A virulent Bacillus cereus strain from deep-sea cold seep induces pyroptosis in a manner that involves NLRP3 inflammasome, JNK pathway, and lysosomal rupture.

Recent studies indicate that the Bacillus species is distributed in deep-sea environments. However, no specific studies on deep-sea Bacillus cereus have been documented. In the present work, we isolated a B. cereus strain, H2, from the deep-sea cold seep in South China Sea. We characterized the pathogenic potential of H2 and investigated H2-induced death of different types of cells. We found that H2 was capable of tissue dissemination and causing acute mortality in mice and fish following intraperitoneal/intramuscular injection. In vitro studies revealed that H2 infection of macrophages induced pyroptosis and activation of the NLRP3 inflammasome pathway that contributed partly to cell death. H2 infection activated p38, JNK, and ERK, but only JNK proved to participate in H2-triggered cell death. Reactive oxygen species (ROS) and intracellular Ca2+ were essential to H2-induced activation of JNK and NLRP3 inflammasome. In contrast, lysosomal rupture and cathepsins were required for H2-induced NLRP3 inflammasome activation but not for JNK activation. This study revealed for the first time the virulence characteristics of deep-sea B. cereus and provided new insights into the mechanism of B. cereus infection.

A novel small heat shock protein of Haliotis discus hannai: characterization, structure modeling, and expression profiles under environmental stresses.

Small heat shock proteins (sHsps) are a class of chaperones with low molecular weight, feathered by a C-terminal α-crystallin domain (ACD). They participate in reestablishing the stability of partially denatured proteins and therefore contribute to cellular homeostasis. In this work, we identified a sHsp homolog (designated as sHsp19) from Haliotis discus hannai, an economically important farmed mollusk in East Asia. sHsp19 possesses a sHsp hallmark domain, which exhibits the typical fold of ACD as revealed by a three-dimensional model constructed through an iterative threading assembly refinement method. The amino acid sequence sHsp19 shares low identities with any other known sHsps, with percentages below 35 %. Besides, sHsp19 shows relatively distant phylogenetic relationships with sHsps of various mollusks, including two other identified sHsps of abalone subspecies. qRT-PCR analysis indicated that the expression of sHsp19 occurred in multiple tissues. Upon exposure to thermal, oxidative, and multiple toxic metal stresses, the level of sHsp19 mRNA was rapidly elevated in a persistent fashion, with the maximum increase up to 170.58-, 405.84-, and 361.96-fold, respectively. These results indicate sHsp is a novel sHsp that possesses the distinguishing structural feature of sHsps but has remote homologies with known sHsps. It is likely to be important in stress adaptation of abalone and may be applied as a bioindicator for monitoring pollution or detrimental changes of environment in abalone culture.

Cathepsin S of Sciaenops ocellatus: Identification, transcriptional expression and enzymatic activity.

Cathepsin S is a member of cysteine cathepsins and belongs to the cathepsin L-like family. In mammals, it is known to participate in various physiological processes and host immune defense. In teleost fish, the function of cathepsin S is less investigated. In the present work, we characterized a cathepsin S homologue (SoCatS) from red drum (Sciaenops ocellatus), a commercially valuable fish in Chinese mariculture. Like all cathepsin S, SoCatS possesses a peptidase domain with four catalytically essential residues (Gln140, Cys146, His285, and Asn305) conserved in the cathepsin S of different organisms. SoCatS shares 60-90% overall sequence identities with known teleost cathepsin S. Phylogenetic profiling indicated that SoCatS is evolutionally close to the cathepsin S of other teleost fish, especially Miichthys miiuy, a member of Sciaenidae family like red drum. SoCatS expression was detected in various tissues and was enhanced by bacterial infection. Purified recombinant SoCatS exhibited apparent peptidase activity with maximum at 50°C and pH 7.5. This activity depended on the catalytic residue Cys146 and was severely reduced by the cathepsin inhibitor E-64. Our results suggest that SoCatS functions as a cysteine protease which is probably involved in the antibacterial immunity of red drum.

Identification, mRNA expression profiling and activity characterization of cathepsin L from red drum (Sciaenops ocellatus).

