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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 TshEt) and examined its function and vaccine potential. TshEt is composed of 1224 residues and has three functional domains typical for autotransporters. Quantitative real-time reverse transcriptase-PCR analysis showed that expression of tshEt 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, TX01Δtsh, was constructed to determine whether TshEt participates in the pathogenicity of E. tarda, Compared to the wild type TX01, TX01Δtsh 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 TX01Δtsh. The passenger domain of TshEt 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 TshEt exerts pleiotropic effects on the pathogenesis of E. tarda., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Unlabelled: 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 TX01Δfur 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 TX01Δfur was rescued by introduction of an exogenous fur gene. iTRAQ-based comparative proteomic analysis of TX01Δfur 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., (Copyright © 2016. Published by Elsevier B.V.)

Hfq is an RNA-binding protein that plays an important role in many cellular processes. In this study, we examined the biological effect of the Hfq of Edwardsiella tarda, a severe fish pathogen with a broad host range that includes humans. To facilitate the study, a markerless hfq in-frame deletion wild type, TXhfq, was constructed. Compared to the wild type TX01, TXhfq exhibited (i) retarded planktonic and biofilm growth, (ii) decreased resistance against oxidative stress, (iii) attenuated overall virulence and tissue dissemination and colonization capacity, (iv) impaired ability to replicate in host macrophages and to block host immune response. Introduction of a trans-expressed hfq gene into TXhfq restored the lost virulence of TXhfq. To identify potential Hfq targets, comparative global proteomic analysis was conducted, which revealed that 20 proteins belonging to different functional categories were differentially expressed in TXhfq and TX01. Quantitative real time RT-PCR analysis showed that the mRNA levels of two thirds of the genes of the identified proteins were consistent with the proteomic results. Since TXhfq is dramatically attenuated in virulence, we further examined its potential as a naturally delivered vaccine administered via the immersion route in a flounder model. The results showed that TXhfq induced effective protection against lethal E. tarda challenge. Taken together, our study indicated that Hfq is required for the normal operation of E. tarda in multiple aspects, and that Hfq probably exerts a regulatory effect on a wide range of target genes at both transcription and post-transcription levels.

Edwardsiella tarda is a Gram-negative bacterial pathogen with a broad host range that includes fish and humans. In this study, we examined the activity and function of the lysozyme inhibitor Ivy (named IvyEt) identified in the pathogenic E. tarda strain TX01. IvyEt possesses the Ivy signature motif CKPHDC in the form of (82)CQPHNC(87) and contains several highly conserved residues, including a tryptophan (W55). For the purpose of virulence analysis, an isogenic TX01 mutant, TXivy, was created. TXivy bears an in-frame deletion of the ivyEt gene. A live infection study in a turbot (Scophthalmus maximus) model showed that, compared to TX01, TXivy exhibited attenuated overall virulence, reduced tissue dissemination and colonization capacity, an impaired ability to replicate in host macrophages, and decreased resistance against the bactericidal effect of host serum. To facilitate functional analysis, recombinant IvyEt (rIvy) and three mutant proteins, i.e., rIvyW55A, rIvyC82S, and rIvyH85D, which bear Ala, Ser, and Asp substitutions at W55, C82, and H85, respectively, were prepared. In vitro studies showed that rIvy, rIvyW55A, and rIvyH85D were able to block the lytic effect of lysozyme on a Gram-positive bacterium, whereas rIvyC82S could not do so. Likewise, rIvy, but not rIvyC82S, inhibited the serum-facilitated killing effect of lysozyme on E. tarda. In vivo analysis showed that rIvy, but not rIvyC82S, restored the lost pathogenicity of TXivy and enhanced the infectivity of TX01. Together these results indicate that IvyEt is a lysozyme inhibitor and a virulence factor that depends on the conserved C82 for biological activity.

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 © 2013 Elsevier Ltd. All rights reserved.)

