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Japanese flounder (Paralichthys olivaceus) is an important economic fish species farmed in China and other countries. It is susceptible to infection by Edwardsiella tarda , a severe fish pathogen with a broad host range. In this study, we employed high-throughput deep sequencing technology to identify, in a global scale, flounder kidney microRNAs (miRNAs) induced by E. tarda at different stages of infection. Differentially expressed miRNAs (DEmiRNAs) and mRNAs (DEmRNAs) exhibiting significantly altered expression levels before and after E. tarda infection were examined. A total of 96 DEmiRNAs were identified, for which 2779 target genes were predicted. Eighty-seven miRNA–mRNA pairs, involving 29 DEmiRNAs and 86 DEmRNAs, showed negative correlations in their expression patterns. GO and KEGG enrichment analysis revealed that the putative target genes of the DEmiRNAs were associated with diverse biological processes, cellular components, and molecular functions. One of the DEmiRNAs, pol-miR-182-5p, was demonstrated to regulate sphingosine-1-phosphate receptor 1 (PoS1PR1) negatively in a manner that depended on the specific interaction between the seed sequence of pol-miR-182-5p and the 3'-UTR of PoS1PR1. Overexpression of pol-miR-182-5p in flounder cells promoted apoptosis and inhibited cellular viability. Knockdown of PoS1PR1 in flounder enhanced E. tarda invasion and dissemination in fish tissues. These results provide new insights into miRNA-mediated anti-bacterial immunity in flounder. • Time-dependent profiles of flounder kidney miRNAs induced by E. tarda was obtained. • Ninety-six differentially expressed miRNAs (DEmiRNAs) were identified. • One DEmiRNA, pol-miR-182-5p, promoted apoptosis and death of flounder cells. • Sphingosine-1-phosphate receptor 1 (PoS1PR1) is a target of pol-miR-182-5p. • PoS1PR1 is required for inhibition of E. tarda dissemination in fish tissues. [ABSTRACT FROM AUTHOR]

This study was to investigate the inhibition effects of para -substituted cinnamic acid derivatives (4-chlorocinnamic acid, 4-ethoxycinnamic acid and 4-nitrocinnamic acid) on tyrosinase catalyzing the substrates, with the purpose of elucidating the inhibition mechanism of the tested derivatives on tyrosinase by the UV–vis spectrum, fluorescence spectroscopy, copper interacting and molecular docking, respectively. The native-PAGE results showed that 4-chlorocinnamic acid (4-CCA), 4-ethoxycinnamic acid (4-ECA) and 4-nitrocinnamic acid (4-NCA) had inhibitory effects on tyrosinase. Spectrophotometric analysis used to determine the inhibition capabilities of these compounds on tyrosinase catalyzing L-tyrosine (L-Tyr) and L-3,4-Dihydroxyphenylalanine (L-DOPA) as well. The IC 50 values and inhibition constants were further determined. Moreover, quenching mechanisms of tested compounds to tyrosinase belonged to static type and a red shift on fluorescence emission peak occurred when 4-NCA added. Copper interacting and molecular docking demonstrated that 4-CCA could not bind directly to the copper, but it could interact with residues in the active center of tyrosinase. Meanwhile, 4-ECA and 4-NCA could chelate a copper ion of tyrosinase. Anti-tyrosinase activities of para -substituted cinnamic acid derivatives would lay scientific foundation for their utilization in designing of novel tyrosinase inhibitors. [ABSTRACT FROM AUTHOR]

In this study, we examined the expression patterns and the functions of the tongue sole Cynoglossus semilaevis CD94, CsCD94. CsCD94 is composed of 209 amino acid residues and shares 43.0–50.2% overall identities with known teleost CD94 sequence. CsCD94 has a C-type lectin-like domain. Expression of CsCD94 occurred in multiple tissues and was upregulated during bacterial infection. Recombinant CsCD94 (rCsCD94) exhibited apparent binding and agglutinating activities against both Gram-positive and Gram-negative bacteria in a Ca 2+ -dependent manner. Treatment of bacteria with rCsCD94 enhanced phagocytosis of the bacteria by peripheral blood leukocytes. Furthermore, incubation of rCsCD94 with bacteria reduced the survival of the bacteria in vitro . Taken together, these results indicate that rCsCD94 is a key factor in the bactericidal and phagocytic effects of tongue sole, and reveal for the first time an essential role of fish CD94 in antibacterial immunity, thereby adding insight into the function of CD94. [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]

The temperature-sensitive hemagglutinin (Tsh), identified as serine protease autotransporters of the Enterobacteriaceae (SPATEs) proteins, is an important virulence factor for avian-pathogenic Escherichia coli (APEC) and uropathogenic E. coli . However, little is known about the role of Tsh as a virulence factor in Edwardsiella tarda , a severe fish pathogen. In this study, we characterized the Tsh of E. tarda (named Tsh Et ) and examined its function and vaccine potential. Tsh Et is composed of 1224 residues and has three functional domains typical for autotransporters. Quantitative real-time reverse transcriptase-PCR analysis showed that expression of tsh Et was upregulated under conditions of high temperature, increased cell density, high pH, and iron starvation and during the infection of host cells. A markerless tsh in-frame mutant strain, TX01Δ tsh , was constructed to determine whether Tsh Et participates in the pathogenicity of E. tarda , Compared to the wild type TX01, TX01Δ tsh exhibited (i) retarded biofilm growth, (ii) decreased resistance against serum killing, (iii) impaired ability to block the host immune response, (iv) attenuated tissue and cellular infectivity. Introduction of a trans -expressed tsh gene restored the lost virulence of TX01Δ tsh . The passenger domain of Tsh Et contains a putative serine protease (PepS) that exhibits apparent proteolytic activity when expressed in and purified from E. coli as a recombinant protein (rPepS). When used as a subunit vaccine to immunize Japanese flounder, rPepS was able to induce effective immune protection. This is the first study of Tsh in a fish pathogen, and the results suggest that Tsh Et exerts pleiotropic effects on the pathogenesis of E. tarda . [ABSTRACT FROM AUTHOR]

Ferric uptake regulator (Fur) is an important transcriptional regulator of Gram-negative bacteria. Edwardsiella tarda is a severe fish bacterial pathogen with a broad host range that includes humans. In this study, we examined the regulatory function of Fur in E. tarda via a proteomic approach. Compared to the wild type TX01, the fur mutant TX01Δ fur exhibited (i) retarded growth, (ii) enhanced siderophore production, (iii) increased acid tolerance, which is in contrast to observations in other bacterial species, (iv) decreased survival against oxidative stress and host serum, (v) impaired ability to inhibit host immune response, (vi) attenuated tissue infectivity and overall virulence. The deficiency of TX01Δ fur was rescued by introduction of an exogenous fur gene. iTRAQ-based comparative proteomic analysis of TX01Δ fur and TX01 identified 89 differentially expressed proteins that cover a wide range of functional categories including those affected by fur mutation. In addition, 16 proteins were identified for the first time to be regulated by Fur in Gram-negative bacteria. These results provide the first protein-based interpretation of the global impact of Fur on the physiology and infectivity of E. tarda . Significance This study demonstrates that in E . tarda , Fur controls multiple aspects of bacterial life, including growth, metabolism, iron acquisition, stress response, and host infection. In line with these observations, proteomics analysis identified a large amount of proteins affected in expression by Fur, which are involved in bacterial physiology and infectivity. Hence, these results link for the first time the pleiotropic effect of Fur with global protein expression and shed new light on the function and regulatory mechanism of Fur in pathogenic bacteria. [ABSTRACT FROM AUTHOR]

Tyrosinase is a key enzyme in post-harvest browning of fruit and vegetable. To control and inhibit its activity is the most effective method for delaying the browning and extend the shelf life. In this paper, the inhibitory kinetics of 4-hydroxy cinnamic acid on mushroom tyrosinase was investigated using the kinetics method of substrate reaction. The results showed that the inhibition of tyrosinase by 4-hydroxy cinnamic acid was a slow, reversible reaction with fractional remaining activity. The microscopic rate constants were determined for the reaction on 4-hydroxy cinnamic acid with tyrosinase. Furthermore, the molecular docking was used to simulate 4-hydroxy cinnamic acid dock with tyrosinase. The results showed that 4-hydroxy cinnamic acid interacted with the enzyme active site mainly through the hydroxy competed with the substrate hydroxy group. The cytotoxicity study of 4-hydroxy cinnamic acid indicated that it had no effects on the proliferation of normal liver cells. Moreover, the results of effects of 4-hydroxy cinnamic acid on the preservation of mushroom showed that it could delay the mushroom browning. These results provide a comprehensive underlying the inhibitory mechanisms of 4-hydroxy cinnamic acid and its delaying post-harvest browning, that is beneficial for the application of this compound. [ABSTRACT FROM AUTHOR]

