The molecular characterization, expression analysis and antimicrobial activity of theromacin from Asian polychaeta (Perinereis linea).

Antimicrobial peptides (AMPs) are molecular factors in innate immunity and are believed to play a key role in invertebrate host defence. We identified theromacin (TM) from an Asian polychaeta, Perinereis linea, using de novo RNA-seq analysis. TM, a typical AMP of invertebrates, is a cysteine-rich AMP with five disulfide bonds consisting of ten cysteine residues. In gene expression analysis, TM genes were constantly upregulated after lipopolysaccharide (LPS) stimulation. In contrast, after peptidoglycan (PGN) stimulation, it was upregulated initially and downregulated after 12 h. We synthesized a peptide based on the macin AMP in the TM amino acid sequence. The synthetic peptide showed antibacterial activity against some Gram-positive and Gram-negative bacteria. Therefore, the AMPs of P. linea might have broad roles in host defence and exhibit different degrees of activity.

Vibrio harveyi Infection Significantly Alters Amino Acid and Carbohydrate Metabolism in Whiteleg Shrimp, Litopenaeus vannamei.

Vibrio harveyi is one of the pathogens that threaten the shrimp farming industry. However, metabolic changes induced by V. harveyi infection in shrimp remain unknown. In this study, we first conducted high resolution-magic angle spinning (HR-MAS) nuclear magnetic resonance (NMR)-based metabolomics studies on gill, hepatopancreas, and haemolymph of V. harveyi-infected white leg shrimp, Litopenaeus vannamei. Using multivariate statistical analysis, we observed a clear separation between the early (3 and 9 h post-injection (hpi)) and late phases (24, 72 and 144 hpi) of the infection in all tissues. Moreover, metabolic changes in response to V. harveyi infection were faster in the haemolymph in the early phase and significantly changed in the late phase of the infection in the gills. Extensive changes were observed in the hepatopancreas, with 24 hpi being the turning point of progression from early to late phase infection in the hepatopancreas. V. harveyi infection increased the energy demand in L. vannamei and the amino acid and carbohydrate metabolism pathways also exhibited significant changes depending on the tissue. Thus, each tissue displayed different metabolic changes, depending on the progress of the infection.

Characterization of gene expression profiles and functional analysis of peptidoglycan recognition protein 2 from rock bream (Oplegnathus fasciatus).

Peptidoglycan recognition protein 2 (PGRP2) is a Zn2+-dependent peptidase that plays important roles in binding to microbial components of the cell membrane, inducing phagocytosis and antimicrobial activity. Rock bream (Oplegnathus fasciatus) PGRP2 (RbPGRP2) was identified in the intestine by next generation sequencing (NGS) analysis. The open reading frame (ORF) the RbPGRP2 cDNA (470 amino acid residues) contains a peptidoglycan recognition protein domain (residues 300 to 446). Alignment analysis revealed that RbPGRP2 shares 37.6-53.5% overall sequence identity with the PGRP2s of other species. Phylogenetic analysis revealed that RbPGRP2 clustered together with PGRP2s from teleosts. In healthy rock bream, RbPGRP2 was found to be ubiquitously expressed in all of the examined tissues, especially in the liver. RbPGRP2 expression was significantly upregulated in all of the examined tissues of rock bream after infection with Edwardsiella piscicida, Streptococcus iniae and red sea bream iridovirus (RSIV) compared with the control. Purified rRbPGRP2 interactions with bacteria and inhibited the growth of bacteria in the presence of Zn2+. These results indicate that RbPGRP2 plays an important role in the innate immune response against bacterial infection.

Differential expression of olive flounder (Paralichthys olivaceus) transcriptome during viral hemorrhagic septicemia virus (VHSV) infection at warmer and colder temperature.

The data presented here are related to the research article entitled "Temperature-dependent immune response of olive flounder (Paralichthys olivaceus) infected with viral hemorrhagic septicemia virus (VHSV)" [1]. In the cited article, we sequenced the whole transcriptome of the olive flounder using Illumina RNA-Seq. Differentially expressed genes (DEG) analysis of VHSV infected head kidney samples showed perturbations in gene expression. Herein we made a comparison of DEGs at early stage of VHSV infection of olive flounder (4 h post infection) in colder (13 °C) and warmer (20 °C) temperatures. The analysis of signaling pathways showed that several major immune pathways were altered. The gene ontology terms associated with the genes differentially expressed are also presented.

Molecular characterization, expression and functional analysis of peptidoglycan recognition protein-SC2 from rock bream, Oplegnathus fasciatus.