Edwardsiella tarda and Vibrio anguillarum are severe fish pathogens. In this study, we aimed at selecting avirulent environmental isolates with application potential in the prevention of E. tarda- and V. anguillarum-associated diseases. For this purpose, we selected and analyzed 2 seawater isolates, P1SW and V3SW, belonging to the genera Pseudomonas and Vibrio, respectively. When administered to turbot Scophthalmus maximus via immersion and oral feeding, P1SW and V3SW at a dose of 2 × 108 colony-forming units caused no mortality, but both strains were able to disseminate into internal organs in a transient, time-dependent manner. When turbot were immunized with P1SW, V3SW, or P1SW plus V3SW (named P1V3) via immersion plus oral routes, the latter with vaccines embedded in sodium alginate microspheres, moderate protection against E. tarda and V. anguillarum was induced by V3SW, and moderate protection against E. tarda was induced by P1SW. Compared to P1SW and V3SW, P1V3 elicited a significantly stronger protection against both E. tarda and V. anguillarum. Immunological analysis showed that (1) P1SW, V3SW, and especially P1V3 activated head kidney macrophages, (2) P1V3 induced significantly higher levels of serum antibodies against E. tarda and V. anguillarum than P1SW and V3SW, and (3) P1V3-induced antibodies were able to bind E. tarda and V. anguillarum and enhance serum bactericidal activity. These results indicate that P1V3 as a naturally delivered vaccine elicited a humoral immune response against both E. tarda and V. anguillarum and, as a result, was cross-protective against E. tarda and V. anguillarum infection.

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 © 2012 Elsevier Ltd. All rights reserved.)

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 © 2012 Elsevier Ltd. All rights reserved.)

Edwardsiella tarda, a Gram-negative bacterium, is a severe fish pathogen that can also infect humans. In this study, we identified, via in vivo-induced antigen technology, an E. tarda antigen, Eta1, and analyzed its function in a Japanese flounder (Paralichthys olivaceus) model. Eta1 is composed of 226 residues and shares homology with putative bacterial adhesins. Quantitative real-time reverse transcriptase (RT)-PCR analysis indicated that when cultured in vitro, eta1 expression was growth phase dependent and reached maximum at mid-logarithmic phase. During infection of flounder lymphocytes, eta1 expression was drastically increased at the early stage of infection. Compared to the wild type, the eta1-defective mutant, TXeta1, was unaffected in growth but exhibited attenuated overall virulence, reduced tissue dissemination and colonization capacity, and impaired ability to invade flounder lymphocytes and to block the immune response of host cells. The lost virulence of TXeta1 was restored when a functional eta1 gene was reintroduced into the strain. Western blot and immunodetection analyses showed that Eta1 is localized to the outer membrane and exposed on the surface of E. tarda and that recombinant Eta1 (rEta1) was able to interact with flounder lymphocytes. Consistent with these observations, antibody blocking of Eta1 inhibited E. tarda infection at the cellular level. Furthermore, when used as a subunit vaccine, rEta1 induced strong protective immunity in flounder against lethal E. tarda challenge. Taken together, these results indicate that Eta1 is an in vivo-induced antigen that mediates pathogen-host interaction and, as a result, is required for optimal bacterial infection.

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 © 2012 Elsevier Ltd. All rights reserved.)

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 © 2012 Elsevier Ltd. All rights reserved.)

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 H(2)O(2) 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 H(2)O(2) 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 © 2011 Elsevier Ltd. All rights reserved.)

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 temperature 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 © 2011 Elsevier B.V. All rights reserved.)

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 © 2011 Elsevier Ltd. All rights reserved.)

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 © 2010 Elsevier Ltd. All rights reserved.)

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 beta-strands with a Ca(2+)-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 2010 Elsevier Ltd. All rights reserved.)

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 2010 Elsevier Ltd. All rights reserved.)

Edwardsiella tarda is a severe aquaculture pathogen that can infect many fish species and cause a systematic disease called edwardsiellosis, which can lead to high mortality under certain conditions. Currently, most vaccine candidates against edwardsiellosis are based on pathogenic E. tarda strains, which can be a concern in some cases. In this study, the vaccine potential of a natural E. tarda isolate, ATCC 15947, was examined in a Japanese flounder model. ATCC 15947 was found to be relatively avirulent to flounder but able to disseminate into and survive transiently in fish tissues following intraperitoneal (i.p.) injection. Fish vaccinated with ATCC 15947 via i.p. injection exhibited a high level of survival rate, which was increased to 100% by a booster injection. Fish vaccinated with ATCC 15947 orally in the form of alginate microspheres showed a moderate survival rate, while fish vaccinated with ATCC 15947 via the immersion route exhibited a low rate of survival. Following oral vaccination, ATCC 15947 could colonize transiently in the gut, liver, and spleen of the vaccinated fish. Both injection and oral vaccination with ATCC 15947 induced production of specific serum antibodies, the levels of which at different time points following vaccination were generally higher in fish vaccinated with ATCC 15947 via injection than in fish vaccinated with ATCC 15947 orally. Compared to control fish, fish vaccinated with ATCC 15947 showed enhanced serum bactericidal activity and significantly increased expression in genes encoding a number of immune-related factors. These results indicated that ATCC 15947 possesses good immunoprotective potential, which may be exploited in the development of E. tarda vaccines., (Copyright 2010 Elsevier Ltd. All rights reserved.)