Tyrosinase (EC 1.14.18.1) catalyzes both the hydroxylation of tyrosine into o -diphenols and the oxidation of o -diphenols into o -quinones that form brown or black pigments. In the present paper, cefotaxime, a cephalosporin antibacterial drug, was tested as an inhibitor of tyrosinase. The results show that cefotaxime inhibits both the monophenolase and diphenolase activities of tyrosinase. For the monophenolase activity, cefotaxime increased the lag time and decreased the steady-state activity with an IC 50 of 3.2 mM. For the diphenolase activity, the inhibition by cefotaxime is reversible and mix-I type with an IC 50 of 0.14 mM. The inhibition constants ( K I and K IS ) were determined to be 0.14 and 0.36 mM, respectively. The molecular mechanism of inhibition of tyrosinase by cefotaxime was determined by fluorescence quenching and molecular docking. The results demonstrated that cefotaxime was a static quencher of tyrosinase and that cefotaxime could dock favorably in the active site of tyrosinase. This research may offer a lead for designing and synthesizing novel and effective tyrosinase inhibitors in the future. [ABSTRACT FROM AUTHOR]

Stomatin-like protein 2 (SLP-2) is a novel and unusual member of the stomatin gene superfamily. In this study, we obtained a full-length SLP-2 (SmSLP-2) cDNA from turbot ( Scopthalmus maximus ) spleen cDNA library. The cDNA sequence of SmSLP-2 contains a 5′-UTR of 107 bp, an ORF of 1050 bp, and a 3′-UTR of 959 bp. The ORF encodes a putative protein of 349 residues, which has a calculated molecular mass of 38.7 kDa. The SmSLP-2 protein possesses a prohibitin-homology (PHB) domain (residues 40 to 198) and shares 72.4–87.6% overall sequence identity with that of the teleost species. The highest expression of SmSLP-2 mRNA was found in the skin, followed by the head kidney, gut, spleen, liver, heart, gill and muscle. Moreover, both viral and bacterial pathogen infection resulted in the up-regulation of SmSLP-2 mRNA in the turbot head kidney and spleen in vivo. Subcellular localization analysis indicated that the SmSLP-2 proteins are mainly located in the peripheral membrane of ZF4 cells. This study also demonstrated that SmSLP-2 modulates IL-2 expression via active NFκB signaling pathway, and is possibly involved in host immune defense against bacterial and viral pathogens. [ABSTRACT FROM AUTHOR]

The efficiency of antigen capture, processing, and presentation by antigen-presenting cells is the key to induce an effective immune response. Cell-penetrating peptides (CPPs) are short peptides that facilitate cellular uptake of various molecular cargoes and have an attractive potential for vaccine delivery. In this study, the Drosophila Antennapedia homeoprotein (Antp) and the human immunodeficiency virus-1 transactivator of transcription (TAT) peptides were fused to the N- or C-terminus of Sia10, a protective antigen of Streptococcus iniae , resulting in four recombinant fusion proteins, i.e., rAntp-Sia10, rSia10-Antp, rTAT-Sia10, and rSia10-TAT. All fusion proteins were expressed and purified, and their ability to penetrate into cells was examined. The results showed that rTAT-Sia10 had the strongest ability to translocate through the cellular membrane into cells. Immunofluorescence microscopy and Western blot assay confirmed that rTAT-Sia10 could penetrate into the head kidney lymphocytes and gill cells of Japanese flounder ( Paralichthys olivaceus ). Immunological analysis showed that rTAT-Sia10 significantly enhanced macrophage activation and peripheral blood leukocyte proliferation, and induced production of specific serum antibodies at 2–8 weeks post-vaccination. Transcriptional analysis showed that vaccination with rTAT-Sia10 up-regulated the expression of the genes encoding IL-1β, IL-8, NKEF, Mx, IgD, IgM, TNFα, MHC I α, MHC IIα, and CD8α. Fish vaccinated with rTAT-Sia10 exhibited significantly higher levels of survival rates (98% at 1 month and 92% at 2 months) compared to fish vaccinated with rSia10 (57% at 1 month and 53% at 2 months). Taken together, these results indicate that TAT-derived peptide has a great potential in the application of bacterial vaccines. [ABSTRACT FROM AUTHOR]

CC chemokines are the largest subfamily of chemokines, which are important components of the innate immune system. To date, sequences of several CC chemokines have been identified in half-smooth tongue sole ( Cynoglossus semilaevis ); however, the activities and functions of these putative chemokines remain unknown. Herein, we characterized a CC chemokine, CsCCL17, from tongue sole, and examined its activity. CsCCL17 contains a 303 bp open reading frame, which encodes a polypeptide of 100 amino acids with a molecular mass of 12 kDa CsCCL17 is phylogenetically related to the CCL17/22 group of CC chemokines and possesses the typical arrangement of four cysteines and an SCCR motif found in known CC chemokines. Under normal physiological conditions, CsCCL17 expression was detected in spleen, liver, heart, gill, head kidney, muscle, brain, and intestine. When the fish were infected by bacterial and viral pathogens, CsCCL17 expression was significantly up-regulated in a time-dependent manner. Chemotactic analysis showed that recombinant CsCCL17 (rCsCCL17) induced migration of peripheral blood leukocytes. A mutagenesis study showed that when the two cysteine residues in the SCCR motif were replaced by serine, no apparent chemotactic activity was observed in the mutant protein rCsCCL17M. rCsCCL17 enhanced the resistance of tongue sole against viral infection, but rCsCCL17M lacked this antiviral effect. Taken together, these findings indicate that CsCCL17 is a functional CC chemokine with the ability to recruit leukocytes and enhance host immune defense in a manner that requires the conserved SCCR motif. [ABSTRACT FROM AUTHOR]

Mushroom ( Agaricus bisporus ) rapidly loses its white skin color and turns brown after harvest rapidly. 100 μmol L −1 4-methoxy cinnamic acid (MCA) was used to extend the shelf life of A. bisporus in this study. Freshly harvested fruit bodies were stored at (20 ± 1) °C and 85% ± 5% relative humidity. The physiological indexes, reactive oxygen metabolism and ultrastructure of A. bisporus were investigated. The results indicated that the weight loss, percentage of open-cap, the degree of browning, relative leakage rate, malondialdehyde (MDA) content and superoxide anion generation rate were reduced in treated group. The respiratory climacteric was delay in treated group. Moreover, they kept high catalase (CAT) and ascorbate peroxidase (APX) activities and endogenous antioxidants (AsA and GSH) content. The mushroom treated with MCA maintained a relatively complete ultrastructure; PPO and POD in tissues contact with phenolic substrates were delayed. These findings suggest that MCA could have potential in maintaining the quality of mushroom. [ABSTRACT FROM AUTHOR]

Highlights: [•] Two CsNF45 isoforms exist in tongue sole as a result of alternative mRNA splicing. [•] Both isoforms were expressed in multi-tissues but differed in expression patterns. [•] Expression of the two isoforms was regulated differently by bacterial and viral infection. [•] Both isoforms were localized in the nucleus and could activate mouse IL-2 promoter. [•] Overexpression of the two isoforms inhibited bacterial and viral infection to different extents. [Copyright &y& Elsevier]

Tyrosinase (EC 1.14.18.1) is the key enzyme of most food enzymatic oxidation. Tyrosinase inhibitors are important in food industry. In the present paper, 2-chlorcinnamic acid and 2,4-dichlorocinnamic acid were synthesized and the inhibitory kinetics on mushroom tyrosinase were investigated. The results showed that both compounds synthesized could inhibit tyrosinase activity. For monophenolase activity, both chlorocinnamic acids could extended the lag time and decrease the steady-state activities, 2-chlorcinnamic acid extended the lag time just by 5%, and 2,4-dichlorcinnamic acid extended the lag time more than by 30.4%. For diphenolase activity, the IC 50 values of 2-chlorcinnamic acid and 2,4-dichlorocinnamic acid were determined to be 0.765 mM and 0.295 mM, respectively. The inhibition kinetics showed that 2-chlorcinnamic acid and 2,4-dichlorocinnamic acid displayed a reversible and uncompetitive mechanism. The inhibition constants were determined to be 0.348 mM and 0.159 mM, respectively. The research may supply the basis for designing new tyrosinase inhibitors. [Copyright &y& Elsevier]