Peptidoglycan recognition proteins are members of the family of pattern recognition receptors (PRRs), that play important roles in the recognition of peptidoglycan and various biological processes. In this study, we have characterized peptidoglycan recognition protein-SC2 (PGRP-SC2) in rock bream (Oplegnathus fasciatus) (RbPGRP-SC2) and analysed its expression in various tissues after pathogen challenge. A sequence alignment revealed that the residues essential to zinc binding of the deduced protein were highly conserved among all the organisms. Phylogenetic analysis revealed that RbPGRP-SC2 is most closely related to the large yellow croaker PGRP-SC2. RbPGRP-SC2 was ubiquitously expressed in all tissues analysed, predominantly distributed in muscle and skin. After challenge with microbial pathogens (Edwardsiella piscicida), Streptococcus iniae or red seabream iridovirus [RSIV]), RbPGRP-SC2 was up-regulated in all the tissues examined, especially in liver. We produced recombinant RbPGRP-SC2 (rRbPGRP-SC2) using an Escherichia coli expression system. The rRbPGRP-SC2 had agglutination activity towards both Gram-negative (E. piscicida) and Gram-positive bacteria (S. iniae). In addition, rRbPGRP-SC2 induced leukocyte apoptosis and promoted leukocyte phagocytosis. These results suggest that the RbPGRP-SC2 plays an important role in the immune system and in maintaining cellular homeostasis of rock bream.

The first report of siglec-3/CD33 gene in a teleost (rock bream, Oplegnathus fasciatus): An analysis of its spatial expression during stimulation to red seabream iridovirus (RSIV) and two bacterial pathogens.

Siglec-3/CD33 is a myeloid-specific inhibitory receptor that is expressed on cells of the immune system, where it is believed to play a regulatory role, modulating the inflammatory and immune responses. We characterized CD33 (RbCD33) in rock bream which is a transmembrane protein with two IG-like domains and a cytoplasmic tail. It has a deduced amino acid sequence of 390 residues and has tyrosine-based signaling motifs in the cytoplasmic tail. The RbCD33 mRNA was highly expressed in peripheral blood leukocytes and was also detected in the muscle, spleen, skin, head kidney, gills, trunk kidney, heart, stomach, brain, intestine and liver by quantitative real-time PCR. A temporal variation in expression of RbCD33 was observed in different tissues after stimulating with E. tarda, S. iniae and red seabream iridovirus (RSIV). In the head kidney tissue, E. tarda and S. iniae induced RbCD33, while a down regulation was observed with RSIV. In addition, in spleen tissue, S. iniae caused a very high induction of RbCD33 in comparison with an E. tarda and RSIV challenge. In the liver and gill tissues, all three pathogens induced a high expression of RbCD33. The expression pattern in various tissues and its high induction after pathogen stimulation suggests that RbCD33 plays an important role in initiating the immune response via the inhibition of signal transduction of the myeloid lineage cells.

Olive flounder CD276 (B7-H3) a coinhibitory molecule for T cells: Responses during viral hemorrhagic septicemia virus (VHSV) stimulation.

Coinhibitory pathways in the B7-CD28 family provide critical inhibitory signals that regulate immune homeostasis, defense and protect tissue integrity. CD276 (B7-H3) is an important immune checkpoint member of this family, which is induced on antigen-presenting cells (APCs), and plays an important role in the inhibition of T-cell function. We have characterized the CD276 gene of olive flounder, Paralichthys olivaceus. OfCD276 has an ORF of 912 bp that codes for 303 amino acids with a predicted molecular mass of 33 kDa. It is a type I transmembrane protein with a single extracellular V- and C-like Ig domains, a transmembrane region, and a highly diverse cytoplasmic tail. This gene was distinctly expressed in gill, spleen, and skin, and sparsely expressed in other tissues. Pathogen stimulation by VHSV revealed that transcription of OfCD276 was induced on early hours in liver and expressed late in head kidney, spleen, intestine and gill tissues. Flow cytometry analysis of leukocytes revealed the percentage of granulocytes and lymphocytes that expressed OfCD276 molecules on their cell surface was 85.1% and 3.1%, respectively. Our study shows a significant role played by this coinhibitory molecule that participate in the regulation of the cell mediated immune response.

The first report of CD2 associated protein gene, in a teleost (Rock bream, Oplegnathus fasciatus): An investigation of the immune response upon infection with several pathogens.

