Characterization of the impact of dietary immunostimulant CpG on the expression of mRNA biomarkers involved in the immune responses in Atlantic salmon (Salmo salar).

Infectious diseases have significantly impacted Atlantic salmon aquaculture worldwide. Modulating fish immunity with immunostimulant-containing functional feeds could be an effective strategy in mitigating disease problems. Previously, we characterized the impact of polyriboinosinic polyribocytidylic acid (pIC) and formalin-killed typical Aeromonas salmonicida bacterin on miRNA expression in Atlantic salmon fed a commercial diet with and without immunostimulant CpG. A set of miRNA biomarkers of Atlantic salmon head kidney responding to pIC and/or bacterin immune stimulations was identified (Xue et al., 2019) [1]. Herein, we report a complementary qPCR study that investigated the impact of the pIC, bacterin and dietary CpG on the expression of immune-relevant mRNAs (n = 31) using the same samples as in the previous study (Xue et al., 2019) [1]. Twenty-six of these genes were predicted target transcripts of the pIC- and/or bacterin-responsive miRNAs identified in the earlier study. The current data showed that pIC and/or bacterin stimulations significantly modulated the majority of the qPCR-analyzed genes involved in various immune pathways. Some genes responded to both stimulations (e.g. tnfa, il10rb, ifng, irf9, cxcr3, campb) while others appeared to be stimulation specific [e.g. irf3, irf7a, il1r1, mxa, mapk3 (pIC only); clra (bacterin only)]. A. salmonicida bacterin stimulation produced a strong inflammatory response (e.g. higher expression of il1b, il8a and tnfa), while salmon stimulated with pIC showed robust interferon responses (both type I and II). Furthermore, the current data indicated significant down-regulation of immune-relevant transcripts (e.g. tlr9, irf5, il1r1, hsp90ab1, itgb2) by dietary immunostimulant CpG, especially among pre-injection and PBS-injected fish. Together with our prior miRNA study, the present research provided complementary information on Atlantic salmon anti-viral and anti-bacterial immune responses and on how dietary CpG may modulate these responses.

Transcriptomic Profiling in Fins of Atlantic Salmon Parasitized with Sea Lice: Evidence for an Early Imbalance Between Chalimus-Induced Immunomodulation and the Host's Defense Response.

Parasitic sea lice (e.g., Lepeophtheirus salmonis) cause costly outbreaks in salmon farming. Molecular insights into parasite-induced host responses will provide the basis for improved management strategies. We investigated the early transcriptomic responses in pelvic fins of Atlantic salmon parasitized with chalimus I stage sea lice. Fin samples collected from non-infected (i.e. pre-infected) control (PRE) and at chalimus-attachment sites (ATT) and adjacent to chalimus-attachment sites (ADJ) from infected fish were used in profiling global gene expression using 44 K microarrays. We identified 6568 differentially expressed probes (DEPs, FDR < 5%) that included 1928 shared DEPs between ATT and ADJ compared to PRE. The ATT versus ADJ comparison revealed 90 DEPs, all of which were upregulated in ATT samples. Gene ontology/pathway term network analyses revealed profound changes in physiological processes, including extracellular matrix (ECM) degradation, tissue repair/remodeling and wound healing, immunity and defense, chemotaxis and signaling, antiviral response, and redox homeostasis in infected fins. The QPCR analysis of 37 microarray-identified transcripts representing these functional themes served to confirm the microarray results with a significant positive correlation (p < 0.0001). Most immune/defense-relevant transcripts were downregulated in both ATT and ADJ sites compared to PRE, suggesting that chalimus exerts immunosuppressive effects in the salmon's fins. The comparison between ATT and ADJ sites demonstrated the upregulation of a suite of immune-relevant transcripts, evidencing the salmon's attempt to mount an anti-lice response. We hypothesize that an imbalance between immunomodulation caused by chalimus during the early phase of infection and weak defense response manifested by Atlantic salmon makes it a susceptible host for L. salmonis.

Identification of a gene encoding a membrane-anchored toll-like receptor 5 (TLR5M) in Oplegnathus fasciatus that responds to flagellin challenge and activates NF-κB.