Abstract: A triplex loop-mediated isothermal amplification (triLAMP) method was developed to detect three major fish pathogens, i.e., Vibrio harveyi, Vibrio anguillarum and Vibrio alginolyticus. Three sets of four species-specific primers were designed with one or two restriction enzyme sites contained in the inner primers of each set. TriLAMP is performed with a combination of all three sets of primers in a single reaction. A subsequent restriction enzyme analysis is used for discriminating the specific Vibrio species which caused the positive reaction. The specificity of this method was validated against a total of 32 strains representing 19 different bacterial species. The sensitivities of the triLAMP assays were 102 to 103 times higher than those of the traditional PCR assays. Via modified processes of sample preparation and visual inspection of the LAMP products with fluorescent staining, the triLAMP method allowed convenient and sensitive detection of V. harveyi, V. anguillarum and V. alginolyticus in the tissues of experimentally infected fish. These results indicate that the triLAMP method developed in this study is a useful tool for sensitive and rapid on-site detection of Vibrio infection in fish. [Copyright &y& Elsevier]

Abstract: Chemokines are classified into several different subfamilies, of which CC chemokines constitute the largest subfamily in teleost. The prominent structural characteristic of CC chemokines is the presence of an Asp-Cys-Cys-Leu (DCCL) motif. To date, cDNA sequences of several CC chemokines have been identified in turbot (Scophthalmus maximus), however, the activity and function of these putative chemokines remain unknown. In this study, we examined the biological effect of the turbot CC chemokine SmCCL19, which has been previously reported as KC70 and shown to be regulated in expression by bacterial infection. To facilitate functional analysis, recombinant SmCCL19 (rSmCCL19) and a mutant form of SmCCL19, SmCCL19M, that bears serine substitutions at the two cysteine residues of the DCCL motif were purified from Escherichia coli. Chemotactic analysis showed that rSmCCL19 induced migration of head kidney leukocytes in a dose-dependent manner, whereas rSmCCL19M caused no apparent cellular migration. To examine the in vivo effect of rSmCCL19, turbot were administered with rSmCCL19 or rSmCCL19M before being inoculated with viral and bacterial pathogens. Subsequent tissue infection analysis showed that the viral and bacterial loads in rSmCCL19-adminsitered fish were significantly reduced, whereas the pathogen loads in rSmCCL19M-adminsitered fish were largely comparable to those in the control fish. Consistent with these observations, significant inductions of immune relevant genes were observed in rSmCCL19-adminsitered fish but not in rSmCCL19M-adminsitered fish. Taken together, these results indicate that SmCCL19 recruits leukocytes and augments host immune defense in a manner that depends on the conserved DCCL motif. [Copyright &y& Elsevier]

Abstract: Megalocytivirus is an important fish pathogen with a broad host range that includes turbot. In this study, proteomic analysis was conducted to examine turbot proteins modulated in expression by megalocytivirus infection. Thirty five proteins from spleen were identified to be differentially expressed at 2days post-viral infection (dpi) and 7dpi. Three upregulated proteins, i.e. heat shock protein 70 (Hsp70), Mx protein, and natural killer enhancing factor (NKEF), were further analyzed for potential antiviral effect. For this purpose, turbot were administered separately with the plasmids pHsp70, pMx, and pNKEF, which express Hsp70, Mx, and NKEF respectively, before megalocytivirus infection. Viral dissemination and propagation in spleen were subsequently determined. The results showed that the viral loads in fish administered with pNKEF were significantly reduced. To examine the potential of Hsp70, Mx, and NKEF as immunological adjuvant, turbot were immunized with a DNA vaccine in the presence of pHsp70, pMx, or pNKEF. Subsequent analysis showed that the presence of pNKEF and pHsp70, but not pMx, significantly reduced viral infection and enhanced fish survival. Taken together, these results indicate that NKEF exhibits antiviral property against megalocytivirus, and that both NKEF and Hsp70 may be used in DNA vaccine-based control of megalocytivirus infection. Biological significance: This study provides the first proteomic picture of turbot in response to megalocytivirus infection. We demonstrated that megalocytivirus infection modulates the expression of turbot proteins associated with various cellular functions, and that one of the upregulated proteins, NKEF, exhibits antiviral effect when overexpressed in vivo, while another upregulated protein, Hsp70, exhibits adjuvant effect when co-immunized with a DNA vaccine. These results add molecular insights into turbot immune response induced by megalocytivirus and provide candidate proteins with application potentials in the control of megalocytivirus-associated disease. [Copyright &y& Elsevier]

Abstract: Vibrio harveyi is a bacterial pathogen that affects marine vertebrates and invertebrates. In this study, we identified 13 outer membrane proteins (OMPs) from a pathogenic V. harveyi strain and analyzed their immunological properties. In vivo immunogenicity analysis showed that antibodies specific to recombinant proteins of the 13 OMPs were detected in the antiserum of V. harveyi-infected rat. When used as subunit vaccines to immunize Japanese flounder (Paralichthys olivaceus), all OMPs were able to elicit specific serum antibody production in the vaccinated fish; however, only two OMPs (OMP173 and OMP214) induced high levels (>70%) of relative percent survival. Pre-incubation of V. harveyi with the antisera of protective OMPs significantly impaired bacterial infectivity against peripheral blood leukocytes (PBL), whereas the antisera of non-protective OMPs had no apparent effect on infection. OMP173 antibodies could bind whole V. harveyi cells and exhibit bactericidal effect in a complement-dependent manner. Passive immunization showed that fish received OMP173 antiserum before being infected with V. harveyi exhibited significantly reduced mortality rate and lower bacterial loads in liver, spleen, and kidney. Finally, treatment of FG cells with OMP173 prior to V. harveyi infection protected the cells from bacterial invasion to a significant extent. Take together, these results indicate that two of the examined OMPs induce protective immunity through production of specific antibodies that block bacterial invasion, and that one OMP is likely to be involved in host cell interaction during the infection process. Thus, the immunoprotectivity of the OMPs is probably associated with functional participations of the OMPs in bacterial infection. [Copyright &y& Elsevier]

Abstract: Edwardsiella tarda is a Gram-negative bacterium that is reckoned one of the most severe fish pathogens. In this study, we analyzed the biological properties of the E. tarda major fimbrial subunit protein, FimA. We found that mutation of fimA resulted in defective biofilm growth, attenuated infectivity against host cells, and impaired ability to disseminate into and colonize host tissues following experimental infection. When used as a subunit vaccine, recombinant FimA (rFimA) elicited a high level of protection in turbot (Scophthalmus maximus) against lethal E. tarda challenge. Immunological analysis showed that rFimA vaccination induced production of specific serum antibodies that bound to live E. tarda via interaction with the FimA on bacterial cells, and that antibody–E. tarda interaction blocked bacterial infection. Furthermore, passive immunization of turbot with anti-rFimA serum before E. tarda infection reduced bacterial loads in fish tissues to significant extents. To examine the adjuvant potential of FimA, turbot were vaccinated with rVhhP2, a protective Vibrio harveyi antigen, in the presence or absence of rFimA. Subsequent analysis showed that the presence of rFimA significantly augmented the protectivity of rVhhP2. ELISA and quantitative real time RT-PCR showed that rFimA significantly increased rVhhP2-specific serum antibody production and enhanced the expression of immune relevant genes. Taken together, these results indicate that FimA is a virulence-associated protein that possesses vaccine as well as adjuvant potentials, and that the immunoprotectivity of FimA is most likely due to its ability to induce specific immune response that inhibits E. tarda infection. [Copyright &y& Elsevier]

Abstract: Disease outbreaks caused by iridoviruses are known to affect farmed flounder (Paralichthys olivaceus) and turbot (Scophthalmus maximus). To facilitate quantitative real time RT-PCR (qRT-PCR) analysis of gene expression in flounder and turbot during viral infection, we in this study examined the potentials of 9 housekeeping genes of flounder and turbot as internal references for qRT-PCR under conditions of experimental infection with megalocytivirus, a member of the Iridoviridae family. The mRNA levels of the 9 housekeeping genes in the brain, gill, heart, intestine, kidney, liver, muscle, and spleen of flounder and turbot were determined by qRT-PCR at 24h and 72h post-viral infection, and the expression stabilities of the genes were determined with geNorm and NormFinder algorithms. The results showed that (i) viral infection induced significant changes in the mRNA levels of the all the examined genes in a manner that was dependent on both tissue type and infection stage; (ii) for a given time point of infection, stability predictions made by the two algorisms were highly consistent for most tissues; (iii) the optimum reference genes differed at different infection time points at least in some tissues; (iv) at both examined time points, no common reference genes were identified across all tissue types. These results indicate that when studying gene expression in flounder and turbot in relation to viral infection, different internal references may have to be used not only for different tissues but also for different infection stages. [Copyright &y& Elsevier]