Cathepsin L is a cysteine protease with a papain-like structure. It is known to be implicated in multiple processes of mammalian immune response to pathogen infection. In teleost fish, the functionality of cathepsin L is less understood. In this work, we characterized a cathepsin L homologue (designated as SoCatL) from red drum Sciaenops ocellatus, an important farmed fish species in China. SoCatL possesses a typical domain arrangement characteristic of cathepsin L, which comprises a proregion and a protease domain with four catalytically essential residues (Gln137, Cys143, His282 and Asn302) conserved in various organisms. SoCatL shares moderate sequence identities with mammalian cathepsin L and relatively high sequence identities with teleost cathepsin L. Phylogenetic analysis revealed that SoCatL is evolutionally close to fish cathepsin L, especially those belonging to the Perciformes order. The homology model of SoCatL was discovered to exhibit a structure resembling human cathepsin L. Transcriptional expression of SoCatL was found ubiquitous in tissues and enhanced after experimental infection with a bacterial pathogen. Recombinant SoCatL purified from Escherichia coli (designated as rSoCatL) displayed apparent proteolytic activity, which was optimal at 50 °C and pH 7.0. The activity of rSoCatL required the catalytic residue Cys143 and was severely reduced by cathepsin inhibitor. These results suggest that SoCatL is a teleost cathepsin L homologue which functions as a cysteine protease and is likely to participate in the host immune response against bacterial infection.

Edwardsiella tarda Sip1: A serum-induced zinc metalloprotease that is essential to serum resistance and host infection.

Edwardsiella tarda is a severe bacterial pathogen to a wide arrange of farmed fish. One salient virulent feature of E. tarda is a remarkable ability to survive in host serum. In this study, in order to identify E. tarda proteins involved in serum resistance, we conducted proteomic analysis to examine the extracellular protein profiles of TX01, a pathogenic E. tarda isolate, in response to serum treatment. Five differentially expressed proteins were identified, one of which was a putative zinc protease (named Sip1). Western blot confirmed extracellular production of Sip1 by E. tarda. Sequence analysis revealed that Sip1 possesses a conserved zinc metalloprotease motif and shares low homology with the putative zinc proteases/aureolysin of several bacterial species. Purified recombinant Sip1 (rSip1) exhibited zinc-dependent proteolytic activity that reached maximum at 40°C and pH 8. Compared to the wild type, the sip1 knockout mutant, TXΔsip1, was dramatically reduced in the ability to cause mortality in the host (Japanese flounder) and to survive in host serum. These lost virulence capacities of TXΔsip1 were restored by complementation with the sip1 gene. Further study showed that rSip1 enhanced the serum resistance of TX01 and TXΔsip1, whereas antibody blocking of the Sip1 produced naturally by TX01 impaired serum resistance. Vaccination study showed that rSip1 as a subunit vaccine was able to induce effective protection in flounder against E. tarda challenge. Taken together, these results indicate that Sip1 is a novel zinc metalloprotease that is essential to serum resistance and host infection.

Evaluation of potential internal references for quantitative real-time RT-PCR normalization of gene expression in red drum (Sciaenops ocellatus).

Quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) has been used extensively for studying gene expression in diverse organisms including fish. In this study, with an aim to identify reliable reference genes for qRT-PCR in red drum (Sciaenops ocellatus), an economic fish species, we determined the expression stability of seven housekeeping genes in healthy and bacterium-infected red drum. Each of the selected candidate genes was amplified by qRT-PCR from the brain, gill, heart, intestine, kidney, liver, muscle, and spleen of red drum infected with or without a bacterial pathogen for 12 and 48 h. The mRNA levels of the genes were analyzed with the geNorm and NormFinder algorithms. The results showed that in the absence of bacterial infection, translation initiation factor 3, NADH dehydrogenase 1, and QM-like protein may be used together as internal references across the eight examined tissues. Bacterial infection caused variations in the rankings of the most stable genes in a tissue-dependent manner. For all tissues, two genes sufficed for reliable normalization at both 12 and 48 h post-infection. However, the optimal gene pairs differed among tissues and, for four of the examined eight tissues, between infection points. These results indicate that when studying gene expression in red drum under conditions of bacterial infection, the optimal reference genes should be selected on the basis of tissue type and, for accurate normalization, infection stage.

CsTrx80, a truncated form of thioredoxin, possesses chemokine-like property and enhances the immune defense of Cynoglossus semilaevis against bacterial infection.

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

First characterization of a teleost Epstein-Barr virus-induced gene 3 (EBI3) reveals a regulatory effect of EBI3 on the innate immune response of peripheral blood leukocytes.

