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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]

Edwardsiella piscicida is a significant pathogenic microorganism, and can lead to systematic infections in multifold hosts, including teleost, amphibians, and mammals. Ail is a surface protein involved in virulence. However, the underlining mechanism is still to be investigated in E. piscicida. This study examined the functions and immunoprotective properties of an Ail molecule from E. piscicida. Ail1 possessed a conserved Ail/Lom superfamily domain and was exposed to the bacterial surface. Mutation of ail1 significantly reduced the survival rate under acid and oxidizing environment, motility, biofilm formation, cellular infection, tissue dissemination, and causing mortality in infected fish. The loss abilities of TX01Δ ail1 were rescued by overexpression of the ail1 gene. Recombinant Ail1 (rAil1) could interact with peripheral blood leukocytes from Japanese flounder and affect E. piscicida infection in vitro. Vaccine trial showed that rAil1 was used as a potential subunit vaccine and could induce effective immune protection against E. piscicida infection. Fish vaccinated with rAil1 produced the specific antibody against rAil1 in serum, which increased the bactericidal activity of serum complement. In addition, rAil1-vaccinated fish displayed the higher ROS and acid phosphatase activity of macrophages. The above findings indicated that Ail1 played a pivotal role in the virulence of E. piscicida and may be explored as a subunit vaccine in aquaculture. • Ail1 is exposed on the surface of Edwardsiella piscicida. • ail1 mutant is affected in bacterial virulence. • ail1 mutant is impaired in oxidative stress and low pH resistance. • Recombinant Ail1 is able to interact with host cell and mediate the infection. • Recombinant Ail1 produced effective protective effect against infection. [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]

CD59 is a complement regulatory protein that inhibits the formation of membrane attack complex of complement. In this study, we examined the expression and activity of tongue sole ( Cynoglossus semilaevis ) CD59 (CsCD59). CsCD59 possesses the conserved structural features of CD59 and shares 33%–46% sequence identities with other fish CD59. Expression of CsCD59 was high in liver, spleen, and muscle, and was stimulated by infection of bacterial pathogens. Recombinant CsCD59 (rCsCD59) exhibited an apparent inhibition effect on the activation of tongue sole serum complement. ELISA and microscopy detected binding of rCsCD59 to a number of Gram-negative and Gram-positive bacteria. Interaction with rCsCD59 did not affect bacterial viability but significantly enhanced bacterial resistance against the killing effect of fish serum. Together these results indicate that fish CD59 may to some degrees facilitate a general escape of bacteria from complement-mediated immunity. [ABSTRACT FROM AUTHOR]

Complement plays an important role in the innate immune system, and it comprises about 35 individual proteins. In mammals, complement is activated via three different pathways, the classical pathway, the alternative pathway, and the lectin pathway. All three activation pathways produce C3-convertase in different forms. C3-convertase cleaves C3 to C3a and C3b and initiates a cascade of cleavage and activation, eventually resulting in the formation of the membrane attack complex. Complement activation results in the generation of activated fragments that are involved in microbial killing, phagocytosis, inflammatory reactions, immune complex clearance, and antibody production. Although the complement system has been studied extensively in mammals, complement is less well understood in teleosts. This review summarizes the current knowledge of the teleost complement components involved in phagocytosis, chemotaxis, and cell lysis. We report the characterized complement components in various teleost species. In addition, we provide a comprehensive compilation of complement regulators, and this information is used to analyze the role of complement regulators in pathogen infection. The influence of complement receptors on the immune responses of teleosts is reviewed. Finally, we propose directions for future study of the molecular evolution, structure, and function of complement components in teleosts. This review provides new insights into the complement system of recognition and defense, and such knowledge is essential for the development of new immune strategies in aquaculture. • This review summarizes current research progress of teleosts complement systems. • The homologue of mammalian complement components are identified in teleosts. • Complement of teleosts plays an important role in immune system. • Regulators of complement activation are studied in teleosts. • Complement receptors may involve in the immune responses of teleosts. [ABSTRACT FROM AUTHOR]