Abstract: Vibrio anguillarum is the etiological agent of vibriosis, an aquaculture disease that affects a wide range of farmed fish. The genome of V. anguillarum contains five flagellin genes, i.e. flaA, flaB, flaC, flaD, and flaE. In this study, we analyzed the vaccine potential and adjuvanticity of FlaA, FlaB, FlaD, and FlaE in a model of Japanese flounder (Paralichthys olivaceus). For this purpose, recombinant FlaA, FlaB, FlaD, and FlaE were expressed in and purified from Escherichia coli. In vivo immunogenicity analysis showed that antibodies against rFlaA, rFlaB, rFlaD, and rFlaE were detected in rat antiserum raised against live V. anguillarum, with the highest antibody level being that against rFlaB. When administered into flounder via intraperitoneal injection, rFlaA, rFlaD, and rFlaE induced comparable relative percent survival (RPS) rates, which were significantly lower than that induced by rFlaB. Specific serum antibodies were induced by all flagellins, however, the antibody level induced by rFlaB was significantly higher than those induced by other three flagellins. Compared to sera from fish vaccinated with rFlaA, rFlaD, and rFlaE, serum from fish vaccinated with rFlaB significantly reduced the infectivity of V. anguillarum against host cells. To examine the potential adjuvant effect of the flagellins, flounder were immunized with rEsa1, a D15-like surface antigen that induces protective immunity as a subunit vaccine, in the presence or absence of rFlaA, rFlaB, rFlaD, and rFlaE respectively. The results showed that rFlaE, but not other three flagellins, significantly increased the RPS of rEsa1. Compared to fish vaccinated with rEsa1, fish vaccinated with rEsa1 plus rFlaE exhibited a significantly higher level of serum antibodies and enhanced expression of the genes involved in innate and adaptive immunity. Taken together, these results indicate that FlaA, FlaB, FlaD, and FlaE have different immunological properties and, as a result, differ in vaccine and adjuvant potentials. [Copyright &y& Elsevier]

Abstract: Streptococcus iniae is an important fish pathogen with a broad host range that includes both marine and freshwater fish species. With an aim to develop effective vaccines against S. iniae, we in this study constructed three monovalent DNA vaccines, i.e., pSagF, pSagG, and pSagI, based on sagF, G, and I, which are components of the streptolysin S cluster. The immunoprotective potentials of these vaccines were examined in a model of Japanese flounder (Paralichthys olivaceus). The results showed that following intramuscular administration, the vaccine plasmids were transported to spleen, kidney, and liver, where the vaccine-encoding transgenes were expressed. Immunocolloidal gold electron microscopy detected production of the vaccine protein in fish vaccinated with each of the vaccine plasmids. Following lethal-dose S. iniae challenge, pSagF-, pSagG-, and pSagI-vaccinated fish exhibited relative percent of survival (RPS) rates of 78%, 65%, and 76% respectively. To examine whether multivalent vaccines composed of different combinations of monovalent vaccines would produce better protections, flounder were vaccinated with FG (pSagF plus pSagG), FI (pSagF plus pSagG), or FGI (pSagF plus pSagG and pSagI). Subsequent challenging study showed that the RPS rates of the fish vaccinated with the divalent and trivalent vaccines were 4%–17% and 13%–26% respectively higher than those of the fish vaccinated with the component monovalent vaccines. Furthermore, FGI exhibited a strong cross protection against both serotype I and serotype II S. iniae, apparently due to, as revealed by sequence analysis, the existence of highly conserved SagF, SagG, and SagI homologs in these serotypes. Immunological analysis showed that all vaccines induced (i) specific serum antibody production, (ii) enhanced complement-mediated bactericidal activity, and (iii) significant induction of a wide range of immune genes. However, the levels of gene expression and serum bactericidal activity induced by FGI were in general more potent than those induced by monovalent vaccines. Taken together, these results indicate that the DNA vaccines based on sagF, G, and I, especially when they are formulated as multivalent vaccines, are highly efficacious against S. iniae infection. [Copyright &y& Elsevier]

Abstract: Iridoviruses are large double-stranded DNA viruses with icosahedral capsid. The Iridoviridae family contains five genera, one of which is Megalocytivirus. Megalocytivirus has emerged in recent years as an important pathogen to a wide range of marine and freshwater fish. In this study, we aimed at developing effective genetic vaccines against megalocytivirus affecting farmed fish in China. For this purpose, we constructed seven DNA vaccines based on seven genes of rock bream iridovirus isolate 1 from China (RBIV-C1), a megalocytivirus with a host range that includes Japanese flounder (Paralichthys olivaceus) and turbot (Scophthalmus maximus). The protective potentials of these vaccines were examined in a turbot model. The results showed that after vaccination via intramuscular injection, the vaccine plasmids were distributed in spleen, kidney, muscle, and liver, and transcription of the vaccine genes and production of the vaccine proteins were detected in these tissues. Following challenge with a lethal-dose of RBIV-C1, fish vaccinated with four of the seven DNA vaccines exhibited significantly higher levels of survival compared to control fish. Of these four protective DNA vaccines, pCN86, which is a plasmid that expresses an 86-residue viral protein, induced the highest protection. Immunological analysis showed that pCN86 was able to (i) stimulate the respiratory burst of head kidney macrophages at 14 d, 21 d, and 28 d post-vaccination, (ii) upregulate the expression of immune relevant genes involved in innate and adaptive immunity, and (iii) induce production of serum antibodies that, when incubated with RBIV-C1 before infection, significantly reduced viral loads in kidney and spleen following viral infection of turbot. Taken together, these results indicate that pCN86 is an effective DNA vaccine that may be used in the control of megalocytivirus-associated diseases in aquaculture. [Copyright &y& Elsevier]

Abstract: Heat shock protein (Hsp) 70 is a molecular chaperone that plays an important role in protein folding and transport. In addition, Hsp70 is also involved in regulation of innate and adaptive immune response. In this study, we examined the biological activity and the immunomodulatory property of an Hsp70 homologue, PoHsp70, from Japanese flounder (Paralichthys olivaceus). Recombinant PoHsp70 purified from Escherichia coli exhibits apparent ATPase activity; however, a mutant PoHsp70, PoHsp70M, that bears mutation of the ATPase-associated domain, was completely abolished in activity. Expression of PoHsp70 was upregulated in a time-dependent manner by vaccination of flounder with a DNA vaccine, pSia10, that expresses a Streptococcus iniae antigen, Sia10. To examine whether PoHsp70 possessed any adjuvant potential, the DNA vaccine plasmids pSia10Hsp70 and pSia10Hsp70M were constructed. pSia10Hsp70 co-expresses Sia10 and PoHsp70, while pSia10Hsp70M co-expresses Sia10 and PoHsp70M. Following vaccination of flounder, production of Sia10 plus PoHsp70 and Sia10 plus PoHsp70M was detected in pSia10Hsp70- and pSia10Hsp70M-vaccinated fish respectively. At one month post-vaccination, comparable levels of serum antibodies were detected in fish vaccinated with pSia10Hsp70, pSia10Hsp70M, and pSia10. Subsequent protection analysis showed that, following S. iniae challenge, pSia10Hsp70 induced a survival rate that was significantly higher than that induced by pSia10, while pSia10Hsp70M induced a survival rate similar to that induced by pSia10. These results indicate that PoHsp70 is an effective adjuvant and that the adjuvanticity of PoHsp70 requires the intrinsic ATPase activity. [Copyright &y& Elsevier]

Pseudomonas fluorescens is a Gram-negative bacterium and a common aquaculture pathogen. In this study, we identified from a pathogenic P. fluorescens strain a TonB-dependent outer membrane receptor, TdrA, as a secreted protein and examined its function and vaccine potential. TdrA is composed of 746 residues and possesses conserved structural domains of TonB-dependent outer membrane receptors. Quantitative real-time reverse transcriptase-PCR analysis showed that expression of tdrA was upregulated under conditions of iron starvation and during infection of host cells. Consistently, iron depletion induced increased production of TdrA protein in the outer membrane. Compared to the wild type, a tdrA-knock out mutant (1) was unable to grow in the absence of iron, (2) exhibited drastically attenuated overall bacterial virulence, and (3) was impaired in the ability to establish lethal infection in host tissues. Purified recombinant TdrA (rTdrA), when used as a subunit vaccine to immunize flounder, was able to induce strong protective immunity, including production of serum-specific antibodies that resulted in effective protection against lethal-dose P. fluorescens challenge. Together, these results indicate that TdrA is an outer membrane receptor and a protective immunogen that is likely to be involved in iron acquisition and, as a result, required for optimal bacterial virulence. [ABSTRACT FROM AUTHOR]