Toll-like receptor 5 (TLR5) recognizes bacterial flagellin and induces the downstream signaling through the myeloid differentiation primary response gene 88 (MyD88) protein to produce proinflammatory cytokines. In this study, we describe a TLR5 membrane form (OfTLR5M) and its adaptor protein MyD88 (OfMyD88) in rock bream, Oplegnathus fasciatus. Both Oftlr5m (6.7 kb) and Ofmyd88 (3.7 kb) genes displayed a quinquepartite structure with five exons and four introns. Protein structure of OfTLR5M revealed the conventional architecture of TLRs featured by an extracellular domain with 22 leucine rich repeats (LRR), a transmembrane domain and an endodomain with TIR motif. Primary OfTLR5M sequence shared a higher homology with teleost TLR5M. The evolutional analysis confirmed that TLR5 identified in the current study is a membrane receptor and the data further suggested the co-evolution of the membrane-anchored and soluble forms of TLR5 in teleosts. Inter-lineage comparison of gene structures in vertebrates indicated that the tlr5m gene has evolved with extensive rearrangement; whereas, the myd88 gene has maintained a stable structure throughout the evolution. Inspection of 5' flanking region of these genes disclosed the presence of several transcription factor binding sites including NF-κB. Quantitative real-time PCR (qPCR) detected Oftlr5m mRNA in eleven tissues with the highest abundance in liver. In vivo flagellin administration strongly induced the transcripts of both Oftlr5m and Ofmyd88 in gills and head kidney tissues suggesting their ligand-mediated upregulation. In a luciferase assay, HEK293T cells transiently transfected with Oftlr5m and Ofmyd88 demonstrated a higher NF-κB activity than the mock control, and the luciferase activity was intensified when cells were stimulated with flagellin. Collectively, our study represents the genomic, evolutional, expressional and functional insights into a receptor and adaptor molecules of teleost origin that are involved in flagellin sensing.

Molecular insights of two STAT1 variants from rock bream (Oplegnathus fasciatus) and their transcriptional regulation in response to pathogenic stress, interleukin-10, and tissue injury.

Signal transducers and activators of transcription 1 (STAT1) is critically involved in mediating cytokine-driven signaling, and triggers the transcription of target genes to activate cellular functions. Although the structural and functional aspects of STAT members have been well described in mammals, only limited information is available for the STAT genes in teleost fishes. In the present study, two variants of STAT1 genes (RbSTAT1 and RbSTAT1L) were identified from rock bream and characterized at the cDNA and genomic sequence levels. RbSTAT1 and RbSTAT1L were found to share a common domain architecture with mammalian STAT1. Phylogenetic analysis revealed that RbSTAT1 shows a common evolutionary trajectory with other STAT1 counterparts, whereas RbSTAT1L showed a separate path, implying that it could be a novel member of the STAT family. The genomic organizations of RbSTAT1 and RbSTAT1L illustrated a similar exon-intron pattern with 23 exons in the coding sequence. Transcription factor-binding sites, which are mostly involved in the regulation of immune responses, were predicted at the putative promoter regions of the RbSTAT1 and RbSTAT1L genes. SYBR Green qPCR analysis revealed the ubiquitous expression of RbSTAT1 and RbSTAT1L transcripts in different fish tissues with the highest level observed in peripheral blood cells. Significantly modulated transcripts were noted upon viral (rock bream iridovirus [RBIV]), bacterial (Edwardsiella tarda and Streptococcus iniae), and pathogen-associated molecular pattern (lipopolysaccharide and poly I:C) stimulations. The WST-1 cell viability assay affirmed the potential antiviral capacity of RbSTAT1 and RbSTAT1L against RBIV. A possible role of RbSTAT1 and RbSTAT1L in the wound healing process was revealed according to their modulated expression in injured fish. In addition, the transcriptional regulation of RbSTAT1 and RbSTAT1L was analyzed by qPCR following stimulation with rock bream interleukin-10. Taken together, these findings suggest that the STAT1-mediated Janus kinase/STAT pathway might at least in part be involved in the regulatory mechanisms underlying the immune defensive roles against microbial pathogens and the wound healing process.

A molluscan calreticulin ortholog from Haliotis discus discus: Molecular characterization and transcriptional evidence for its role in host immunity.

