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In the complement system, C3 is a central component in complement activation, immune defense and immune regulation. In all pathways of complement activation, the pivotal step is conversion of the component C3 to C3b and C3a, which is responsible to eliminate the pathogen and opsonization. In this study, we examined the immunological properties of C3 and its activated fragment C3a from Japanese flounder (Paralichthys olivaceus) (PoC3 and PoC3a), a teleost species with important economic value. PoC3 is composed of 1655 amino acid residues, contains the six domains and highly conserved GCGEQ sequence of the C3 family. We found that PoC3 expression occurred in nine different tissues and was upregulated by bacterial challenge. In serum, PoC3 was able to bind to a broad-spectrum of bacteria, and purified native PoC3 could directly kill specific pathogen. When PoC3 expression in Japanese flounder was knocked down by siRNA, serum complement activity was significantly decreased, and bacterial replication in fish tissues was significantly increased. Recombinant PoC3a (rPoC3a) exhibited apparent binding capacities to bacteria and Japanese flounder peripheral blood leukocytes (PBL) and induce chemotaxis of PBL. Japanese flounder administered rPoC3a exhibited enhanced resistance against bacterial infection. Taken together, these results indicate that PoC3 is likely a key factor of complement activation, and PoC3 and PoC3a are required for optimal defense against bacterial infection in teleost. [ABSTRACT FROM AUTHOR]

In mammals, membrane-associated complement regulatory proteins (MCRP) can protect host cells from the damaging of the activated complement. In teleost, few studies on the function of MCRP have been documented. In the present report, we identified a MCRP (named CsMCRP) from the teleost fish tongue sole Cynoglossus semilaevis and examined its immune function. CsMCRP shares moderate sequence identities with fish DAF-like molecules. CsMCRP was predicted to be a transmembrane protein with three short consensus repeats located in the extracellular region. CsMCRP expression occurred in nine different tissues, especially blood, and in peripheral blood leukocytes (PBL). Recombinant CsMCRP inhibited complement activation and interacted with bacterial pathogen, the latter in a highly selective manner. Antibody blocking the CsMCRP on PBL significantly inhibited bacterial infection of PBL. These results indicate that teleost CsMCRP is both a regulator of complement activation and a cellular receptor involved in bacterial invasion. [ABSTRACT FROM AUTHOR]

Complement C1q domain containing protein (C1qDC) is a vital recognition molecule and has an important effect on immunity. The C1qDCs exhibit opsonic activity in fish, while the mechanisms of C1qDCs in activation complement still remain unclear. This study explored immunological characteristics of a C1qDC from Japanese flounder (Paralichthys olivaceus) (PoC1qDC). PoC1qDC consists of 296 amino acid residues, possessing a collagen domain and a C1q domain. According to our results, PoC1qDC was expressed in 9 diverse tissue samples and showed up-regulation after bacterial challenge. Recombinant PoC1qDC (rPoC1qDC) activated normal serum bactericidal and hemolytic activities by interaction with Japanese flounder IgM, but not enhanced the complement activity of C3-depeleted serum. rPoC1qDC was significantly bound to various bacterial species and agglutination activity against Edwardsiella piscicida and Streptococcus iniae. Furthermore, rPoC1qDC showed direct interaction with peripheral blood leucocytes while enhancing phagocytic and chemotactic activity. When PoC1qDC was overexpressed in Japanese flounder before E. piscicida infection, bacterial replication was significantly inhibited in fish tissues. Consistently, when PoC1qDC expression in Japanese flounder was knocked down, bacterial replication was significantly enhanced. The above findings first suggested the role of PoC1qDC in teleost in mediating complement activation by interaction with IgM, which can positively influence bacterial infection. • PoC1qDC possesses a collagen domain and a C1q domain. • Bacterial infection enhanced PoC1qDC expression in spleen, kidney and blood. • Recombinant PoC1qDC mediated complement activation by interaction with IgM. • Recombinant PoC1qDC could bind to bacteria and exhibited agglutination activity. • PoC1qDC is required for optimal defense against bacterial infection. [ABSTRACT FROM AUTHOR]

NK-lysin is an antimicrobial protein produced by cytotoxic T lymphocytes and natural killer cells. In this study, we examined the biological property of a peptide, NKLP27, derived from tongue sole (Cynoglossus semilaevis) NK-lysin. NKLP27 is composed of 27 amino acids and shares little sequence identity with known NK-lysin peptides. NKLP27 possesses bactericidal activity against both Gram-negative and Gram-positive bacteria including common aquaculture pathogens. The bactericidal activity of NKLP27 was dependent on the C-terminal five residues, deletion of which dramatically reduced the activity of NKLP27. During its interaction with the target bacterial cells, NKLP27 destroyed cell membrane integrity, penetrated into the cytoplasm, and induced degradation of genomic DNA. In vivo study showed that administration of tongue sole with NKLP27 before bacterial and viral infection significantly reduced pathogen dissemination and replication in tissues. Further study revealed that fish administered with NKLP27 exhibited significantly upregulated expression of the immune genes including those that are known to be involved in antibacterial and antiviral defense. These results indicate that NKLP27 is a novel antimicrobial against bacterial and viral pathogens, and that the observed effect of NKLP27 on bacterial DNA and host gene expression adds new insights to the action mechanism of fish antimicrobial peptides. [ABSTRACT FROM AUTHOR]