Abstract: Bacterial sialidases are a group of glycohydrolases that are known to play an important role in invasion of host cells and tissues. In this study, we examined in a model of Japanese flounder (Paralichthys olivaceus) the potential function of NanA, a sialidase from the fish pathogen Edwardsiella tarda. NanA is composed of 670 residues and shares low sequence identities with known bacterial sialidases. In silico analysis indicated that NanA possesses a sialidase domain and an autotransporter domain, the former containing five Asp-boxes, a RIP motif, and the conserved catalytic site of bacterial sialidases. Purified recombinant NanA (rNanA) corresponding to the sialidase domain exhibited glycohydrolase activity against sialic acid substrate in a manner that is pH and temperature dependent. Immunofluorescence microscopy showed binding of anti-rNanA antibodies to E. tarda, suggesting that NanA was localized on cell surface. Mutation of nanA caused drastic attenuation in the ability of E. tarda to disseminate into and colonize fish tissues and to induce mortality in infected fish. Likewise, cellular study showed that the nanA mutant was significantly impaired in the infectivity against cultured flounder cells. Immunoprotective analysis showed that rNanA in the form of a subunit vaccine conferred effective protection upon flounder against lethal E. tarda challenge. rNanA vaccination induced the production of specific serum antibodies, which enhanced complement-mediated bactericidal activity and reduced infection of E. tarda into flounder cells. Together these results indicate that NanA plays an important role in the pathogenesis of E. tarda and may be exploited for the control of E. tarda infection in aquaculture. [Copyright &y& Elsevier]

Abstract: The immunoglobulin superfamily (IgSF) is a large group of cell surface proteins that include various immunoregulatory receptors such as novel immune type receptors (NITRs), which are a family of diversified proteins found exclusively in bony fish. In this study, we identified and analyzed an IgSF protein, SoIgSF1, from red drum (Sciaenops ocellatus). SoIgSF1 is composed of 225 amino acid residues and moderately related to teleost NITRs. In silico analysis indicated that SoIgSF1 is a type I transmembrane glycoprotein and contains an N-terminal signal peptide sequence, a single extracellular immunoglobulin V domain, a transmembrane region, and a cytoplasmic region. However, unlike most NITRs, the cytoplasmic region of SoIgSF1 exhibits no consensus inhibitory or stimulatory signaling sequences but has two tyrosine-containing motifs that conform to the right-half sequence of the immunoreceptor tyrosine-based inhibitory motif (ITIM). Quantitative real time RT-PCR analysis showed that SoIgSF1 expression occurred mainly in immune organs and was drastically induced by viral and bacterial infection. Immunofluorescence microscopy indicated that viral infection of head kidney (HK) leukocytes induced surface expression of SoIgSF1, which was able to interact with antibodies against recombinant SoIgSF1. Antibody cross-linking of SoIgSF1 on HK leukocytes inhibited the expression of immune relevant genes and promoted viral and bacterial infection. Taken together, these results indicate that SoIgSF1, though lacking canonical intracellular signaling motifs, is involved in regulation of host immune response during pathogen infection possibly by functioning as a negative signaling receptor through a novel mechanism. [Copyright &y& Elsevier]

Abstract: Tyrosinase (EC 1.14.18.1), a copper-containing multifunctional oxidase, was known to be a key enzyme for biosynthesis in fungi, plants and animals. In this work, the inhibition properties α-naphthol and β-naphthol toward the activity of tyrosinase have been evaluated, and the effects of α-naphthol and β-naphthol on monophenolase and diphenolase activity of tyrosinase have been investigated. The results showed that both α-naphthol and β-naphthol could potently inhibit both monophenolase activity and diphenolase activity of mushroom tyrosinase, and that β-naphthol exhibited stronger inhibitory effect against tyrosinase than α-naphthol. For monophenolase activity, β-naphthol could not only lengthen the lag time but also decrease the steady-state activity, while α-naphthol just only decreased the steady-state activity. For diphenolase activity, both α-naphthol and β-naphthol displayed revisible inhibition. Kinetic analyses showed that both α-naphthol and β-naphthol were competetive inhibitors. [Copyright &y& Elsevier]

Abstract: Vibrio harveyi is a Gram-negative bacterial pathogen that can infect a wide range of marine animals. In previous studies, we have reported a virulent V. harveyi strain, T4D. In the present study, an attenuated mutant of T4D, T4DM, was obtained by selection of rifampicin resistance. Compared to the wild type, T4DM was different in whole-cell protein profile and much slower in growth rate when cultured in stress conditions caused by iron depletion. Virulence analysis showed that compared to T4D, T4DM exhibited a dramatically increased median lethal dose, impaired tissue dissemination capacity, defective hemolytic activity, and significantly reduced resistance against the killing effect of host serum. To examine the potential of T4DM as a live attenuated vaccine, Japanese flounder (Paralichthys olivaceus) were vaccinated with T4DM via intraperitoneal injection or immersion. The results showed that at one and two months post-vaccination, fish administered with T4DM via both approaches, in particular that of immersion, were effectively protected against not only V. harveyi but also Vibrio alginolyticus, another important fish pathogen. Microbiological analysis showed that following immersion vaccination, T4DM was recovered from the internal organs of the vaccinated fish in a time-dependent manner within the first 6 days post-vaccination. Serum antibodies against V. harveyi and V. alginolyticus were detected in T4DM-vaccinated fish, and, compared to serum from control fish, serum from T4DM-vaccinated fish was significantly enhanced in bactericidal activity. These results indicate that T4DM is an attenuated strain with residual infectivity and that T4DM can induce effective cross-species protection against both V. harveyi and V. alginolyticus when used as a live immersion vaccine. [Copyright &y& Elsevier]

Abstract: T lymphocyte activation requires a combination of signals, one of which is provided by interaction between CD28 and its ligands on antigen presenting cells. Although CD28-like sequences have been identified in a few teleosts, the function of fish CD28 is virtually unknown. In this study, we cloned and analyzed a CD28 gene, CsCD28, from half-smooth tongue sole (Cynoglossus semilaevis). The deduced amino acid sequence of CsCD28 contains 229 residues and shares 20.2%–40.3% overall sequence identities with known fish CD28 sequences. CsCD28 possesses structural features conserved in mammalian and teleost CD28, which include the MYPPPY motif in the extracellular immunoglobulin-like domain and the YXN motif in the cytoplasmic domain. The CsCD28 gene is 2746 bp and composed of four exons and three introns, which in organization resemble those of mammalian and trout CD28. Quantitative real time RT-PCR analysis showed that CsCD28 expression occurred predominately in kidney, spleen, gut, and gill. CsCD28 is localized on the surface of head kidney lymphocytes, and antibody ligation of CsCD28 induced significant levels of cellular proliferation. Taken together, these results indicate that CsCD28 is similar to mammalian CD28 in genetic and protein structures and possibly plays a role in T cell activation. [Copyright &y& Elsevier]

Abstract: Chemokines are small cytokines that, based on their structural differences, are classified into four groups, one of which is called CXC chemokines. In this study, we identified a CXC chemokine, CsCXCe1, from half-smooth tongue sole (Cynoglossus semilaevis) and analyzed its function. The deduced amino acid sequence of CsCXCe1 contains 115 residues and is phylogenetically distinct from known CXC chemokines. CsCXCe1 possesses the conserved RCXC motif in the form of RCWC but lacks the ELR sequence that is found in some CXC chemokines. Expression of CsCXCe1 as determined by quantitative real time RT-PCR occurred abundantly in immune organs and was upregulated by bacterial and viral infection in time dependent manners. Purified recombinant CsCXCe1 (rCsCXCe1) exhibited comparable chemotactic activities against tongue sole and turbot (Scophthalmus maximus) peripheral blood leukocytes (PBL). Microscopic analysis identified lymphocytes as the major cellular population in PBL that responds to rCsCXCe1. Mutational study showed that when the two cysteine residues in the RCWC motif of CsCXCe1 were substituted by serine, the chemoattractive activity of CsCXCe1 was completely lost. Further study showed that treatment of PBL with rCsCXCe1 (i) stimulated cellular proliferation and respiratory burst activity, (ii) upregulated the expression of a wide spectrum of immune relevant genes, and (iii) enhanced cellular resistance against bacterial infection. Taken together, these results indicate that CsCXCe1 is likely a new type of CXC chemokine that exerts chemotactic and immunostimulatory effects on PBL. [Copyright &y& Elsevier]