Calreticulin (CALR), a Ca(2+) binding chaperone of the endoplasmic reticulum (ER) and mainly involved in Ca(2+) storage and signaling. In this study, we report the molecular characterization and immune responses of CALR homolog from disk abalone (AbCALR). The full length AbCALR cDNA (1837 bp) had an ORF of 1224 bp. According to the multiple alignments analysis, N- and P-domains were highly conserved in all the selected members of CALRs. In contrast, the C-domain which terminated with the characteristic ER retrieval signal (HDEL) was relatively less conserved. The phylogenetic analysis showed that all the selected molluscan homologs clustered together. Genomic sequence of AbCALR revealed that cDNA sequence was dispersed into ten exons interconnected with nine introns. AbCALR mRNA expression shows the significant (P < 0.05) up-regulation of AbCALR transcripts in hemocytes upon bacterial (Listeria monocytogenes and Vibrio parahaemolyticus), viral (Viral haemorrhagic septicaemia virus; VHSV) and immune stimulants (LPS and poly I:C) challenges at middle and/or late phases. These results collectively implied that AbCALR is able to be stimulated by pathogenic signals and might play a potential role in host immunity.

Molecular, genomic, and expressional delineation of a piscidin from rock bream (Oplegnathus fasciatus) with evidence for the potent antimicrobial activities of Of-Pis1 peptide.

The piscidin family comprises a group of antimicrobial peptides (AMPs) that are vital components of teleost innate immunity. Piscidins protect the host from pathogens, through multifaceted roles as immunomodulators and anti-infective peptides. The present study reports the identification, and characterization of a putative piscidin homolog, Of-Pis1, from rock bream (Oplegnathus fasciatus). A combined genomic and transcriptomic approach revealed that the Of-Pis1 gene comprises 1396 nucleotides (nt), four exons, and three introns. The cDNA with the 213 nt open reading frame encoded a 70-amino acid preprotein consisting of a signal peptide, a mature peptide, and a prodomain. Predicted mature Of-Pis1 was assumed to be a membrane-active AMP, based on the prediction of an amphipathic α-helical conformation with a net charge of +4. In addition, Of-Pis1 demonstrated significant similarities with other piscidin family members in terms of gene structure, sequence homology, and evolutionary relationship. Examination by quantitative real-time PCR (qPCR) of basal transcription of Of-Pis1 in the tissues of naïve rock bream, revealed predominant transcript levels in the gills, followed by the spleen, intestine, skin, and head kidney. In gill tissues, the temporally induced mRNA expression of Of-Pis1, upon in vivo injection trials with lipopolysaccharide (LPS); polyinosinic:polycytidylic acid (poly I:C); and pathogens, including Edwardsiella tarda, Streptococcus iniae, and rock bream iridovirus (RBIV), was weak. In contrast, in vivo flagellin administration led to a robust upregulation of Of-Pis1 in different tissues. Antimicrobial potency was determined by employing recombinant (rOf-Pis1), and synthetic (pOf-Pis1) peptides, in in vitro assays. Recombinant overexpression inhibited the growth of bacteria expressing the rOf-Pis1 protein in a growth delay assay. The broad antimicrobial spectrum of pOf-Pis1 was evidenced by its potent activity against an array of microbes, including bacteria, fungi, and parasitic species. In addition, pOf-Pis1 showed no significant hemolytic toxicity against human erythrocytes. Collectively, the data presented in the current study improve our understanding of the piscidin AMP family, and the contribution of Of-Pis1 to the rock bream immunity.

Antimicrobial response of galectin-1 from rock bream Oplegnathus fasciatus: Molecular, transcriptional, and biological characterization.

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 ß-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.

Thioredoxin domain-containing protein 12 from Oplegnathus fasciatus: Molecular characterization, expression against immune stimuli, and biological activities related to oxidative stress.

