditions and may contribute cysteine protease inhibitory activity to modulate the immune response., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

In this study, we describe the identification and characterization of manganese superoxide dismutase, an important antioxidant enzyme acting as the chief reactive oxygen species (ROS) scavenger, from rock bream Oplegnathus fasciatus (Of-mMnSOD) at genomic- and transcriptional-levels as well as the biological activity of recombinant protein. The Of-mMnSOD protein portrayed distinct MnSOD family features including signature motifs, metal association sites and the typical active site topology. It was also predicted to be localized in mitochondrial matrix. The Of-mMnSOD had a quinquepartite genome organization encompassing five exons interrupted by four introns. Comparison of its sequence and gene structure with that of other lineages emphasized its strong conservation among different vertebrates. The Of-mMnSOD was ubiquitously transcribed in different rock bream tissues with higher levels in blood cells and metabolically active tissues. Transcription of Of-mMnSOD was kinetically modulated in response to investigational challenges using mitogens (lipopolysaccharide and poly I:C) and live-pathogens (Edwardsiella tarda and rock bream irido virus) in blood cells and liver tissue. The purified recombinant Of-mMnSOD possessed potential antioxidant capacity and actively survived over a range of pH (7.5-11) and temperature (15-40 °C) conditions. Collectively, findings of this study suggest that Of-mMnSOD combats against oxidative stress and cellular damages induced by mitogen/pathogen-mediated inflammation, by detoxifying harmful ROS (O(2)(●-)) in rock bream., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Superoxide dismutases (SODs), antioxidant metalloenzymes, represent the first line of defense in biological systems against oxidative stress caused by excessive reactive oxygen species (ROS), in particular O(2)(•-). Two distinct members of SOD family were identified from Manila clam Ruditapes philippinarum (abbreviated as RpMnSOD and RpCu/ZnSOD). The structural analysis revealed all common characteristics of SOD family in both RpSODs from primary to tertiary levels, including three MnSOD signatures and two Cu/ZnSOD signatures as well as invariant Mn(2+)- and Cu/Zn(2+)-binding sites in RpMnSOD and RpCu/ZnSOD, respectively. Putative RpMnSOD and RpCu/ZnSOD proteins were predicted to be localized in mitochondrial matrix and cytosol, respectively. They shared 65.2% and 63.9% of identity with human MnSOD and Cu/ZnSOD, respectively. Phylogentic evidences indicated the emergence of RpSODs within molluscan monophyletic clade. The analogous spatial expression profiles of RpSODs demonstrated their higher mRNA levels in hemocytes and gills. The experimental challenges with poly I:C, lipopolysaccharide and Vibrio tapetis illustrated the time-dependent dynamic expression of RpSODs in hemocytes and gills. The recombinant RpMnSOD was expressed in a prokaryotic system and its antioxidant property was studied. The rRpMnSOD exhibited its optimum activity at 20 °C, under alkaline condition (pH 9) with a specific activity of 3299 U mg(-1). These outcomes suggested that RpSODs were constitutively expressing inducible proteins that might play crucial role(s) in innate immunity of Manila clam., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Two type I interferon (IFN) genes, designated as rbIFN1 and rbIFN2, have been cloned and characterized in rock bream. They are both comprised of 5 exons and 4 introns, and are closely linked on the rock bream chromosome in a unique head-to-head configuration. Both genes encode 183 amino acid (aa) precursor with a putative 17 aa signal peptide in the N-terminal. Only one amino acid divergence is present between two IFNs. Compared with the type I IFNs in higher vertebrates, two rock bream IFNs possess conserved alpha helical structure and share approximately 20% identity in aa sequence. The highest aa sequence homology (83.2%) was found with European seabass IFNs. Phylogenetic analysis grouped two rock bream IFNs into the subgroup-d of two-cysteine containing IFNs. The gene synteny analysis revealed that they are orthologous with the zebrafish IFNφ4 on chromosome-12 and paralogous to each other, which are likely derived from a gene duplication event