Abstract: Edwardsiella tarda and Streptococcus iniae are important aquaculture pathogens that affect many species of farmed fish. In this study, we analyzed the expression, activity, and immunoprotective potential of E. tarda heat shock protein DnaK. We found that dnaK expression was upregulated under conditions of heat shock, oxidative stress, and infection of host cells. Recombinant DnaK (rDnaK) purified from Escherichia coli exhibited ATPase activity and induced protection in Japanese flounder (Paralichthys olivaceus) against lethal E. tarda challenge. On the basis of these results and our previous observation that a protective S. iniae antigen Sia10 which, when expressed heterogeneously in E. coli DH5α, is secreted into the extracellular milieu, we constructed a chimeric antigen by fusing DnaK to Sia10. The resulting fusion protein Sia10-DnaK was expressed in DH5α via the plasmid pTDK. Western blot analysis indicated that Sia10-DnaK was detected in the culture supernatant of DH5α/pTDK. When flounder were vaccinated with live DH5α/pTDK, strong protection was observed against both E. tarda and S. iniae. ELISA analysis detected specific serum antibody production in fish vaccinated with rDnaK and DH5α/pTDK. Taken together, these results indicate that rDnaK is an intrinsic ATPase with immunoprotective property and that Sia10-DnaK delivered by a live bacterial host is an effective bivalent vaccine candidate against E. tarda and S. iniae 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: The Rab family proteins belong to the Ras-like GTPase superfamily and play important roles in intracellular membrane trafficking. To date no studies on fish Rab have been documented, though rab-like sequences have been found in a number of teleosts. In this study, we identified and analyzed a Rab homologue, SoRab1, from red drum, Sciaenops ocellatus. The cDNA of SoRab1 contains a 5′- untranslated region (UTR) of 358 bp, an open reading frame (ORF) of 612 bp, and a 3′-UTR of 265 bp. The ORF encodes a putative protein of 203 residues, which shares 92–99% overall sequence identities with the Rab1 from fish, human, and mouse. SoRab1 possesses a typical Rab1 GTPase domain with the conserved G box motifs and the switch I and switch II regions. Recombinant SoRab1 purified from Escherichia coli exhibits apparent GTPase activity. Quantitative real time RT-PCR analysis showed that SoRab1 expression was detected in a number of tissues, with the lowest expression found in blood and highest expression found in muscle. Bacterial and lipopolysaccharide challenges significantly upregulated SoRab1 expression in liver, kidney, and spleen in time-dependent manners. Transient overexpression of SoRab1 in primary hepatocytes reduced intracellular bacterial infection, whereas interference with SoRab1 expression by RNAi enhanced intracellular bacterial invasion. These results provide the first indication that a fish Rab1 GTPase, SoRab1, regulates intracellular bacterial infection and thus is likely to play a role in bacteria-induced host immune defense. [Copyright &y& Elsevier]

Abstract: DNA-binding protein from starved cells (Dps) is a member of ferritin-like proteins that exhibit properties of nonspecific DNA binding and iron oxidation and storage. Although studies of Dps from many bacterial species have been reported, no investigations on Dps from fish pathogens have been documented. In this study, we examined the biological function of two Dps proteins, Dps1 and Dps2, from Edwardsiella tarda, an important fish bacterial pathogen that can also infect humans. Dps1 and Dps2 are, respectively, 163- and 174-residue in length and each contains the conserved ferroxidase center of Dps. Expression of dps1 and dps2 was growth phase-dependent and reached high levels in stationary phase. Purified recombinant Dps1 and Dps2 were able to mediate iron oxidation by H2O2 and bind DNA. Compared to the wild type strain, (i) the dps1 mutant (TXDps1) and the dps2 mutant (TXDps2) were unaffected in growth, while the dps2 mutant with interfered dps1 expression (TXDps2RI) exhibited a prolonged lag phase; (ii) TXDps1, TXDps2, and especially TXDps2RI were significantly reduced in H2O2 and UV tolerance and impaired in the capacity to invade into host tissues and replicate in head kidney macrophages; (iii) TXDps1, TXDps2, and TXDps2RI induced stronger macrophage respiratory burst activity and thus were defective in the ability to block the bactericidal response of macrophages. Taken together, these results indicate that Dps1 and Dps2 are functional analogues that possess ferroxidase activity and DNA binding capacity and are required for optimum oxidative stress resistance and full bacterial virulence. [Copyright &y& Elsevier]

Abstract: High mobility group box 1 protein (HMGB1) is a chromatin-associated nonhistone protein that is involved in nucleosome formation and transcriptional regulation. In addition, HMGB1 is also known as an extracellular cytokine that triggers inflammation and immune responses. HMGB1-like sequences have been identified in a number of fish species, however, the function of piscine HMGB1 remains uninvestigated. In this study, we reported the identification and analysis of SoHMGB1, an HMGB1 homologue from red drum (Sciaenops ocellatus). SoHMGB1 is 206 residues in length and contains two basic HMG boxes and a highly acidic C-terminal domain. SoHMGB1 shares 71–87% overall sequence identities with the HMGB1 counterparts from human, rat, and several fish species. Quantitative real time RT-PCR analysis showed that constitutive SoHMGB1 expression was detected in various tissues, with the lowest and highest levels found in kidney and muscle respectively. Bacterial challenge upregulated SoHMGB1 expression in head kidney (HK) and HK macrophages and induced extracellular secretion of SoHMGB1 by the activated macrophages. Recombinant SoHMGB1 (rSoHMGB1) purified from yeast exhibited no direct antimicrobial effect but was significantly stimulatory on the proliferation, activation, and bactericidal activity of HK macrophages. Taken together, these results indicate for the first time that a fish HMGB1, SoHMGB1, can function as a secreted cytokine in the event of bacterial infection and promote innate defense through the activation of macrophages. [Copyright &y& Elsevier]

Abstract: CXCL8, or interleukin-8, is a CXC chemokine that promotes neutrophil migration in response to inflammatory stimuli. In this study, we identified and analyzed a CXCL8 orthologue, SmCXCL8, from turbot (Scophthalmus maximus). The deduced amino acid sequence of SmCXCL8 is 99-residue in length and shares 52–83% overall identities with the lineage 1 CXCL8 of a number of teleost. SmCXCL8 possesses a CXC chemokine domain that contains the conserved CXC motif preceded by the tripeptide sequence EMH. Purified recombinant SmCXCL8 (rSmCXCL8) induced chemotaxis in peripheral blood neutrophils and, to lesser extents, head kidney (HK) lymphocytes and macrophages in a dose-dependent manner. Mutation of the EMH motif by alanine substitution reduced the chemoattractive effect of rSmCXCL8. Expression of SmCXCL8 as determined by quantitative real time RT-PCR (qRT-PCR) was detected mainly in immune organs under normal physiological conditions and was upregulated by experiment challenges with bacterial pathogen and poly(I:C). In addition, SmCXCL8 expression was also induced to significant extents following vaccination of turbot with a subunit vaccine. When rSmCXCL8 was added to the cell cultures of peripheral blood leukocytes and HK lymphocytes and macrophages, it stimulated the proliferation of these cells and enhanced cellular resistance against intracellular bacterial survival. qRT-PCR analysis showed that rSmCXCL8 induced the expression of TNF-α and suppressor of cytokine signaling 3 in HK lymphocytes in different time frames. On the other hand, SmCXCL8 expression was also upregulated by TNF-α. Taken together, these results indicate that SmCXCL8 is a functional CXC chemokine with immunomodulatory effect and plays a role in inflammatory response induced by bacterial infection. [Copyright &y& Elsevier]

Abstract: Hsp90 is a molecular chaperone that is involved in diverse cellular processes including protein folding/repairing and signal transduction. Edwardsiella tarda is a serious fish pathogen that affects fish aquaculture worldwide. The aim of this study was to investigate the potential importance of HtpG, the prokaryotic homologue of Hsp90, in the pathogenesis of E. tarda. E. tarda HtpG is 627-residue in length and contains domain structures that are conserved among Hsp90 family members. Quantitative real time RT-PCR analysis indicated that expression of htpG is induced by heat shock and oxidative stress. Recombinant HtpG (rHtpG) purified from Escherichia coli exhibits apparent ATPase activity, which is optimal at 40°C. Mutation of htpG (i) affects bacterial growth at elevated tempertaure and renders the cells more sensitive to stress induced by reactive oxygen species, (ii) causes dramatic reduction in blood dissemination and general bacterial virulence, (iii) weakens the ability of E. tarda to block head kidney macrophage activation and to resist against the bactericidal effect of macrophages, and (iv) upregulates the expression of pro-inflammatory cytokines in macrophages. Taken together, these results indicate that HtpG is a biologically active protein that is required for E. tarda to cope with various stress conditions especially that encountered in vivo the host system during infection. [Copyright &y& Elsevier]