Abstract: Thioredoxin (Trx) is a redox protein that plays an important role in anti-oxidative stress and redox signaling. In a recent report, we observed that the Trx of tongue sole (Cynoglossus semilaevis), CsTrx1, is a reductase and an antioxidant with immunostimulatory property. In the current work, we studied the biological effect of a truncated CsTrx1, CsTrx80, which is composed of the N-terminal 80 residues of CsTrx1. We found that recombinant CsTrx80 (rCsTrx80) purified from Escherichia coli exhibited no insulin disulfide reductase activity; however, unlike rCsTrx1, which showed no apparent chemotactic activity, rCsTrx80 was able to bind to the head kidney monocytes of tongue sole and caused cellular migration in a dose-dependent manner. Mutation of the two cysteine residues in the highly conserved CXXC motif had no effect on the chemotactic activity of rCsTrx80. Like rCsTrx1, rCsTrx80 induced the expression of immune relevant genes in the target cells, but the induction patterns differed from that induced by rCsTrx1. In vivo study showed that when tongue sole were administered with rCsTrx80 before being infected with a bacterial pathogen, bacterial dissemination and colonization in fish tissues were significantly reduced compared to control fish. Taken together, these results indicate for the first time that a Trx80 derived from fish Trx possesses chemotactic and immunoregulatory properties which promote host resistance against bacterial infection. [Copyright &y& Elsevier]

In mammals, complement factor I (CFI) is a serine protease in serum and plays a pivotal role in the regulation of complement activation. In the presence of cofactor, CFI cleaves C3b to iC3b, and further degrades iC3b to C3c and C3d. In teleost, the function of CFI is poorly understood. In this study, we examined the immunological property of CFI from Japanese flounder (Paralichthys olivaceus) (PoCFI), a teleost species with important economic value. PoCFI is composed of 597 amino acid residues and possesses a trypsin-like serine protease (Tryp) domain. We found that PoCFI expressions occurred in nine different tissues and were upregulated by bacterial challenge. Recombinant PoCFI-Tryp (rPoCFI-Tryp) inhibited complement activation and degraded C3b in serum. rPoCFI-Tryp exhibited apparent binding capacities to a board-spectrum of bacteria and inhibited bacterial growth. These results provide the first evidence to indicate that CFI in teleost negatively regulates complement activation via degradation C3b, and probably plays a role in host immune defense against bacterial infection. • PoCFI possesses a trypsin serine protease (PoCFI-Tryp) domain. • PoCFI expression occurred in multiple tissues and was most abundant in liver. • Pathogen infection enhanced PoCFI expression in spleen, kidney and blood. • Recombinant PoCFI-Tryp (rPoCFI-Tryp) inhibited complement activation. • rPoCFI-Tryp was able to bind to bacteria and inhibited bacterial growth. [ABSTRACT FROM AUTHOR]

Chemokines are a large family of chemotactic cytokines. Based on the arrangement of the first two cysteine residues, chemokines are divided into four groups, one of which is the CC chemokine group. In this study, we characterized a CC chemokine, CsCCL21, from half-smooth tongue sole ( Cynoglossus semilaevis ), and analyzed its activity. CsCCL21 contains two conserved N-terminal cysteine residues in a NCCL motif and is phylogenetically related to the CCL19/21/25 subgroup of CC chemokines. CsCCL21 was constitutively expressed in nine tissues and significantly upregulated by bacterial and viral infection. The recombinant CsCCL21 (rCsCCL21) induced migration of peripheral blood leukocytes. When the two conserved cysteine residues in the NCCL motif were mutated, the chemotactic activity of rCsCCL21 was abolished. rCsCCL21 enhanced the resistance of tongue sole against bacterial infection, but the mutant protein with NCCL mutation lacked this antibacterial effect. Taken together, these results suggest that CsCCL21 is a functional CC chemokine with the ability to recruit leukocytes and is involved in antibacterial immunity in a manner that requires the conserved NCCL motif. [ABSTRACT FROM AUTHOR]