Thioredoxin domain-containing protein 12 (TXNDC12) is a small, disulfide-containing protein that belongs to the thioredoxin (TXN) superfamily. In the present study, we identified and characterized a TXNDC12-like gene, designated OfTXNDC12, from rock bream, Oplegnathus fasciatus. OfTXNDC12 consists of seven exons interrupted by six introns. Comparative genomic structural analysis revealed that the TXNDC12 of vertebrates is a structurally conserved gene. The coding sequence of OfTXNDC12 comprises 522 bp, which encodes 173 amino acid residues with the conserved thioredoxin active site motif, CGAC, and a probable C-terminal ER retrieval motif, GDEL. Transcriptional analysis of OfTXNDC12 showed the highest concentrations of the mRNA transcript in the liver, implying that it has a significant role in the liver under normal physiological conditions. In comparison, injection of lipopolysaccharide, Edwardsiella tarda, Streptococcus iniae, polyinosinic:polycytidylic acid (poly[I:C]) and rock bream iridovirus mostly triggered greater upregulation of OfTXNDC12 transcript levels in liver than in gill tissue, supporting its potential functional importance in the liver. Insulin disulfide reduction assay showed that the recombinant fusion protein (rOfTXNDC12) possesses significant thioredoxin activity. Treatment of LNCaP cells with the recombinant protein along with H2O2 revealed that rOfTXNDC12 increased the viability of cells and further supported its putative antioxidant capacity. Taken together, the results from our study suggest that OfTXNDC12 encodes for a potent antioxidant involved in redox regulation that shows significant responses to immune stimuli.

First comparative characterization of three distinct ferritin subunits from a teleost: Evidence for immune-responsive mRNA expression and iron depriving activity of seahorse (Hippocampus abdominalis) ferritins.

Ferritins play an indispensable role in iron homeostasis through their iron-withholding function in living beings. In the current study, cDNA sequences of three distinct ferritin subunits, including a ferritin H, a ferritin M, and a ferritin L, were identified from big belly seahorse, Hippocampus abdominalis, and molecularly characterized. Complete coding sequences (CDS) of seahorse ferritin H (HaFerH), ferritin M (HaFerM), and ferritin L (HaFerL) subunits were comprised of 531, 528, and 522 base pairs (bp), respectively, which encode polypeptides of 177, 176, and 174 amino acids, respectively, with molecular masses of ∼20-21 kDa. Our in silico analyses demonstrate that these three ferritin subunits exhibit the typical characteristics of ferritin superfamily members including iron regulatory elements, domain signatures, and reactive centers. The coding sequences of HaFerH, M, and L were cloned and the corresponding proteins were overexpressed in a bacterial system. Recombinantly expressed HaFer proteins demonstrated detectable in vivo iron sequestrating (ferroxidase) activity, consistent with their putative iron binding capability. Quantification of the basal expression of these three HaFer sequences in selected tissues demonstrated a gene-specific ubiquitous spatial distribution pattern, with abundance of mRNA in HaFerM in the liver and predominant expression of HaFerH and HaFerL in blood. Interestingly, the basal expression of all three ferritin genes was found to be significantly modulated against pathogenic stress mounted by lipopolysaccharides (LPS), poly I:C, Streptococcus iniae, and Edwardsiella tarda. Collectively, our findings suggest that the three HaFer subunits may be involved in iron (II) homeostasis in big belly seahorse and that they are important in its host defense mechanisms.

Three novel C1q domain containing proteins from the disk abalone Haliotis discus discus: Genomic organization and analysis of the transcriptional changes in response to bacterial pathogens.

The globular C1q (gC1q) domain containing proteins, commonly referred as C1q domain containing (C1qDC) proteins, are an essential family of proteins involved in various innate immune responses. In this study, three novel C1qDC proteins were identified from the disk abalone (Haliotis discus discus) transcriptome database and designated as AbC1qDC1, AbC1qDC2, and AbC1qDC3. The cDNA sequences of AbC1qDC1, AbC1qDC2, and AbC1qDC3 consisted of 807, 1305, and 660 bp open reading frames (ORFs) encoding 269, 435, and 220 amino acids (aa), respectively. Putative signal peptides and the N-terminal gC1q domain were identified in all three AbC1qDC proteins. An additional predicted motif region, known as the coiled coil region (CCR), was identified next to the signal sequence of AbC1qDC2. The genomic organization of the AbC1qDCs was determined using a bacterial artificial chromosome (BAC) library. It was found that the CDS of AbC1qDC1 was distributed among three exons, while the CDSs of AbC1qDC2 and AbC1qDC3 were distributed between two exons. Sequence analysis indicated that the AbC1qDC proteins shared <40% identity with other counterparts from different species. According to the neighbor-joining phylogenetic tree, the proteins were grouped within an invertebrate group with high evolutionary distances, which suggests that they are new members of the C1qDC family. Higher expression of AbC1qDC1 and AbC1qDC2 was detected in hepatopancreas, muscle, and mantle tissues compare to the other tissues analyzed, using reverse transcription, followed by quantitative real-time PCR (qPCR) using SYBR Green, whereas AbC1qDC3 was predominantly expressed in gill tissues, followed by muscles and the hepatopancreas. The temporal expression of AbC1qDC transcripts in gills after bacterial (Vibrio parahaemolyticus and Listeria monocytogenes) and lipopolysaccharide stimulation indicated that AbC1qDCs can be strongly induced by both Gram-negative and Gram-positive bacterial species with different response profiles. The results of this study suggest that AbC1qDCs are involved in immune responses against invading bacterial pathogens.