Abstract: Vibrio harveyi is a severe aquaculture pathogen that can infect a large number of fish species. In previous studies, we have identified two protective immunogens, DegQ and Vhp1, from pathogenic V. harveyi strains. In this study, we examined the potentials of DegQ and Vhp1 as monovalent and bivalent DNA vaccines in a Japanese flounder (Paralichthys olivaceus) model. For this purpose, the DNA vaccine plasmids pDegQ, pVhp1, and pDV were constructed. pDegQ and pVhp1 express, respectively, degQ and vhp1, while pDV expresses degQ and vhp1 as a bicistronic mRNA transcript (degQ–vhp1). Following vaccination, all vaccine plasmids were detected in muscle, spleen, and kidney at 7 and 14 days post-vaccination. At the same time points and in the same tissues, degQ, vhp1, and degQ–vhp1 mRNA transcripts were detected in, respectively, pDegQ-, pVhp1-, and pDV-vaccinated fish. Production of DegQ and Vhp1 proteins was found in the muscle tissues of pDegQ- and pVhp1-vaccinated fish respectively, while both DegQ and Vhp1 proteins were detected in pDV-vaccinated fish. Compared to control fish, fish vaccinated with pDegQ and pVhp1 exhibited, respectively, 64.1% and 56.5% relative percent survival rates following lethal V. harveyi challenge, while fish vaccinated with pDV exhibited a survival rate that is more than 20% higher than those induced by pDegQ and pVhp1. Furthermore, in addition to V. harveyi, pDV also elicited dramatic cross protection against Vibrio parahaemolyticus. Immunological analysis showed that all vaccine plasmids induced the production of specific serum antibodies and the expression of a wide range of immune genes; however, the induction folds effected by pDV were in general higher than those effected by pDegQ and pVhp1. These results indicate that DNA vaccines, such as that represented by pDV, is a good vaccination option to combat V. harveyi-related vibriosis. [Copyright &y& Elsevier]

Abstract: Edwardsiella tarda and Vibrio harveyi are Gram-negative bacterial pathogens that affect a wide range of cultured fish. In previous studies, we have reported an E. tarda live vaccine ATCC15947 and a V. harveyi subunit vaccine DegQ. On the basis of these studies, in the present study we developed a cross protective vaccine against both E. tarda and V. harveyi by constructing a recombinant ATCC15947, Et15VhD, that expresses and secrets V. harveyi DegQ as a soluble antigen. Laboratory studies in a turbot (Scophthalmus maximus) model showed that Et15VhD elicited significant protections against E. tarda and V. harveyi when administered via intraperitoneal injection, oral feeding, immersion, and oral plus immersion, respectively. Microbiological analysis indicated dissemination and transient colonization of Et15VhD in fish tissues following vaccination. Since, compared to injection, oral plus immersion is a practically more acceptable vaccination procedure in aquaculture, we conducted a mock field trial to further examine the potential of Et15VhD as an oral plus immersion vaccine. The results showed that during the period before artificial bacterial challenge, mortality was observed in both the vaccinated group and the control group; however, the mortality of Et15VhD-vaccinated fish was significantly lower than that of the control fish. Following experimental challenge with E. tarda and V. harveyi at one and two months post-vaccination, Et15VhD-vaccinated fish exhibited dramatically increased survival rates compared to control fish. Serum antibody analysis indicated specific antibody production in Et15VhD-vaccinated fish. Taken together, these results demonstrate that Et15VhD induces strong protective immunity against E. tarda and V. harveyi under both laboratory and mock field conditions, which suggests a potential for Et15VhD to be used in aquaculture. [Copyright &y& Elsevier]

Abstract: Cystatins form a large family of cysteine protease inhibitors found in a wide arrange of organisms. Studies have indicated that mammalian cystatins play important roles under both physiological and pathological conditions. However, much less is known about fish cystatins. In this report, we described the identification and analysis of a cystatin B homologue, SmCytB, from turbot Scophthalmus maximus. The open reading frame of SmCytB is 300bp, which encodes a 99-residue protein that shares high levels of sequence identities with the cystatin B of a number of fish species and contains the conserved cysteine protease inhibitor motif of cystatin B. Constitutive expression of SmCytB is high in muscle, brain, heart and liver, and low in spleen, blood, gill and kidney. Bacterial infection upregulates SmCytB expression in kidney, spleen, liver and brain but not in muscle or heart. Functional analysis showed that recombinant SmCytB purified from Escherichia coli exhibits apparent cysteine protease inhibitor activity. Transient overexpression of SmCytB in head kidney macrophages enhances macrophage bactericidal activity probably through a nitric oxide-independent mechanism. These results indicate that SmCytB is involved in the immune defense of turbot against bacterial infection. [Copyright &y& Elsevier]

Abstract: C2 domains are protein structural modules found in many eukaryotic proteins involved in signal transduction, membrane trafficking, and immune defense. Most of the studied C2 domain-containing proteins are multi-domained in structure, in which the C2 domain is an independently folded motif and plays an essential role in calcium-dependent membrane-targeting. Although C2 domains isolated from intact proteins have been studied for biological functions, no study on natural proteins containing C2 domain only has been documented. In this study, we identified a Scophthalmus maximus protein SmC2P1 that is comprised of a single C2 domain and lacks any other apparent domain structures. The deduced amino acid sequence of SmC2P1 contains 129 residues and shares 36–38% identities with the C2 domains of the perforins of several fish species. Like typical C2 domains, SmC2P1 is predicted to organize into eight β-strands with a Ca2+-binding site located in inter-strand loops. SmC2P1 expression was detected, in deceasing order, in liver, spleen, blood, brain, muscle, kidney, gill, and heart. Experimental challenge of turbot with a bacterial pathogen significantly upregulated SmC2P1 expression in kidney in a time-dependent manner. Recombinant SmC2P1 purified from yeast exhibits no hemolytic activity but binds to pathogen-infected kidney lymphocytes in the presence of calcium. Furthermore, interaction of recombinant SmC2P1 with bacterium-infected lymphocytes reduced bacterial survival. These results indicate that SmC2P1 is a functional protein that is involved in host immune defense against bacterial infection. [Copyright &y& Elsevier]

Abstract: NAGase (EC.3.2.1.52) from crustaceans has the important roles in immunity, molting and digestion of chitinous foods. In this paper, the effects of citric acid on the activity of NAGase from Litopenaeus vannamei for the hydrolysis of pNP-NAG have been studied. The results showed that appropriate concentrations of citric acid could lead to reversible inhibition on NAGase and IC 50 was estimated to be 5.00 ± 0.35 mM. Using the plots of Lineweaver–Burk, the inhibition of NAGase by citric acid belongs to competitive type, the inhibitory equilibrium constant for citric acid binding with free NAGase, K I, is 3.26 ± 0.25 mM. The inhibitory kinetics of citric acid on NAGase in the appropriate concentrations of citric acid has been studied using the kinetic method of substrate reaction. The time course of NAGase for the hydrolysis of pNP-NAG in the presence of different concentrations of citric acid showed that at each citric acid concentration, the rate decreased with increasing time until a straight line was approached. The results show that the inhibition of NAGase by citric acid is a slow, reversible reaction with fractional remaining activity. The microscopic rate constants are determined for the reaction on citric acid with NAGase. [ABSTRACT FROM AUTHOR]

Abstract: CD83 is a transmembrane glycoprotein of the immunoglobulin (Ig) superfamily and a surface marker for fully matured dendritic cells (DCs) in humans and mice. In teleosts, DC-like cells and their molecular markers are largely unknown. In this report, we described the identification and expressional analysis of a CD83 homologue, SmCD83, from turbot Scophthalmus maximus. The open reading frame of SmCD83 is 639 bp, which is preceded by a 5′-untranslated region (UTR) of 87 bp and followed by a 3′-UTR of 1111 bp. The SmCD83 gene is 4716 bp in length, which contains five exons and four introns. The deduced amino acid sequence of SmCD83 shares 40–50% overall identities with the CD83 of several fish species. Like typical CD83, SmCD83 possesses an Ig-like extracellular domain, a transmembrane domain, and a cytoplasmic domain. The conserved disulfide bond-forming cysteine residues and the N-linked glycosylation sites that are preserved in CD83 are also found in SmCD83. Expressional analysis showed that constitutive expression of SmCD83 was high in gill, blood, spleen, muscle, and kidney and low in heart and liver. Bacterial infection and poly(I:C) treatment enhanced SmCD83 expression in kidney in time-dependent manners. Likewise, bacterial challenge caused significant induction of SmCD83 expression in cultured macrophages. Vaccination of turbot with a bacterin and a purified recombinant subunit vaccine-induced significant SmCD83 expression during the first week following vaccination. These results demonstrate that SmCD83 expression correlates with microbial challenge and antigen stimulation, which suggests the possibility that there may exist in turbot DC-like antigen-presenting cells that express SmCD83 upon activation by antigen uptake. In addition, these results also suggest that SmCD83 may serve as a marker for activated macrophages in turbot. [ABSTRACT FROM AUTHOR]