Edwardsiella tarda is a severe aquaculture pathogen that can infect many important fish species cultured worldwide. The aim of this study was to evaluate the vaccine potential of an E. tarda antigen, Eta21, which was identified from a pathogenic E. tarda strain via the method of in vivo-induced antigen technology (IVIAT). Eta21 is 510-amino acid in length and shares approximately 58% sequence identity with a putative peptidase of several bacterial species. eta21 was subcloned into Escherichia coli, and recombinant Eta21 was purified as a histidine-tagged protein. When used as a subunit vaccine, purified recombinant Eta21 was effective against lethal E. tarda challenge in a Japanese flounder model. In order to improve the immunoprotective efficacy of Eta21, the chimera AgaV-Eta21 was constructed, which consists of Eta21 fused in-frame to the secretion domain of AgaV, an extracellular beta-agarase. E. coli DH5alpha harboring plasmid pTAET21, which constitutively expresses agaV-eta21, was able to produce and secret AgaV-Eta21 into the extracellular milieu. Vaccination of Japanese flounder with live DH5alpha/pTAET21 elicited immunoprotection that is significantly higher in level than that induced by vaccination with purified recombinant Eta21. Vaccination with DH5alpha/pTAET21 and recombinant Eta21 both induced the production of specific serum antibodies at four to eight weeks post-vaccination. Taken together, these results demonstrate that Eta21, especially that delivered by DH5alpha/pTAET21, is an effective vaccine candidate against E. tarda infection.

Edwardsiella tarda is an opportunistic pathogen that can infect humans, animal, and fish. Two E. tarda antigens, Eta6 and FliC, which are homologues to an ecotin precursor and the FliC flagellin, respectively, were identified by in vivo-induced antigen technology from a pathogenic E. tarda strain isolated from diseased fish. When used as a subunit vaccine, purified recombinant Eta6 was moderately protective against lethal challenge of E. tarda in a Japanese flounder model, whereas purified recombinant FliC showed no apparent immunoprotectivity. Similarly, DNA vaccines based on eta6 and fliC in the form of plasmids pEta6 and pFliC induced, respectively, moderate and marginal protection against E. tarda infection. To improve the vaccine efficacy of eta6, a chimeric DNA vaccine, pCE6, was constructed, which encodes Eta6 fused in-frame to FliC. pCE6 was found to induce significantly higher level of protection than pEta6. Likewise, another chimeric DNA vaccine, pCE18, which expresses FliC fused to a previously identified E. tarda antigen Et18, elicited significantly stronger protective immunity than the DNA vaccine based on et18 alone. Fish immunized with pEta6 and pCE6 produced specific serum antibodies and exhibited significantly enhanced expression of the genes encoding elements that are involved in both innate and adaptive immune responses. Furthermore, the induction magnitudes of most of these genes were significantly higher in pCE6-vaccinated fish than in pEta6-vaccinated fish.

Edwardsiella tarda is a pathogen with a broad host range that includes human and animals. The E. tarda hemolysin (Eth) system, which comprises EthA and EthB, is a noted virulence element that is widely distributed in pathogenic isolates of E. tarda. Previous study has shown that the expression of ethB is regulated by iron, which suggests the possibility that the ferric uptake regulator (Fur) is involved in the regulation of ethB. The work presented in this report supports the previous findings and demonstrates that ethB expression was decreased under conditions when the E. tarda Fur (Fur(Et)) was overproduced, and enhanced when FurEt was inactivated. We also identified a second ethB regulator, EthR, which is a transcription regulator of the GntR family. EthR represses ethB expression by direct interaction with the ethB promoter region. In addition to ethB, EthR also modulates, but positively, luxS expression and AI-2 production by binding to the luxS promoter region. The expression of ethR itself is subject to negative autoregulation; interference with this regulation by overexpressing ethR during the process of infection caused (i) drastic changes in ethB and luxS expressions, (ii) vitiation in the tissue dissemination and survival ability of the bacterium, and (iii) significant attenuation of the overall bacterial virulence. These results not only provide new insights into the regulation mechanisms of the Eth hemolysin and LuxS/AI-2 quorum sensing systems but also highlight the importance of these systems in bacterial virulence.