CD2 is expressed on the surfaces of virtually all T cells and natural killer (NK) cells. In mammals, the CD2 molecule is 50 kDa. The cytoplasmic tail of CD2 interacts with CD2-associated protein (CD2AP), which plays an important role in mediating the trigger signal in outer magnetic pole cells. In this study, we identified CD2AP from rock bream and investigated its gene expression. The ORF of CD2AP (1950 bp) encodes 650 amino acids (aa). CD2AP has a Src homology 3 (SH3) domain. Quantitative real-time PCR analysis revealed that CD2AP shows higher expression in the gills and skin. Under experimental challenge, CD2AP gene expression was increased as relative to the control after 7 days. This result will improve our understanding of blood vessels in teleost fish, and will provide a basis for the study of CD2-related genes.

Molecular characterization of a T cell co-stimulatory receptor, CD28 of rock bream (Oplegnathus fasciatus): Transcriptional expression during bacterial and viral stimulation.

CD28 is a co-stimulatory receptor that provides a critical second signal alongside T cell receptors for the activation of naive T cells. We characterized the CD28 gene of rock bream, which has a deduced amino acid sequence of 221 residues with an extracellular Ig-superfamily V domain, transmembrane region, and cytoplasmic tail. The conservation in domain structures and other motifs shows that it is highly likely that RbCD28 is a homologue of mammalian CD28 and may have related co-stimulatory functions. RbCD28 is constitutively expressed in most tissues that were analysed, with a relatively higher expression in teleost lymphoid organs, such as spleens, gills, trunk kidneys and skin. Unlike human CD28, RbCD28 is highly expressed in skin and gill-associated lymphoid organs. Although gills showed constitutive expression of RbCD28 in control animals, after a pathogen challenge, induction of CD28 was low, particularly in RSIV and E. tarda infection. Whereas induction of RbCD28 was observed in kidney during E. tarda and S. iniae infection, downregulation was observed during RSIV infection. In the case of the liver, E. tarda caused an initial upregulation of RbCD28. RbCD28 activation of T cells in the spleen was limited to S. iniae infection. Activation of RbCD28 observed in lymphoid organs during infection of various pathogens shows its key role as a co-stimulatory receptor of T cells.

Protective responses of two paralogs of granulocyte colony stimulating factor (GCSF) in rock bream, Oplegnathus fasciatus during bacterial and viral infection.

Granulocyte colony stimulating factor (GCSF) has a key role in the production of neutrophilic granulocytes during normal hematopoietic development and release of neutrophils into the blood circulation. In this study we have identified and characterized two paralogs of GCSF (RbGCSF) in rock bream. Although RbGCSF-1 and RbGCSF-2 share low sequence conservation, its domains and protein structure still share significant similarity. Basal levels of RbGCSF-1 gene expression was high in peripheral blood leukocytes (PBLs), spleen and intestine whereas the RbGCSF-2 was highly expressed in PBLs and kidney, of healthy animals. A significant induction of RbGCSFs were observed after the challenge with Streptococcus iniae in kidney, spleen and gills during initial hours of infection. Whereas Edwarsiella tarda infection caused a reasonable expression in kidney. Red seabream iridovirus caused induction of RbGCSF-1 transcription only in gills during initial hours. This higher expression of RbGCSF in early hours may be its response to induce emergency hematopoiesis, due to shortage of neutrophils to combat the surge in pathogens. The difference in induction of RbGCSF paralogs during infection may constitute to its different roles or overlapping functions.

Molecular cloning and expression analysis of a new lily-type lectin in the rock bream, Oplegnathus fasciatus.

A new lily-type lectin RbLTL was identified from rock bream (Oplegnathus fasciatus) and its expression analysed. In this study, a new lily-type lectin gene (RbLTL) was cloned from rock bream using expressed sequence tag (EST) analysis. The full-length RbLTL cDNA was encoding a 117-amino acid protein. The deduced amino acid sequence of RbLTL contained all of the conserved features crucial for its fundamental structure, including B-lectin domain and three d-mannose binding sites. RbLTL mRNA was predominately expressed in the gills, with reduced expression noted in intestine tissue. Expression analysis of time series sampled fertilized eggs revealed that expression gradually increased 1, 3, 12, and 24 h: However, expression decreased at 36 h. RbLTL expression was differentially up-regulated in rock bream gills challenged with Streptococcus iniae, Edwardsiella tarda and RSIV. Our results revealed that novel rock bream lily-type lectin may be an important molecule involved in pattern recognition and pathogen elimination in the innate immunity of rock bream.

Piscidin: Antimicrobial peptide of rock bream, Oplegnathus fasciatus.