Abstract: Hsp70 proteins are a family of molecular chaperones that are involved in many aspects of protein homeostasis. In this study, an Hsp70 homologue (SoHsp70) was identified from red drum Sciaenops ocellatus and analyzed at molecular level. The open reading frame of SoHsp70 is 1920 bp and intronless, with a 5′-untranslated region (UTR) of 399 bp and a 3′-UTR of 241 bp. The deduced amino acid sequence of SoHsp70 shares 84–92% overall identities with the Hsp70s of a number of fish species. In silico analysis identified in SoHsp70 three conserved Hsp70 domains involved in nucleotide and substrate binding. The coding sequence of SoHsp70 was subcloned into Escherichia coli, from which recombinant SoHsp70 was purified and, upon ATPase assay, found to exhibit apparent ATPase activity. Expressional analysis showed that constitutive expression of SoHsp70 was detectable in heart, liver, spleen, kidney, brain, blood, and gill. Experimental challenges with poly(I:C) and bacterial pathogens of Gram-positive and Gram-negative nature induced SoHsp70 expression in kidney to different levels. Stress-responsive analysis of SoHsp70 expression in primary cultures of red drum hepatocytes showed that acute heat shock treatment elicited a rapid induction of SoHsp70 expression which appeared after 10 min and 30 min of treatment. Exposure of hepatocytes separately to iron, copper, mercury, and hydrogen peroxide significantly upregulated SoHsp70 expression in time-dependent manners. Vaccination of red drum with a Streptococcus iniae bacterin was also found to induce SoHsp70 expression. Furthermore, recombinant SoHsp70 enhanced the immunoprotective effect of a subunit vaccine. Taken together, these results suggest that SoHsp70 is a stress-inducible protein that is likely to play a role in immunity and in coping with environmental and biological stresses. [ABSTRACT FROM AUTHOR]

Abstract: Bacterial flagellin is known to induce potent immune response in vertebrate systems via the toll-like receptor (TLR) 5. As a result, flagellin has been studied extensively as a vaccine adjuvant. In a previous study, we examined the vaccine and adjuvant potentials of the flagellin (FliC) of the fish pathogen Edwardsiella tarda. We found that E. tarda FliC induced low protective immunity by itself but could function as a molecular adjuvant and potentiate the specific immune response induced by the E. tarda antigen Eta6. Since FliC is a large protein and organized into distinct structural domains, we wondered whether the immunostimulating effect observed with the full-length protein could be localized to a certain region. To investigate this question, we in the present study dissected the FliC protein into several segments according to its structural features: (i) N163, which consists of the conserved N-terminal 163 residues of FliC; (ii) M160, which consists of the variable middle 160 residues; (iii) C94, which consists of the conserved C-terminal 94 residues; (iv) NC257, which is an artificial fusion of N163 and C94. To examine the adjuvanticity of the FliC fragments, DNA vaccine plasmids expressing FliC fragments in fusion with Eta6 were constructed and used to immunize Japanese flounder. The results showed that N163 produced the best adjuvant effect, which, in respect to improvement in the relative percent survival of the vaccinated fish, was comparable to that of the full-length FliC. None of the other FliC fragments exhibited apparent immunopotentiating effect. Further analysis showed that N163 enhanced the production of serum specific antibodies and, like full-length FliC, significantly upregulated the expression of the genes that are possibly involved in innate and adaptive immunity. These results indicate that N163 is the immunodominant region of FliC and suggest that E. tarda FliC may induce immune responses in Japanese flounder via mechanisms alternative to that involving TLR5. [Copyright &y& Elsevier]

Abstract: Peroxiredoxins (Prxs) are a group of antioxidant proteins that protect cells from oxidative damage caused by various peroxides. To date, six different isoforms of peroxiredoxin (Prx1 to Prx6) have been identified, of which, Prx6 belongs to the 1-Cys Prx subfamily. Although Prx6 of several fish species have been reported at sequence level, there are very few documented studies on the potential function of fish Prx6. In this report, we describe the identification and analysis of a Prx6 homologue, SmPrx6, from turbot Scophthalmus maximus. The full length cDNA of SmPrx6 contains a 5′- untranslated region (UTR) of 60 bp, an open reading frame of 666 bp, and a 3′-UTR of 244 bp. The deduced amino acid sequence of SmPrx6 shares 81–87% overall identities with known fish Prx6. In silico analysis identified in SmPrx6 a conserved Prx6 catalytic motif, PVCTTE, and the catalytic triads putatively involved in peroxidase and phospholipase A2 activities. Expression of SmPrx6 was detected in most fish organs, with the highest expression levels found in blood and heart and the lowest level in spleen. Experimental challenges with bacterial pathogens and poly(I:C) upregulated SmPrx6 expression in liver and spleen in a manner that is dependent on the challenging agent and the tissue type. Treatment of cultured primary hepatocytes with H2O2 enhanced SmPrx6 expression in a dose-dependent manner. Recombinant SmPrx6 expressed in and purified from Escherichia coli exhibited thiol-dependent antioxidant activity and could protect cultured hepatocytes from H2O2-induced oxidative damage. Taken together, these results indicate that SmPrx6 is a Prx6 homologue with antioxidative property and is likely to be involved in both cellular maintenance and protective response during host immune defense against bacterial infection. [Copyright &y& Elsevier]

Abstract: Ferritins are conserved iron storage proteins that exist in most living organisms and play an essential role in iron homeostasis. In this study, we reported the identification and analysis a ferritin M subunit, SmFerM, from turbot Scophthalmus maximus. The full length cDNA of SmFerM contains a 5′-untranslated region (UTR) of 232bp, an open reading frame (ORF) of 531bp, and a 3′-UTR of 196bp. The ORF encodes a putative protein of 176 amino acids, which shares extensive sequence identities with the M ferritins of several fish species. In silico analysis identified in SmFerM both the ferroxidase center of mammalian H ferritins and the iron nucleation site of mammalian L ferritins. Quantitative real time reverse transcriptase-PCR analysis indicated that SmFerM expression was highest in muscle and lowest in heart and responded positively to experimental challenges with bacterial pathogens and poly(I:C). Exposure of cultured turbot hepatocytes to treatment of stress inducers (iron, copper, and H2O2) significantly upregulated the expression of SmFerM in a dose dependent manner. Iron chelating analysis showed that recombinant SmFerM purified from Escherichia coli exhibited apparent iron binding activity. These results suggest that SmFerM is a functional M ferritin and is likely to play a role in iron sequestration and protection against oxidative stress and microbial infection. [Copyright &y& Elsevier]

Abstract: C-type lectins are calcium-dependent carbohydrate-binding proteins that play important roles in innate immunity. In this study, a C-type lectin homologue (SmLec1) was identified from turbot (Scophthalmus maximus) and analyzed at expression and functional levels. The open reading frame of SmLec1 is 504 bp, with a 5′-untranslated region (UTR) of 101 bp and a 3′-UTR of 164 bp. The deduced amino acid sequence of SmLec1 shares 34%–38% overall identities with the C-type lectins of several fish species. In silico analysis identified in SmLec1 conserved C-type lectin features, including a carbohydrate-recognition domain, four disulfide bond-forming cysteine residues, and the mannose-type carbohydrate-binding motif. In addition, SmLec1 possesses a putative signal peptide sequence and is predicted to be localized in the extracellular. Expression of SmLec1 was highest in liver and responded positively to experimental challenges with fish pathogens. Recombinant SmLec1 (rSmLec1) purified from yeast was able to agglutinate the Gram-negative fish pathogen Listonella anguillarum but not the Gram-positive pathogen Streptococcus iniae. The agglutinating ability of rSmLec1 was abolished in the presence of mannose and ethylenediaminetetraacetic acid and by elevated temperature (65 °C). Further analysis showed that rSmLec1 could stimulate kidney lymphocyte proliferation and enhance the killing of bacterial pathogen by macrophages. Taken together, these results suggest that SmLec1 is a unique mannose-binding C-type lectin that possesses apparent immunomodulating property and is likely to be involved in host defense against bacterial infection. [Copyright &y& Elsevier]