Identification and molecular characterization of peroxiredoxin 6 from Japanese eel (Anguilla japonica) revealing its potent antioxidant properties and putative immune relevancy.

Peroxiredoxins (Prdx) are thiol specific antioxidant enzymes that play a pivotal role in cellular oxidative stress by reducing toxic peroxide compounds into nontoxic products. In this study, we identified and characterized a peroxiredoxin 6 counterpart from Japanese eel (Anguilla japonica) (AjPrdx6) at molecular, transcriptional and protein level. The identified full-length coding sequence of AjPrdx6 (669 bp) coded for a polypeptide of 223 aa residues (24.9 kDa). Deduced protein of AjPrdx6 showed analogy to characteristic structural features of 1-cysteine peroxiredoxin sub-family. According to the topology of the generated phylogenetic reconstruction AjPrdx6 showed closest evolutionary relationship with Salmo salar. As detected by Quantitative real time PCR (qPCR), AjPrdx6 mRNA was constitutively expressed in all the tissues examined. Upon the immune challenges with Edwardsiella tarda, lipopolysaccharides and polyinosinic:polycytidylic acid, expression of AjPrdx6 mRNA transcripts were significantly induced. The general functional properties of Prdx6 were confirmed using purified recombinant AjPrdx6 protein by deciphering its potent protective effects on cultured vero cells (kidney epithelial cell from an African green monkey) against H2O2-induced oxidative stress and protection against oxidative DNA damage elicited by mixed function oxidative (MFO) system. Altogether, our findings suggest that AjPrdx6 is a potent antioxidant protein in Japanese eels and its putative immune relevancy in pathogen stress mounted by live-bacteria or pathogen associated molecular patterns (PAMPs).

Molecular characterization, genomic structure and expressional profiles of a CXC chemokine receptor 4 (CXCR4) from rock bream Oplegnathus fasciatus.

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.

Gene structure, molecular characterization and transcriptional expression of two p38 isoforms (MAPK11 and MAPK14) from rock bream (Oplegnathus fasciatus).

The p38 kinases are one of the four subgroups of mitogen-activated protein kinase (MAPK) superfamily which are involved in the innate immunity. The p38 subfamily that includes four members namely p38α (MAPK14), p38ß (MAPK11), p38γ (MAPK12) and p38δ (MAPK13), regulates the activation of several transcription factors. In this study, a p38ß (OfMAPK11) homolog and a p38α (OfMAPK14) homolog of Oplegnathus fasciatus were identified at genomic level. Results clearly showed that both MAPK11 and MAPK14 are well-conserved at both genomic structural- and amino acid (aa)-levels. Genomic sequences of OfMAPK11 (∼ 15.6 kb) and OfMAPK14 (∼ 13.4 kb) had 12 exons. A comparison of exon-intron structural arrangement of these genes from different vertebrate lineages indicated that all the exon lengths are highly conserved, except their terminal exons. Full-length cDNAs of OfMAPK11 (3957 bp) and OfMAPK14 (2504 bp) encoded corresponding proteins of 361 aa and 360 aa, respectively. Both OfMAPK proteins harbored a Ser/Thr protein kinases catalytic domain (S_TKc domain) which includes an activation loop with a dual phosphorylation site (TGY motif) and several specific-binding sites for ATP and substrates. Molecular modeling of the activation loop and substrate binding sites of rock bream MAPKs revealed the conservation of crucial residues and their orientation in 3D space. Transcripts of OfMAPKs were ubiquitously detected in eleven tissues examined, however at different levels. The modulation of OfMAPKs' transcription upon pathogen-associated molecular patterns (PAMPs: flagellin, lipopolysaccharide and poly I:C) and pathogens (Edwardsiella tarda, Streptococcus iniae and rock bream iridovirus) was investigated. Among the seven examined tissues, the flagellin-challenge upregulated the mRNA level of both OfMAPKs in the head kidney. Meanwhile, modulation of OfMAPK mRNA expression in the liver upon other immune-challenges varied in a time-dependent manner. Collectively, these results suggest that OfMAPKs are true members of p38 subfamily, which might be induced by different immune stimuli.