The piscidin family consists of antimicrobial peptides (AMPs) that are mainly found in fish and are crucial effectors of fish innate immune responses. The piscidin family typically has broad-spectrum antimicrobial activity and can modulate immune responses. In this study, we cloned rock bream piscidin (Rbpisc) and investigated its gene expression and biological activity (including antimicrobial and cytotoxic activities). The coding region of Rbpisc consisted of 213 base pairs (bp) encoding 70 amino acid residues. The tertiary structure predicted for Rbpisc includes an amphipathic helix-loop-helix structure. The Rbpisc gene was highly expressed in the gills of healthy fish. The gene expression of Rbpisc increased in the gills after pathogen infection, while the expression was down-regulated in other tissues. A synthetic peptide based on the AMP 12 domain amino acid sequence of Rbpisc appeared to have broad-spectrum antimicrobial activity against various bacteria. However, the synthetic peptide exhibited weak haemolytic activity against fish erythrocytes. These results suggest that Rbpisc might play an important role in the innate immune responses of rock bream.

Coagulation factor II from rock bream (Oplegnathus fasciatus): First report on the molecular biological function and expression analysis in the teleost.

The rapid haemostasis of fish prevents bleeding or infection that could be caused by physical properties of the aquatic environment. Additionally, the innate immune system is the first line of defence against infection and is responsible for the recognition of pathogen-associated molecular patterns, which are important for the activation of acquired immune responses. Coagulation factor II (CFII) is an important factor in the coagulation system and is involved in recognition and interaction with various bacterial and extracellular proteins. In this study, we identified and characterised the gene encoding CFII in rock bream (Oplegnathus fasciatus) (RbCFII) and analysed its expression in various tissues after a pathogen challenge. The full-length RbCFII cDNA (2079 bp) contained an open reading frame of 1854 bp encoding 617 amino acids. Alignment analysis revealed that a gamma-carboxyglutamic acid-rich domain, two kringle domains, and a trypsin-like serine protease domain of the deduced protein were well conserved. RbCFII was ubiquitously expressed in all tissues examined but, predominantly detected in the liver and skin. RbCFII expression was dramatically up-regulated in the kidney, spleen and liver after infection with Edwardsiella tarda, Streptococcus iniae, or red seabream iridovirus. The recombinant protein RbCFII (rRbCFII) produced using an Escherichia coli expression system was able to bind all examined bacteria. Interestingly, rRbCFII has agglutination activities towards E. coli and E. tarda, while no agglutination was shown toward Vibrio ordalii and S. iniae. These findings indicate that rRbCFII performs an immunological function in the immune response, and might be involved in innate immunity as well as blood coagulation.

First description of programmed cell death10 (PDCD10) in rock bream (Oplegnathus fasciatus): Potential relations to the regulation of apoptosis by several pathogens.

In this study, we isolated and characterized programmed cell death10 (PDCD10), which is known to be related to apoptosis, from rock bream (Oplegnathus fasciatus). The full-length rock bream PDCD10 (RbPDCD10) cDNA (1459 bp) contains an open reading frame of 633 bp that encodes 210 amino acids. Furthermore, multiple alignments revealed that the six of the α-helix bundles were well conserved among the other PDCD10 sequences tested. RbPDCD10 was significantly expressed in the liver, RBC (red blood cell), gill, intestine, trunk kidney and spleen. RbPDCD10 gene expression was also examined in several tissues, including the kidney, spleen, liver, and gill, under bacterial and viral challenges. Generally, all of the examined tissues from the fish that were infected with Edwardsiella tarda and the red sea bream iridovirus (RSIV) exhibited significant up-regulations of RbPDCD10 expression compared to the controls. However, RbPDCD10 expression exhibited dramatic down-regulations in all of the examined tissues following injections of Streptococcus iniae, which is major bacterial pathogen that is responsible for mass mortality in rock bream. Our results revealed that rock bream PDCD10 may be involved in the apoptotic regulation of rock bream immune responses.

Gene expression and functional characterization of serum amyloid P component 2 in rock bream, Oplegnathus fasciatus.