Activation of the complement system leads to the cleavage of component factor C5 into C5a and C5b. C5a can induce chemotaxis and inflammatory responses in mammals. The function of C5a in fish is poorly understood. In this study, we report the identification and analysis of a C5 homologue, CsC5, from tongue sole ( Cynoglossus semilaevis ). CsC5 is composed of 1683 amino acid residues that include an anaphylatoxin homologous domain. Expression of CsC5 could be detected in a variety of tissues and was up-regulated by bacterial or viral pathogen infection. Purified recombinant CsC5a (rCsC5a) could bind to peripheral blood leukocytes (PBL) and stimulate PBL chemotaxis, proliferation, respiratory burst, acid phosphatase activity, and phagocytosis. Tongue sole administered rCsC5a exhibited enhanced resistance against bacterial and viral infections. These results indicate that CsC5a is an anaphylatoxin with a role in innate immune defense against bacterial and viral infections. [ABSTRACT FROM AUTHOR]

Tumor necrosis factor (TNF) is one of the most important cytokines involved in inflammation, apoptosis, cell proliferation, and stimulation of the immune system. The TNF gene has been cloned in teleost fish; however, the in vivo function of fish TNF is essentially unknown. In this study, we report the identification of a TNF homologue, CsTNF1, from tongue sole ( Cynoglossus semilaevis ) and analysis of its expression and biological effect. CsTNF1 is composed of 242 amino acid residues and possesses a TNF domain and conserved receptor binding sites. Expression of CsTNF1 was detected in a wide range of tissues and up-regulated in a time-dependent manner by experimental challenge with bacterial and viral pathogens. Bacterial infection of peripheral blood leukocytes (PBL) caused extracellular secretion of CsTNF1. Purified recombinant CsTNF1 (rCsTNF1) was able to bind to PBL and stimulate the respiratory burst activity of PBL. In contrast, rCsTNF1M1 and rCsTNF1M2, the mutant CsTNF1 bearing substitutions at the receptor binding site, failed to activate PBL. Fish administered with rCsTNF1, but not with rCsTNF1M1 and rCsTNF1M2, exhibited enhanced expression of IL-1, IL-6, IL-8, IL-27, TLR9 and G3BP in a time-dependent manner and augmented resistance against bacterial and viral infection. These results provide the first evidence that the receptor binding sites are essential to a fish TNF, and that CsTNF1 is involved in the innate immune defense of fish against microbial pathogens. [ABSTRACT FROM AUTHOR]

Chemokines are a large, diverse group of small cytokines that can be classified into several families, including the CC chemokine family, which plays a pivotal role in host defense by inducing leukocyte chemotaxis under physiological and inflammatory conditions. Here we studied 9 CC chemokines from half-smooth tongue sole ( Cynoglossus semilaevis ). Phylogenetic analysis divided these chemokines into four groups. The tissue specific expression patterns of the 9 chemokines under normal physiological conditions varied much, with most chemokines highly expressed in immune organs, while some other chemokines showing high expression levels in non-immune organs. In addition, the 9 chemokines exhibited similar or distinctly different expression profiles in response to the challenge of virus and intracellular and extracellular bacterial pathogens. These results indicate that in tongue sole, CC chemokines may be involved in different immune responses as homeostatic or inflammatory chemokines. [ABSTRACT FROM AUTHOR]

Megalocytivirus, a DNA virus belonging to the Iridoviridae family, is a severe pathogen to a wide range of marine and freshwater fish. In this study, using turbot ( Scophthalmus maximus ) as a host model, we examined the immunoprotective property of one megalocytivirus gene, ORF75, in the form of DNA vaccine (named pORF75). Immunofluorescence microscopy and RT-PCR analysis showed that P444, the protein encoded by ORF75, was naturally produced in the tissues of turbot during megalocytivirus infection, and that the vaccine gene in pORF75 was expressed in fish cells transfected with pORF75 and in the tissues of turbot immunized with pORF75. Following vaccination of turbot with pORF75, a high level of survival (73%) was observed against a lethal megalocytivirus challenge. Consistently, viral replication in the vaccinated fish was significantly inhibited. Immune response analysis showed that pORF75-vaccinated fish (i) exhibited upregulated expression of the genes involved in innate and adaptive immunity, (ii) possessed specific memory immune cells that showed significant response to secondary antigen stimulation, and (iii) produced specific serum antibodies which, when co-introduced into turbot with megalocytivirus, blocked viral replication. Furthermore, whole-genome transcriptome analysis revealed that ORF75 knockdown altered the transcription of 43 viral genes. Taken together, these results indicate that ORF75 encoded a highly protective immunogen that is also a global transcription regulator of megalocytivirus. [ABSTRACT FROM AUTHOR]