A CXC chemokine gene, CXCL12, from rock bream, Oplegnathus fasciatus: Molecular characterization and transcriptional profile.

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.

Insights into molecular profiles and genomic evolution of an IRAK4 homolog from rock bream (Oplegnathus fasciatus): immunogen- and pathogen-induced transcriptional expression.

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.

Molecular genomic- and transcriptional-aspects of a teleost TRAF6 homolog: Possible involvement in immune responses of Oplegnathus fasciatus against pathogens.

Tumor necrosis factor receptor (TNFR)-associated factor 6 (TRAF6) is a crucial docking molecule for TNFR superfamily and Interleukin-1 receptor/Toll-like receptor (IL-1R/TLR) superfamily. As an adaptor protein in pathogen-induced signaling cascades, TRAF6 modulates both adaptive- and innate-immunity. In order to understand the immune responses of teleost TRAF6, Oplegnathus fasciatus TRAF6-like gene (OfTRAF6) was identified and characterized. Genomic length of OfTRAF6 (4 kb), obtained by means of a genomic BAC library, spanned seven exons which represented a putative coding sequence of 1716 bp and encoded 571 amino acids (aa) with an estimated molecular weight of 64 kDa. This putative protein demonstrated the classical tetra-domain architecture composed of a zinc finger RING-type profile, two zinc finger TRAF-type profiles, a coiled-coil region and a MATH domain. While the sequence similarity with human TRAF6 was 66.5%, OfTRAF6 shared a higher overall similarity with teleost homologs (∼75-92%). Phylogeny of TRAF-family was examined and TRAF6-subfamily appeared to be the precursor of other subfamilies. In addition, the clustering pattern confirmed that OfTRAF6 is a novel member of TRAF6subfamily. Based on comparative genomic analysis, we found that vertebrate TRAF6 exhibits two distinct structures in teleost and tetrapod lineages. An intron-loss event has probably occurred in TRAF6 gene during the evolution of tetrapods from teleosts. Inspection of putative OfTRAF6 promoter revealed the presence of several immune responsive transcription factor binding sites. Real-time qPCR assay detected OfTRAF6 transcripts in eleven juvenile fish tissues with higher levels in peripheral blood cells followed by liver. Putative role of OfTRAF6 in response to flagellin, LPS, poly I:C, pathogenic bacteria (Edwardsiella tarda and Streptococcus iniae) and rock bream iridovirus (RBIV) was profiled in different tissues and OfTRAF6 revealed up-regulated transcript levels. Altogether, these findings implicate that OfTRAF6 is not only involved in flagellin-induced signaling cascade, but also contributes to the antibacterial- and antiviral-responses.

Three novel clade B serine protease inhibitors from disk abalone, Haliotis discus discus: Molecular perspectives and responses to immune challenges and tissue injury.

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.

Two variants of selenium-dependent glutathione peroxidase from the disk abalone Haliotis discus discus: Molecular characterization and immune responses to bacterial and viral stresses.

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.

A homolog of Kunitz-type serine protease inhibitor from rock bream, Oplegnathus fasciatus: Molecular insights and transcriptional modulation in response to microbial and PAMP stimulation, and tissue injury.

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.

Antibacterial activity and immune responses of a molluscan macrophage expressed gene-1 from disk abalone, Haliotis discus discus.

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.