followed by an inversion. A number of cis-regulatory elements associated with immune response including 15 IRF and 6 NF-κB binding sites are predicted in the shared 4.5 kb 5'-flanking region. Highest constitutive expression of two IFNs was detected in blood cells and skin. Their expression in blood cells and head kidney was up-regulated by lipopolysaccharide, poly I:C, Edwardsiella tarda, Streptococcus iniae and iridovirus. Furthermore, recombinant rbIFN1 protein produced by E. coli induced a rapid and transient expression of the interferon inducible Mx gene in head kidney cells. These results suggest that two duplicated type I IFN genes are involved in rock bream host response to both viral and bacterial pathogens., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

The complement component 9 (C9) is a single-chain glycoprotein that mediates formation of the membrane attack complex (MAC) on the surface of target cells. Full-length C9 sequence was identified from a cDNA library of rock bream (Oplegnathus fasciatus), and its genomic sequence was obtained by screening and sequencing of a bacterial artificial chromosome (BAC) genomic DNA library of rock bream. The rock bream complement component 9 (Rb-C9) gene contains 11 exons and 10 introns and is composed of a 1782 bp complete open reading frame (ORF) that encodes a polypeptide of 593 amino acids. Sequence analysis revealed that the Rb-C9 protein contains two thrombospondin type-1domains, a low-density lipoprotein receptor domain class A, a membrane attack complex & perforin (MACPF) domain, and an epidermal growth factor (EGF)-like domain. Important putative transcription factor binding sites, including those for NF-κB, SP-1, C/EBP, AP-1 and OCT-1, were found in the 5' flanking region. Phylogenetic analysis revealed a close proximity of Rb-C9 with the orthologues in puffer fish, and Japanese flounder. Quantitative real-time RT-PCR analysis confirmed that Rb-C9 was constitutively expressed in all the examined tissues isolated from healthy rock bream, with highest expression occurring in liver. Pathogen challenge, including Edwardsiella tarda, Streptococcus iniae, lipopolysaccharide endotoxin and rock bream iridovirus led to up-regulation of Rb-C9 in liver but no change in peripheral blood cells. The observed response to bacterial and viral challenges and high degree of evolutionary relationship to respective orthologues, confirmed that Rb-C9 is an important immune gene, likely involved in the complement system lytic pathway of rock bream., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

The interferon regulatory factor (IRF) members IRF4 and IRF8 contribute to B-lymphocyte development and can act as regulators of immunoglobulin (Ig) light chain gene transcription. These two IRFs are closely interrelated and are expressed at high levels in the lymphoid and myeloid cells of the immune system. In this study, the complete cDNA and genomic sequences of rock bream IRF4 (RbIRF4) and IRF8 (RbIRF8) were identified by homology screening of a multi-tissue normalized cDNA library and a BAC library, respectively, which had been established using Roche 454 GS-FLX™ technology. The full-length RbIRF4 cDNA is composed of 3442 bp and encodes a polypeptide of 462 amino acids; the genomic DNA is 9262 bp in length, consisting of eight exons and seven introns. The full-length RbIRF8 cDNA is composed of 2186 bp and encodes a 422 amino acid polypeptide; the genomic DNA is 4120 bp in length, consisting of nine exons and eight introns. The deduced amino acid sequences of RbIRF4 and RbIRF8 include a conserved DNA-binding domain (DBD) encompassing a tryptophan pentad-repeat and an IRF-association domain (IAD). Several putative transcription factor binding sites were also identified in 5' flanking region of both RbIRF4 and RbIRF8, and include those of immune-related factors. Quantitative real time PCR analysis of healthy rock bream detected the highest expression levels of RbIRF4 and RbIRF8 in lymphomyeloid-rich tissues. In addition, viral (rock bream iridovirus) and bacterial (Edwardsiella tarda and Streptococcus iniae) infection stimulated RbIRF4 and RbIRF8 expressions in head kidney and spleen. These results suggest not only that RbIRF4 and RbIRF8 may have a protective function against virus and bacteria pathogen invasion in rock bream, but also that IRFs may be immunomodulatory