In mammals, CD83 is a surface marker on mature dendritic cells and vital to lymphocyte activation. In teleost, studies on the function of CD83 are very limited. In this study, we examined the potential involvement of turbot ( Scophthalmus maximus ) CD83, SmCD83, in vaccine-induced immunity. For this purpose, turbot were immunized with pORF75, a DNA vaccine against megalocytivirus, in the presence or absence of pSmCD83, a plasmid that constitutively expresses SmCD83. Immune response and protection analysis showed that the presence of pSmCD83 significantly (i) enhanced the activation of head kidney macrophages (HKM) and immune gene expression, (ii) inhibited viral replication in fish tissues following megalocytivirus challenge and increased the survival of the vaccinated fish, and (iii) stimulated production of specific serum antibody and the cytotoxicity of peripheral blood leukocytes. To further examine the effect of SmCD83, pORF75 was administered into turbot in which SmCD83 was knocked down. Subsequent analysis showed that in fish with SmCD83 knockdown, vaccine-induced HKM activation and antibody production were severely reduced, and, consistently, the protectivity of pORF75 was drastically decreased. Taken together, these results indicate for the first time that teleost CD83 is required for the induction of protective immune response by DNA vaccine. [ABSTRACT FROM AUTHOR]

Edwardsiella tarda is a Gram-negative bacterium with a broad host range that includes a wide variety of farmed fish as well as humans. E. tarda has long been known to be able to survive in host serum, but the relevant mechanism is unclear. In this study, we investigated the fundamental question, i.e. whether E. tarda activated serum complement or not. We found that (i) when incubated with flounder serum, E. tarda exhibited a high survival rate (87.6%), which was slightly but significantly reduced in the presence of Mg 2+ ; (ii) E. tarda -incubated serum possessed strong hemolytic activity and bactericidal activity, (iii) compared to the serum incubated with a complement-sensitive laboratory Escherichia coli strain, E. tarda -incubated serum exhibited much less chemotactic activity, (iv) in contrast to the serum incubated with live E. tarda , the serum incubated with heat-inactivated E. tarda exhibited no apparent hemolytic capacity. Taken together, these results indicate for the first time that E. tarda circumvents serum attack by preventing, to a large extent, complement activation via the alternative pathway, and that heat-labile surface structures likely play an essential role in the complement evasion of E. tarda . [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]

Streptococcus iniae is a Gram-positive bacterium and a severe pathogen to a wide range of economically important fish species. In addition, S. iniae is also a zoonotic pathogen and can cause serious infections in humans. In this study, we identified from a pathogenic S. iniae strain a putative bacteriocin, Sil, and examined its biological activity. Sil is composed of 101 amino acid residues and shares 35.6% overall sequence identity with the lactococcin 972 of Lactococcus lactis. Immunoblot analysis showed that Sil was secreted by S. iniae into the extracellular milieu. Purified recombinant Sil (rSil) exhibited a dose-dependent inhibitory effect on the growth of Bacillus subtilis but had no impact on the growths of other 16 Gram-positive bacteria and 10 Gram-negative bacteria representing 23 different bacterial species. Treatment of rSil by heating at 50°C abolished the activity of rSil. rSil bound to the surface of B. subtilis but induced no killing of the target cells. Cellular study revealed that rSil interacted with turbot (Scophthalmus maximus) head kidney monocytes and inhibited the innate immune response of the cells, which led to enhanced cellular infection of S. iniae. Antibody blocking of the extracellular Sil produced by S. iniae significantly attenuated the infectivity of S. iniae. Consistent with these in vitro observations, in vivo study showed that administration of turbot with rSil prior to S. iniae infection significantly increased bacterial dissemination and colonization in fish tissues. Taken together, these results indicate that Sil is a novel virulence-associated bacteriostatic and an immunoregulator that promotes S. iniae infection by impairing the immune defense of host fish. [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]