Edwardsiella tarda is one of the leading marine pathogens that can infect a wide range of cultured marine species. In this study, the acrR-acrAB cluster was cloned from TX1, a pathogenic E. tarda strain isolated from diseased fish. AcrR and AcrAB were found to be involved in resistance against acriflavine and methyl viologen, which positively regulate the expression of acrAB. AcrR negatively regulates its own expression and the expression of the acrAB operon, most likely by interacting with a 24-bp operator site that overlaps the putative promoter of acrA (P(acrA)). The repressive effect of AcrR on P(acrA) could be relieved by acriflavine, methyl viologen, and ethidium bromide, the presence of each of which enhanced transcription from P(acrA). Interruption of the regulated expression of acrR by introducing into TX1 a plasmid that overexpresses acrR affected growth under stress conditions, AI-2 production, and bacterial virulence. In addition, mutational analyses identified a constitutively active AcrR mutant (named N215), which exhibits full repressor activity but is impaired in its ability to interact with the inducer. Overexpression of N215 produced the same kind of but moderately stronger effect on TX1 compared to that produced by overexpression of the wild-type acrR.

Edwardsiella tarda is a gram-negative pathogen with a broad host range that includes humans, animals, and fish. Recent studies have shown that the LuxS/autoinducer type 2 (AI-2) quorum sensing system is involved in the virulence of E. tarda. In the present study, it was found that the E. tarda LuxS mutants bearing deletions of the catalytic site (C site) and the tyrosine kinase phosphorylation site, respectively, are functionally inactive and that these dysfunctional mutants can interfere with the activity of the wild-type LuxS. Two small peptides, 5411 and 5906, which share sequence identities with the C site of LuxS, were identified. 5411 and 5906 proved to be inhibitors of AI-2 activity and could vitiate the infectivity of the pathogenic E. tarda strain TX1. The inhibitory effect of 5411 and 5906 on AI-2 activity is exerted on LuxS, with which these peptides specifically interact. The expression of 5411 and 5906 in TX1 has multiple effects (altering biofilm production and the expression of certain virulence-associated genes), which are similar to those caused by interruption of luxS expression. Further study found that it is very likely that 5411 and 5906 can be released from the strains expressing them and, should TX1 be in the vicinity, captured by TX1. Based on this observation, a constitutive 5411 producer (Pseudomonas sp. strain FP3/pT5411) was constructed in the form of a fish commensal isolate that expresses 5411 from a plasmid source. The presence of FP3/pT5411 in fish attenuates the virulence of TX1. Finally, it was demonstrated that fish expressing 5411 directly from tissues exhibit enhanced resistance against TX1 infection.

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]

Abstract: Edwardsiella tarda is a severe aquaculture pathogen that can infect many different fish species cultured worldwide. Et49 is a major E. tarda antigen with weak immunoprotective potential. In this study, using Et49 as an example vaccine, the adjuvanticity of Freund''s incomplete adjuvant (FIA), aluminum hydroxide, and aluminum phosphate adjuvant were evaluated in a Japanese flounder model. The results showed that the presence of FIA, aluminum hydroxide, and aluminum phosphate adjuvant increased the relative percent of survival of Et49-vaccinated fish by 47%, 19%, and 35%, respectively. Fish vaccinated with FIA-adjuvanted Et49 exhibited longer persistence of vaccine at the injection site and more severe intra-abdominal lesions than fish vaccinated with aluminum-adjuvanted Et49. Both aluminum adjuvants and, to a lesser degree, FIA augmented the production of specific serum antibodies, which reached the highest levels at 6 and 7 weeks post-vaccination. Passive immunization of Japanese flounder with sera from fish vaccinated with aluminum- and FIA-adjuvanted Et49 induced no protection against lethal E. tarda challenge. Examination of the transcription profile of immune-related genes showed that vaccination with aluminum-adjuvanted Et49 significantly enhanced the expression of the genes that are associated mainly with humoral immunity, whereas vaccination with FIA-adjuvanted Et49 induced the expression of a much broader spectrum of genes that are likely to be involved in humoral and innate cellular immunity. These results provide new insights to the action mechanisms of FIA and aluminum adjuvants in Japanese flounder and may be useful for the selection of adjuvant for vaccine formulations intended for Japanese flounder. [Copyright &y& Elsevier]