Takifugu rubripes is a highly valued cultured fish in Asia, while pathogen infections can result in severe diseases and lead to substantial economic losses. Toll-like receptors (TLRs), as pattern recognition receptors, play a crucial role on recognition pathogens and initiation innate immune response. However, the immunological properties of teleost-specific TLR23 remain largely unknown. In this study, we investigated the biological functions of TLR23 (TrTLR23) from T. rubripes , found that TrTLR23 existed in various organs. Following bacterial pathogen challenge, the expression levels of TrTLR23 were significantly increased in immune related organs. TrTLR23 located on the cellular membrane and specifically recognized pathogenic microorganism. Co-immunoprecipitation and antibody blocking analysis revealed that TrTLR23 recruited myeloid differentiation primary response protein (MyD88), thereby mediating the activation of the ERK signaling pathway. Furthermore, in vivo showed that, when TrTLR23 is overexpressed in T. rubripes , bacterial replication in fish tissues is significantly inhibited. Consistently, when TrTLR23 expression in T. rubripes is knocked down, bacterial replication is significantly enhanced. In conclusion, these findings suggested that TrTLR23 played a critical role on mediation TLR23-MyD88-ERK axis against bacterial infection. This study revealed that TLR23 involved in the innate immune mechanism, and provided the foundation for development disease control strategies in teleost. • TrTLR23 is phylogenetically distantly related to teleost-specific TLR23. • Bacterial infection enhanced TrTLR23 expression in liver, spleen, and kidney. • TrTLR23 located on the cellular membrane and specifically recognized pathogen. • TrTLR23 recruited myeloid MyD88 and activated the ERK signaling pathway. • TrTLR23 is required for optimal defense against bacterial infection. [ABSTRACT FROM AUTHOR]

Abstract: Hepcidin is an antimicrobial peptide and a regulator of iron homeostasis. In turbot (Scophthalmus maximus), two types of hepcidins have been identified, which share approximately 50% sequence identity. In this study, we examined the antimicrobial potentials of the two hepcidins in the form of synthesized peptides, SmHep1P and SmHep2P. We found that SmHep1P and SmHep2P exhibited apparent bactericidal activities against both Gram-positive and Gram-negative bacteria in a dose-dependent manner. The bactericidal effect of SmHep1P was stronger against Gram-positive bacteria, while the bactericidal effect of SmHep2P was stronger against Gram-negative bacteria. Fluorescence and electron microscopy showed that both peptides were able to bind to the target bacterial cells and alter the surface structure of the cells. In vitro studies showed that SmHep1P and SmHep2P reduced bacterial invasion into cultured fish cells. In vivo studies showed that turbot administered with SmHep1P and SmHep2P exhibited significantly enhanced resistance against bacterial and viral infection. In both in vivo and in vitro studies, the antimicrobial activities of SmHep2P were in most cases significantly stronger than those of SmHep1P. Together these results indicate that the two hepcidins of turbot most likely possess antimicrobial properties and play a role in the innate immune defense against bacterial and viral pathogens. [Copyright &y& Elsevier]

In this study, we examined the function of a Japanese flounder (Paralichthys olivaceus) microRNA (miRNA), pol-miR-363-3p. We found that pol-miR-363-3p targets an ubiquitin-specific protease (USP), USP32. USP is a family of deubiquitinating enzymes essential to the functioning of the ubiquitin proteasome system. In mammals, USP32 is known to be associated with cancer and immunity. In fish, the function of USP32 is unknown. We found that flounder USP32 (PoUSP32) expression was detected in the major tissues of flounder, particularly intestine. In vitro and in vivo studies showed that pol-miR-363-3p directly regulated PoUSP32 in a negative manner by interaction with the 3′UTR of PoUSP32. Overexpression of pol-miR-363-3p or interference with PoUSP32 expression in flounder cells significantly blocked Streptococcus iniae infection. Consistently, in vivo knockdown of pol-miR-363-3p or overexpression of PoUSP32 enhanced dissemination of S. iniae in flounder tissues, whereas in vivo knockdown of PoUSP32 inhibited S. iniae dissemination. In addition, pol-miR-363-3p knockdown also significantly promoted the tissue dissemination of the viral pathogen megalocytivirus, which, as well as S. iniae , regulated pol-miR-363-3p expression. Together these results revealed an important role of pol-miR-363-3p in flounder immune defense against bacterial and viral infection. • PoUSP32 was a target gene of pol-miR-363-3p and regulated negatively by the latter. • pol-miR-363-3p expression was regulated by Streptococcus iniae and megalocytivirus. • PoUSP32 knockdown/pol-miR-363-3p overexpression blocked S. iniae infection in vitro. • In vivo pol-miR-363-3p knockdown promoted S. iniae and megalocytivirus infection. • In vivo PoUSP32 overexpression/knockdown affected S. iniae invasion in flounder. [ABSTRACT FROM AUTHOR]