Highlights: [•] CsEBI3 expression was detected in multiple tissues and induced by pathogen infection. [•] CsEBI3 was secreted by peripheral blood leukocytes (PBL) upon bacterial stimulation. [•] rCsEBI3 enhanced the immune response and bacterial resistance of PBL. [•] Antibody blocking of CsEBI3 on PBL reduced cell resistance to bacterial invasion. [•] The immunoregulatory function of rCsEBI3 depends on the FN3 domain of the protein. [Copyright &y& Elsevier]

Abstract: Pentraxins are a family of evolutionarily conserved proteins that play an important part in innate immunity. C-reactive protein (CRP) is a member of the pentraxin family and in humans is known to be the major acute phase protein. In this work, we report the identification and analysis of a CRP, CsCRP, from half-smooth tongue sole (Cynoglossus semilaevis). CsCRP is composed of 228 amino acid residues and possesses a Pentraxin/CRP domain. Expression of CsCRP occurred in a wide range of tissues and was upregulated by pathogen infection in kidney, spleen, blood, and, in particular, liver. Following bacterial infection, CsCRP level in blood rose rapidly within 12 h and was approximately 3.8 fold of that of the basal level. Purified recombinant CsCRP (rCsCRP) was able to interact with Gram-negative and Gram-positive bacteria including those of pathogenic nature in a dose-dependent manner. When peripheral blood leukocytes (PBL) were infected with bacterial pathogen in the presence of rCsCRP, the respiratory burst and phagocytic capacity of the cells were increased to significant extents. Taken together, these results indicate that CsCRP is an acute phase protein that plays a role in innate immune defense against bacterial infection. [Copyright &y& Elsevier]

Abstract: Invasin is an outer membrane protein that is known to mediate entry of enteric bacteria into mammalian cells. In this study, we analyzed the function and immunoprotective potential of the invasin Inv1 from Edwardsiella tarda, a serious fish pathogen that can also infect humans. In silico analysis indicated that Inv1 possesses a conserved N-terminal DUF3442 domain and a C-terminal group 1 bacterial Ig-like domain. Subcellular localization analysis showed that Inv1 is exposed on cell surface and could be recognized by specific antibodies. Mutation of inv1 had no effect on bacterial growth but attenuates overall bacterial virulence and impaired the ability of E. tarda to attach and invade into host cells. Consistent with these observations, antibody blocking of Inv1 inhibited E. tarda infection of host cells. To examine the immunoprotective potential of Inv1, recombinant Inv1 (rInv1) corresponding to the DUF3442 domain was purified and used to vaccinate Japanese flounder (Paralichthys olivaceus). The results showed that rInv1 induced strong protection against lethal-dose challenge of E. tarda. ELISA analysis showed that rInv1-vaccinated fish produced specific serum antibodies that could enhance the serum bactericidal activity against E. tarda. Taken together, these results indicate that Inv1 is a surface-localized virulence factor that is involved in host infection and can induce effective immunoprotection when used as a subunit vaccine. [Copyright &y& Elsevier]

Abstract: Peptidoglycan recognition proteins (PGRPs) are a family of innate immune molecules that recognize bacterial peptidoglycan. PGRPs are highly conserved in invertebrates and vertebrates including fish. However, the biological function of teleost PGRP remains largely uninvestigated. In this study, we identified a PGRP homologue, SoPGLYRP-2, from red drum (Sciaenops ocellatus) and analyzed its activity and potential function. The deduced amino acid sequence of SoPGLYRP-2 is composed of 482 residues and shares 46–94% overall identities with known fish PGRPs. SoPGLYRP-2 contains at the C-terminus a single zinc amidase domain with conserved residues that form the catalytic site. Quantitative RT-PCR analysis detected SoPGLYRP-2 expression in multiple tissues, with the highest expression occurring in liver and the lowest expression occurring in brain. Experimental bacterial infection upregulated SoPGLYRP-2 expression in kidney, spleen, and liver in time-dependent manners. To examine the biological activity of SoPGLYRP-2, purified recombinant proteins representing the intact SoPGLYRP-2 (rSoPGLYRP-2) and the amidase domain (rSoPGLYRP-AD) were prepared from Escherichia coli. Subsequent analysis showed that rSoPGLYRP-2 and rSoPGLYRP-AD (i) exhibited comparable Zn2+-dependent peptidoglycan-lytic activity and were able to recognize and bind to live bacterial cells, (ii) possessed bactericidal effect against Gram-positive bacteria and slight bacteriostatic effect against Gram-negative bacteria, (iii) were able to block bacterial infection into host cells. These results indicate that SoPGLYRP-2 is a zinc-dependent amidase and a bactericide that targets preferentially at Gram-positive bacteria, and that SoPGLYRP-2 is likely to play a role in host innate immune defense during bacterial infection. [Copyright &y& Elsevier]