The major histocompatibility complex (MHC) is a highly polymorphic region of the vertebrate genome that plays a critical role in initiating immune responses towards invading pathogens. It is well known that MHC I molecules play a central role in the immune response to viruses. However, rare literatures were reported the role of MHC I in the resistance to intracellular bacteria. Sequences of MHC Iα were identified in multiple teleost species, including Japanese flounder (Paralichthys olivaceus), however, the immunological function of MHC Iα remain largely unknown. In this study, we examined the expression profile and biological activity of an MHC Iα homologue, PoMHC Iα, from P. olivaceus. Structural analysis showed that PoMHC Iα possesses conserved structural characteristics of MHC Iα proteins, including MHC_I domain, IGc1 domain, transmembrane region. Expression of PoMHC Iα was upregulated in a time-dependent manner by extracellular and intracellular bacterial pathogens and viral pathogen infection. Different expression patterns were exhibited in response to the infection of different types of microbial pathogens in different immune tissues. Recombinant PoMHC Iα increased the capability of host cells to defense against intracellular pathogen Edwardsiella tarda infection and enhanced the expression of immune related genes. The knockdown of PoMHC Iα attenuated the ability of cells to eliminate E. tarda , which was sustained by the in vivo results that overexpression of PoMHC Iα promoted the host defense against invading E. tarda. Antigen uptake assay indicated PoMHC Iα participated in cells antigen presentation. Collectively, this study is the first report that MHC Iα plays an important role in immune defense against intracellular bacterial pathogen in teleost. Taken together, these findings add new insights into the biological function of teleost MHC Iα and emphasize the importance of MHC I gene products for the control of E. tarda infection. • Expression of PoMHC Iα was upregulated during bacterial and viral pathogens infection. • rPoMHC Iα enhanced cellular resistance against intracellular pathogen infection. • The knockdown of PoMHC Iα attenuated the ability of host cells to eliminate pathogenic bacteria. • Overexpression of PoMHC Iα promoted the host defense against invading pathogen. • rPoMHC Iα and overexpression of PoMHC Iα enhanced immune related genes expression. [ABSTRACT FROM AUTHOR]

Granulocyte colony stimulating factor (GCSF) is a key regulator of neutrophil production, and plays a vital role in immune response of mammals and teleost against pathogen. Sequences of GCSF were identified in several teleost species, however, the function and activity of GCSF in teleost remain largely unknown. In this study, we examined the biological activity and the immunomodulatory property of a GCSF homologue, PoGCSF, from Japanese flounder (Paralichthys olivaceus). Structural analysis showed that PoGCSF possesses conserved structural characteristics of GCSF proteins, including a signal peptide and a typical IL-6 domain. The expression of PoGCSF was upregulated in a time-dependent manner by extracellular and intracellular bacterial pathogens and viral pathogen. Different expression patterns were exhibited in response to the infection of different types of microbial pathogens in different immune tissues. Recombinant PoGCSF increased the capability of host cells to defense against pathogen infection and enhanced the expression of immune related genes. The knockdown of PoGCSF attenuated the ability of host cells to eliminate pathogenic bacteria. In vivo results showed that overexpression of PoGCSF promoted the host defense against invading pathogenic microorganism. Collectively, this study is the first report about the immunoregulatory property and anti-infectious immunity of GCSF in teleost. These findings suggested that PoGCSF serves as an immune-related cytokine and plays an important role in the immune defense system of Japanese flounder. • Expression of PoGCSF was upregulated during bacterial and viral pathogens infection. • rPoGCSF increased the capability of host cells to defense against pathogen infection. • rPoGCSF enhanced the expression of immune related genes. • The knockdown of PoGCSF attenuated the ability of host cells to eliminate pathogenic bacteria. • Overexpression of PoGCSF promoted the host clearance of pathogen. [ABSTRACT FROM AUTHOR]