Mammalian serum amyloid P component (SAP) recognizes a wide range of exogenous pathogenic substances and activates a complementary pathway leading to pathogen clearance. To determine the potential roles of SAP in the fish immune system, SAP (RbSAP2) gene was cloned from ESTs analysis of rock bream (Oplegnathus fasciatus), which consisted of a signal peptide and pentraxin domain. Phylogenetic analysis revealed that the RbSAP2 gene was classified with other known fish SAPs. RbSAP2 was highly expressed in the liver of healthy rock bream. Overall, pathogen exposure led to an induction of RbSAP2 in the liver and spleen, although this effect was not observed in the spleen following infection with Edwardsiella tarda. A high concentration of recombinant RbSAP2 (rRbSAP2) showed lower growth Streptococcus iniae than control in the absence of Ca(2+), whereas E. tarda growth was decreased by high concentration of rRbSAP in the presence of the Ca(2+). These results suggest that RbSAP plays an important role in the immune response against invading pathogens.

First molecular characterisation and expression analysis of a teleost thioredoxin-interacting protein (TXNIP) gene from rock bream (Oplegnathus fasciatus).

Thioredoxin-interacting protein (TXNIP) is an important regulator of glucose metabolism that functions by inhibiting cellular glucose uptake. The full-length rock bream (Oplegnathus fasciatus) TXNIP (RbTXNIP) cDNA (2499 bp) contains an open reading frame of 1188 bp encoding 396 amino acids. Furthermore, multiple alignments showed that the arrestin domain was well conserved among the other TXNIP sequences tested. RbTXNIP was predicted to contain a PxxP and PPxY motif. Phylogenetic analysis indicated that RbTXNIP is most closely related to Fugu rubripes TXNIP. RbTXNIP was expressed significantly in the RBC, intestine, and spleen. RbTXNIP mRNA expression was also examined in several tissues under conditions of bacterial and viral challenge. Generally, all tissues examined from fish infected with Streptococcus iniae, Edwardsiella tarda and red sea bream iridovirus (RSIV) showed significant downregulation in RbTXNIP expression compared to controls. However, RbTXNIP expression showed significant upregulation in the spleen and kidney after injection of recombinant rock bream TRx1 protein. These findings provide a molecular foundation for functional studies and applications in teleosts.

Expression analysis and biological activity of moronecidin from rock bream, Oplegnathus fasciatus.

The piscidin-family, one of antimicrobial peptides (AMPs) mainly distributed in fish, is crucial effectors of fish innate immune response. Piscidin-family typically has broad-spectrum antimicrobial activity and the ability to modulate the immune response. In this study, we identified moronecidin (Rbmoro) included in piscidin-family from rock bream and investigated its gene expression using quantitative real-time PCR and biological activity (including antimicrobial and cytotoxic activity). The coding region of Rbmoro was 204 bp encoding 67 amino acid residues. Tertiary structure prediction of Rbmoro showed an amphipathic α-helical structure. Rbmoro gene was widely expressed in different tissues of healthy fish. Additionally, Rbmoro gene expression was induced in all tested tissues after infection with Edwardsiella tarda, Streptococcus iniae and red seabream iridovirus. We synthesized mature peptide of Rbmoro based on amino acid sequence of its AMP 12 domain, and the synthetic peptide appeared broad-spectrum antimicrobial activity to various bacteria. However, the synthetic peptide has weak haemolytic activity against fish erythrocytes. These results suggest that Rbmoro might play an important role in innate immune response of rock bream.

Molecular cloning and expression analysis of interleukin (IL)-15 and IL-15 receptor α from rock bream, Oplegnathus fasciatus.

Mammalian interleukin (IL)-15 plays an important role in the activation of CD8(+) T cells and natural killer (NK) cells along with its receptor α (IL-15Rα). To understand the potential roles of IL-15 and IL-15Rα in fish, we identified IL-15 and IL-15Rα cDNA from rock bream (Oplegnathus fasciatus) and investigated their gene expression profiles after bacterial and viral infection. Coding regions of rock bream (Rb) IL-15 and RbIL-15Rα cDNAs were 534 and 402 bp encoding 177 and 133 amino acid residues, respectively. The sushi domain of IL-15Rα was highly conserved between rock bream and other species. Unlike other IL-15Rαs, RbIL-15Rα does not have a transmembrane region. Gene expression of RbIL-15 and RbIL-15Rα was widely expressed in different tissues of healthy fish, especially immune-related tissues. RbIL-15 and RbIL-15Rα were highly induced in the kidney and spleen after infection with Edwardsiella tarda, Streptococcus iniae and red seabream iridovirus. Gene expression patterns of RbIL-15 and RbIL-15Rα were similar in the kidney and spleen after pathogen infection. However, these genes were differentially induced in the liver after pathogen infection. These results suggest that the different responses of RbIL-15 and RbIL-15Rα to pathogen infection may be induced by different tissues or cell types.