Abstract: Vibrio harveyi is an important aquaculture pathogen that can infect a large number of marine animals. In this study, we examined three V. harveyi strains (VJ1, VJ2, and VJ3) isolated in the years of 2008, 2009, and 2010 respectively from diseased Japanese flounder (Paralichthys olivaceus) in a fish farm in China that had been inflicted with disease outbreaks. VJ1, VJ2, and VJ3 exhibited comparable virulence in a flounder model and induced symptoms in experimentally infected fish that are indistinguishable from those of naturally diseased fish. 16S rDNA and multilocus sequence analysis indicated that VJ1, VJ2, and VJ3 share 98–100% sequence identities with known V. harveyi isolates in 16S rDNA and four housekeeping genes. VJ1, VJ2, and VJ3 have distinct and identical AFLP banding patterns, which differ profoundly from those of two environmental V. harveyi isolates. VJ1, VJ2, and VJ3 exhibited comparable growth profiles and hemolytic activities and possess the vhh hemolysin gene. Compared to an environmental isolate, VJ1, VJ2, and VJ3 showed comparable and significantly stronger resistances against the bactericidal effect of host serum and induced higher levels of respiratory burst activity in head kidney macrophages. However, VJ1, VJ2, and VJ3 were unable to proliferate in macrophages. When incubated with a cultured cell line of flounder, VJ1, VJ2, and VJ3 induced apoptosis characterized by the occurrence of DNA fragmentation, apoptotic bodies, and elevated caspase 3 activity. Taken together, these results indicate that VJ1, VJ2, and VJ3 are probably clones of the same V. harveyi lineage that has persisted during the examined three-year time frame and likely caused recurrent disease outbreaks via a virulence mechanism involving serum resistance and triggering apoptosis in host cells. [Copyright &y& Elsevier]

The bacterial outer membrane proteins are important components of bacterial cell. Edwardsiella tarda is a vital bacterial pathogen to a wide range of aquaculture animals. In this study, we examined the function of an outer membrane protein of E. tarda (named Omp1 Et). Omp1 Et was found to localize on the surface of E. tarda , and recombinant Omp1 Et (rOmp1 Et) was able to interact with flounder peripheral blood leukocytes. Compared to the wild type, the omp1 Et knockout TX01Δ omp1 was significantly reduced in motility, biofilm formation, attachment and invasion into host cells, dissemination in host tissues, and inducing host mortality. Furthermore, TX01Δ omp1 was significantly impaired in the ability to survive under low pH and oxidizing conditions. When introduced into flounder as a subunit vaccine, rOmp1 Et induced strong immune protection against E. tarda challenge. Taken together, these results indicate that Omp1 Et is a surface-located virulence factor of E. tarda and rOmp1 Et can induce effective immune-protection as a submit vaccine. • Omp1 Et is localized on the surface of Edwardsiella tarda. • The Omp1 Et -defective mutant, TX01Δ omp1 , is attenuated in overall bacterial virulence. • TX01Δ omp1 is impaired in the ability to attach and invade into host cells. • TX01Δ omp1 exhibites reduced survival under low pH and oxidizing condition. • Recombinant Omp1 Et induces strong protective immunity in flounder. [ABSTRACT FROM AUTHOR]