factors of teleost fish., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Ferritins are iron chelating proteins, which involve in iron metabolism and sequestration, contributing to the iron homeostasis in living organisms. In the present study, one ferritin subunit which was identified as H-like subunit was completely characterized in cDNA and protein levels from Manila clam (Ruditapes philippinarum); (RpFeH). The full length cDNA of RpFeH was 776 bp and it consisted of open reading frame of 513 bp, encoding a 171 amino acid peptide with a calculated molecular mass of 19.6 kDa and isoelectric point of 5.23. The amino acid sequence resembled the characteristic features of typical ferritin H subunits, including seven metal ligands in ferroxidase center, two iron binding region signatures and potential bio-mineralization residue (Thy(27)). Moreover, it was possible to identify an iron response element in 5' untranslated region, showing an agreement with previously reported ferritin H like subunits. The generated 3 dimensional tertiary structure of RpFeH showed a substantial consistency with the human ferritin H subunit, reinforcing its validity. Recombinant RpFeH was overexpressed in Escherichia coli BL21 (DE3) and purified. The recombinant protein showed a detectable iron binding activity according to the results obtained in our iron chelating activity assay. Furthermore it showed a noticeable anti-bacterial activity through suppressing the growth of Vibrio tapetis bacteria. The quantitative real time PCR detected ubiquitous RpFeH expression in tissues examined. However expression was found to be elevated in hemocyte and gill tissues. Transcriptional profile of Manila clam gill tissue, challenged with V. tapetis demonstrated a prolonged significant (P < 0.05) transcriptional upregulations from 12 h post injection to 48 h post injection. Our findings suggest that RpFeH functions as an iron chelating protein subunit in Manila clam while contributing to the innate immune responses against bacterial infections, via its iron withholding function.

Heat shock proteins (HSPs) production in cell is inducible by many physical and chemical stressors, providing adaptive significance for organisms when faced with environmental changes. In this study, we characterized a novel small HSP gene from disk abalone, designated as HdHSP20, and investigated its temporal expression by different environmental stimuli. The full-length genome sequence of HdHSP20 is composed of three exons and two introns. The 5' flanking region contains multiple putative transcription factor binding sites related to stress response. The open reading frame of the HdHSP20 cDNA is 480 bp and encodes 160 amino acid residues with 18.76 kDa molecular mass. The deduced amino acid sequence shares highest similarity with HSP20 genes from other invertebrates. HdHSP20 also shows several structural signatures of small HSP, including the conserved α-crystallin domain, the absence of cysteine residues, a high number of Glx/Asx residues and the compact β-sandwich structure in the C-terminal region. Overexpression of recombinant HdHSP20 protein conveyed enhanced thermotolerance to Escherichia coli cells, suggesting its functional activity in the cellular chaperone network. qRT-PCR measurements of HdHSP20 mRNA level have shown rapid and drastic induction by extreme temperatures, extreme salinities, heavy metals and the microbial infections. Collectively, our results suggest that HdHSP20 gene is likely involved in the stress resistant mechanisms in disk abalone. Its expression may serve as a potential biomarker capable to indicate a stress state in abalone due to extreme environmental change and pathogen infection., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Caspase 3 is a prominent mediator of apoptosis and participates in the cell death signaling cascade. In this study, caspase 3 was identified (Rbcasp3) and characterized from rock bream (Oplegnathus fasciatus). The full-length cDNA of Rbcasp3 is 2683 bp and contains an open reading frame of 849 bp, which encodes a 283 amino acid protein with a calculated molecular mass of 31.2 kDa and isoelectric point of 6.31. The amino acid sequence resembles the conventional caspase 3 domain architecture, including crucial amino acid residues in the catalytic site and binding pocket. The genomic length of Rbcasp3 is 7529 