Jin-Sol Bae

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.

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.

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.

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

Abstract 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. HighlightsCD28 a T cell costimulatory receptor is identified in Rock bream.Conservation in domain and motifs shows that RbCD28 is a homolog of mammalian CD28.It was highly expressed in various teleost lymphoid organs including gill and skin.E. tarda, S. iniae and RSIV infection caused differential expression of RbCD28 in lymphoid organs.Activation of RbCD28 in lymphoid organs during infection shows its key role as a costimulatory receptor of T cells.

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.

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.

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

&NA; 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.

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

&NA; 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. HighlightsTwo GCSF paralogs of rock bream were identified and characterized.Basal levels of GCSF‐1 and GCSF‐2 expression were high in tissues with immuno‐hematopoietic related functions.High induction of GCSF paralogs in kidney, spleen and gills of rock bream after the challenge with Streptococcus iniae.Possibilities of difference in roles or overlapping functions for GCSF paralogs were observed.

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

ABSTRACT 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. HighlightsPeptidoglycan recognition protein‐SC2 from rock bream was identified and characterized.RbPGRP‐SC2 was highly expressed in muscle and skin of healthy rock bream.RbPGRP‐SC2 was up‐regulated in the fish infected with pathogens, especially in liver.rRbPGRP‐SC2 can agglutinate pathogens, induce leukocyte apoptosis, and promote phagocytosis of pathogens.

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

ABSTRACT 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 33kDa. 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. HIGHLIGHTSCD276, a type I transmembrane protein with an ORF of 912bp coding for 303aa.It was expressed in gill, spleen, skin, and sparsely expressed in other tissues.VHSV challenge induced this gene in liver, head kidney, spleen, intestine and gills.Flow cytometry revealed that CD276 was majorly expressed in granulocyte cells.