Bacillus subtilis subsp. subtilis G7 was isolated from a deep-sea hydrothermal vent and is pathogenic to pathogenic to fish (Japanese flounder) and mice. G7 is able to survive in host sera and phagocytes. In this study, we investigated the underlying mechanism of G7 serum resistance. We found that (i) the remaining complement activity was very low in G7-incubated flounder serum but high in G7-incubated mouse serum; (ii) cleaved C3 and C5 components were detected on flounder serum-incubated G7 but not on mouse serum-incubated G7; (iii) abundant uncleaved C5 was localized in G7-incubated mouse, but not flounder, serum; (iv) G7-incubated flounder, but not mouse, serum exhibited strong chemotactic activity; (v) pre-treatment with low-dose lysozyme abolished the serum resistance of G7. Hence, G7 activates flounder complement but is protected from complement-mediated destruction by its cell wall structure, while G7 prevents the activation of mouse complement. These results indicate that G7 employs different mechanisms to avoid the complement killing of different hosts. • G7 activates flounder complement leading to the cleavage of C3 and C5. • The cell wall of G7 confers protection against the killing by flounder complement. • G7 prevents the activation of mouse complement via unknown mechanism. • G7 employs different mechanisms to evade the complement killing of different hosts. [ABSTRACT FROM AUTHOR]

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]

In teleost fish, the immune functions of mannan-binding lectin (MBL) associated protein (MAP) and MBL associated serine protease (MASP) are scarcely investigated. In the present study, we examined the biological properties both MAP (CsMAP34) and MASP (CsMASP1) molecules from tongue sole (Cynoglossus semilaevis). We found that CsMAP34 and CsMASP1 expressions occurred in nine different tissues and were upregulated by bacterial challenge. CsMAP34 protein was detected in blood, especially during bacterial infection. Recombinant CsMAP34 (rCsMAP34) bound C. semilaevis MBL (rCsBML) when the latter was activated by bacteria, while recombinant CsMASP1 (rCsMASP1) bound activated rCsBML only in the presence of rCsMAP34. rCsMAP34 stimulated the hemolytic and bactericidal activities of serum complement, whereas anti-CsMAP34 antibody blocked complement activities. Knockdown of CsMASP1 in C. semilaevis resulted in significant inhibition of complement activities. Furthermore, rCsMAP34 interacted directly with peripheral blood leukocytes (PBL) and enhanced the respiratory burst, acid phosphatase activity, chemotactic activity, and gene expression of PBL. These results indicate for the first time that a teleost MAP acts one hand as a regulator that promotes the lectin pathway of complement activation via its ability to recruit MBL to MASP, and other hand as a modulator of immune cell activity. [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]

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]

Interleukin 8 (IL8) is a CXC chemokine that plays a crucial role on promoting inflammatory response and immune regulation. In teleost, IL8 can induce the migration and activation of immune cells. However, the biological functions of IL8 are still unknown in Takifugu rubripes. In this study, we examined the biological characteristics of TrIL8 in T. rubripes. TrIL8 is composed of 98 residues and contained a chemokine CXC domain. We found that the TrIL8 expression was detected in diverse organs and significantly increased by Vibrio harveyi or Edwardsiella tarda challenge. The recombinant TrIL8 (rTrIL8) exhibited significantly the binding capacities to 8 tested bacteria. In addition, rTrIL8 could bind to peripheral blood leukocytes (PBL), and increased the expression of immune gene, resistance to bacterial infection, respiratory burst, acid phosphatase activity, chemotactic activity, and phagocytic activity of PBL. In the presence of rTrIL8, T. rubripes was enhanced the resistance to V. harveyi infection. These results indicated that TrIL8 is a chemokine and involved in the activation of immune cells against bacterial infection in teleost. • TrIL8 possesses a chemokine CXC domain. • Bacterial infection enhanced TrIL8 expression in liver, spleen, and kidney. • Recombinant TrIL8 (rTrIL8) induced chemotaxis of peripheral blood leukocytes (PBL). • rTrIL8 could bind to PBL and enhanced the immune activity of cells. • rTrIL8 enhanced the resistance to bacterial infection. [ABSTRACT FROM AUTHOR]