bp, and encompasses six exons interrupted by five introns. Phylogenetic analysis affirmed that Rbcasp3 represents a complex group in fish that has been shaped by gene duplication and diversification. Many putative transcription factor binding sites were identified in the predicted promoter region of Rbcasp3, including immune factor- and cancer signal-inducible sites. Rbcasp3, excluding the pro-domain, was expressed in Escherichia coli. The recombinant protein showed a detectable activity against the mammalian caspase 3/7-specific substrate DEVD-pNA, indicating a functional role in physiology. Quantitative real time PCR assay detected Rbcasp3 expression in all examined tissues, but with high abundance in blood, liver and brain. Transcriptional profiling of rock bream liver tissue revealed that challenge with lipopolysaccharides (LPS) caused prolonged up-regulation of Rbcasp3 mRNA whereas, Edwardsiella tarda (E. tarda) stimulated a late-phase significant transcriptional response. Rock bream iridovirus (RBIV) up-regulated Rbcasp3 transcription significantly at late-phase, however polyinosinic-polycytidylic acid (poly(I:C)) induced Rbcasp3 significantly at early-phase. Our findings suggest that Rbcasp3 functions as a cysteine-aspartate-specific protease and contributes to immune responses against bacterial and viral infections., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

In this study, we describe the molecular characterization, immune responses of rock bream, Oplegnathus fasciatus peroxiredoxin 6 cDNA (RbPrx6) and DNA protection activity of its recombinant protein. The full-length cDNA sequence of RbPrx6 was identified after pyrosequencing of rock bream cDNA library. RbPrx6 consists of 663 bp open reading frame (ORF) that codes for a putative protein of 221 amino acids with predicted molecular mass of 27 kDa. It showed characteristic peroxiredoxin super-family domain similar to vertebrate Prx counterparts. In the pair-wise comparison, RbPrx6 showed the highest amino acid identity (92.8%) to Scophthalmus maximus Prx6. Real-time RT-PCR analysis revealed that constitutive expression of RbPrx6 transcripts in eleven tissues selected from un-challenged fish showing the highest level in liver. Synthetic polyinosinic:polycytidylic acid (poly I:C) and iridovirus containing supernatant, up-regulated the RbPrx6 mRNA in liver. Purified recombinant RbPrx6 protein was able to protect supercoiled plasmid DNA from damages that is induced by metal-catalyzed generation of reactive oxygen species. Our results suggest that RbPrx6 may play an important role in regulating oxidative stress by scavenging of ROS, involving immune reactions and minimizing the DNA damage in rock bream., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

A disk abalone Haliotis discus discus 4.2 K cDNA microarray was designed by selecting abalone expressed sequence tags (ESTs). Transcriptional profiles in gills and hemocytes were analyzed upon abalone challenged with viral hemorrhagic septicemia virus (VHSV) in order to select candidates for screening of immune response genes. Among the 4188 genes analyzed, 280 (6.6%) transcripts were changed their expression level in gills and hemocytes against VHSV challenge compared to control animals. Total of 88 and 65 genes were up-regulated in gills and hemocytes, respectively. These genes can be grouped under various immune-functional categories such as transcription factors (Krüppell-like factor; ETS-family transcription factor), inflammatory and apoptosis related genes (TNF superfamily members, Fas ligand), IFN regulatory proteins (IFN-44 like, interferon gamma-inducible lysosomal thiol reductase) and detoxification proteins (glutathione peroxidase). In contrast, 25 and 102 genes were shown down-regulation in gills and hemocytes, respectively. Among the differentially expressed transcripts, considerably higher numbers of ESTs were represented as either hypothetical (unknown) proteins or no GenBank match suggesting those may be novel genes associated with internal defense of abalone., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Angiotensinogen (AGT) is the precursor of the renin-angiotensin system and contributes to osmoregulation, acute-phase and immune responses. A full-length cDNA of the AGT (2004 bp with a 1389 bp coding region) was isolated from rock bream (Rb), Oplegnathus fasciatus. The encoded polypeptide of 463 amino acids had a predicted molecular mass of 51.6 kDa. RbAGT possessed a deduced signal peptide of 22 residues upstream of a putative angiotensin I sequence ((23)NRVYVHPFHL(32)). RbAGT possessed a specific domain profile and a signature motif which are characteristics of the serpin family. Sequence homology and phylogenetic analysis indicated that RbAGT was evolutionarily closest to AGT of Rhabdosargus sarba. The mRNA expression profile of RbAGT was determined by quantitative RT-PCR and it demonstrated a constitutive and tissue-specific expression with the highest transcript level in the liver. Significantly up-regulated RbAGT expression was elicited by systemic injection of a lipopolysaccharide, rock bream iridovirus (RBIV) and bacteria (Edwardsiella tarda and Streptococcus iniae), revealing its pathogen inducibility. RbAGT manifested a down-regulated response to systemic injury, contemporaneously with two other serpins, protease nexin-1 (PN-1), and heparin cofactor II (HCII). In addition, a synchronized expression pattern was elicited by RbAGT and RbTNF-α in response to injury, suggesting that TNF-α might be a potential modulator of AGT transcription., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Thioredoxin (TRx) is a ubiquitous protein involved in the regulation of multiple biological processes. The TRx-2 isoform is exclusively expressed in mitochondria, where it contributes to mitochondrial redox state maintenance. In the present study, a novel thioredoxin-2 gene was identified in the Manila clam, Ruditapes philippinarum. The full-length sequence of RpTRx-2 (1561 bp) consists of a 498 bp coding region encoding a 166 amino acid protein. The N-terminal region of RpTRx-2 harbors a mitochondrial localization signal (56 amino acids), while the C-terminal portion contains the characteristic (89)WCGPC(93) catalytic active site. Phylogenetic analysis revealed that RpTRx-2 is closest to its ortholog from abalone. The broad distribution pattern of RpTRx-2 mRNA in healthy animal tissues implicates a generally significant function in normal clam physiology. The transcription level of RpTRx-2, however, is highest in hemocytes. Lipopolysaccharide and Vibrio tapetis bacterium caused up-regulation of the RpTrx-2 transcript levels in gill and hemocytes. Interestingly, clam manganese superoxide dismutase (MnSOD) mRNA levels in hemocytes elicited a corresponding response to these immune challenges. RpTRx-2 was recombinantly expressed in Escherichia coli BL21 (DE3) and used in insulin disulfide reduction assay as well as metal-catalyzed oxidation assay to elucidate its antioxidant property by reducing substrate and protecting super-coiled DNA from oxidative damage through free radical scavenging, respectively. Collectively, our data indicated that RpTRx-2, a mitochondrial TRx-2 family member, is an antioxidant enzyme that may be involved in antibacterial defense of clams., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Myeloid differentiation factor 88 (MyD88) is a universal adaptor protein which is required for signal transduction of TLR/IL-1R family. In this study, a novel molluscan MyD88 family member protein (named as RpMyD88) was identified from manila clam, Ruditapes philippinarum. It was identified using BLAST algorithm from GS-FLX™ sequencing data. The cDNA of RpMyD88 consists of 1416 bp open reading frame (ORF) encoding 471 amino acid residues. The RpMyD88 contains death domain and Toll/interleukin-1 receptor (TIR) domain which are typical features of MyD88 family proteins. The predicted amino acid sequence of RpMyD88 shares 27% identity with scallop MyD88. The expression level of RpMyD88 mRNA was investigated in healthy and challenged clams by quantitative real-time RT-PCR. The RpMyD88 gene expression is ubiquitous in all selected tissues. The RpMyD88 mRNA was strongly expressed in hemocyte, gill and mantle. In contrast, it was weakly expressed in siphon, foot and adductor muscle. RpMyD88 was up-regulated in gill and hemocyte after immune challenge with both Vibrio tapetis and LPS challenge. All results considered, sequence characterization, comparison and gene expression data suggesting that MyD88-dependent signaling pathway is presence in manila clam and RpMyD88 plays an important role in innate immune response against bacteria., (Copyright © 2011 Elsevier Ltd. All rights reserved.)