Epstein-Barr virus-induced gene 3 (EBI3) encodes a protein that in mammals is known to be a subunit of interleukin (IL)-27 and IL-35, both which regulate cytokine production and inflammatory response. To date, no studies on fish EBI3 have been documented. In this work, we report the identification of an EBI3 homologue, CsEBI3, from tongue sole (Cynoglossus semilaevis) and analysis of its expression and biological effect. CsEBI3 is composed of 245 amino acid residues and possesses a Fibronectin type 3 (FN3) domain that is preserved in lower and higher vertebrates. Expression of CsEBI3 was detected in a wide range of tissues, in particular those of immune relevant organs, and upregulated in a time-dependent manner by experimental challenge with bacterial and viral pathogens. Bacterial infection of peripheral blood leukocytes (PBL) enhanced CsEBI3 expression and caused extracellular secretion of CsEBI3. Purified recombinant CsEBI3 (rCsEBI3) stimulated the respiratory burst activity of PBL and upregulated the expression of IL-1ß, IL-8, Myd88, interferon-induced gene 15, CD28, and chemokines. In contrast, rCsEBI3M, a mutant CsEBI3 that lacks the FN3 domain failed to activate PBL and induced much weaker expression of the immune genes. Treatment of PBL with rCsEBI3, but not with the mutant rCsEBI3M, enhanced cellular resistance against bacterial invasion, whereas antibody blocking of CsEBI3 on PBL significantly reduced cellular resistance against bacterial infection. Taken together, these results indicate for the first time that a teleost EBI3 possesses immunoregulatory property in a manner that is dependent on the conserved FN3 domain, and that CsEBI3 is involved in the innate immune defense of PBL against microbial pathogens.

Immunological study of the outer membrane proteins of Vibrio harveyi: insights that link immunoprotectivity to interference with bacterial infection.

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.

Edwardsiella tarda Ivy, a lysozyme inhibitor that blocks the lytic effect of lysozyme and facilitates host infection in a manner that is dependent on the conserved cysteine residue.

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.

Complete genome sequence and transcription profiles of the rock bream iridovirus RBIV-C1.

The family Iridoviridae consists of 5 genera of double-stranded DNA viruses, including the genus Megalocytivirus, which contains species that are important fish pathogens. In a previous study, we isolated the first rock bream iridovirus from China (RBIV-C1) and identified it as a member of the genus Megalocytivirus. In this report, we determined the complete genomic sequence of RBIV-C1 and examined its in vivo expression profiles. The genome of RBIV-C1 is 112333 bp in length, with a GC content of 55% and a coding density of 92%. RBIV-C1 contains 4584 simple sequence repeats, 89.8% of which are distributed among coding regions. A total of 119 potential open reading frames (ORFs) were identified in RBIV-C1, including the 26 core iridovirus genes; 41 ORFs encode proteins that are predicted to be associated with essential biological functions. RBIV-C1 exhibits the highest degree of sequence conservation and colinear arrangement of genes with orange-spotted grouper iridovirus (OSGIV) and rock bream iridovirus (RBIV). The pairwise nucleotide identities are 99.49% between RBIV-C1 and OSGIV and 98.69% between RBIV-C1 and RBIV. Compared to OSGIV, RBIV-C1 contains 11 insertions, 13 deletions, and 103 single nucleotide mutations. Whole-genome transcription analysis showed that following experimental infection of rock bream with RBIV-C1, all but 1 of the 119 ORFs were expressed at different time points and clustered into 3 hierarchical groups based on their expression patterns. These results provide new insights into the genetic nature and gene expression features of megalocytiviruses.

Environmental isolates P1SW and V3SW as a bivalent vaccine induce effective cross-protection against Edwardsiella tarda and Vibrio anguillarum .

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.

Expression characterization and activity analysis of a cathepsin B from Pacific abalone Haliotis discus hannai.

Cathepsin B (EC 3.4.22.1) is a member of lysosomal cysteine protease and has a papain-like fold. In mammals, it is involved in protein degradation and other physiological processes including immune response. However, little is known about the function of cathepsin B in mollusks. In this study, we identified and analyzed a cathepsin B homolog (HdCatB) from Pacific abalone (Haliotis discus hannai), an economically important mollusk species cultured in East Asia. HdCatB is composed of 336 amino acid residues and its mature form is predicted to start at residue 86. HdCatB possesses typical domain architecture of cathepsin B and contains a propeptide region and a cysteine protease domain, the latter containing the four active site residues (Q108, C114, H282, and N302) that are conserved in many different organisms. HdCatB shares 40-60% overall sequence identities with the cathepsin Bofa number of vertebrates and invertebrates and is phylogenetically very close to mollusk cathepsin B. Quantitative real time RT-PCR analysis revealed that HdCatB expression occurred in multiple tissues and was upregulated by bacterial infection. Recombinant HdCatB purified from Escherichia coli exhibited apparent protease activity, which was optimal at 45 °C and pH 6.0. These results indicate that HdCatB is a bioactive protease that is likely to be implicated in the immune response of abalone during bacterial infection.

Identification and characterization of a cell surface scavenger receptor cysteine-rich protein of Sciaenops ocellatus: bacterial interaction and its dependence on the conserved structural features of the SRCR domain.

The scavenger receptor cysteine-rich (SRCR) proteins are secreted or membrane-bound receptors with one or multiple SRCR domains. Members of the SRCR superfamily are known to have diverse functions that include pathogen recognition and immunoregulation. In teleost, although protein sequences with SRCR structure have been identified in some species, very little functional investigation has been carried out. In this study, we identified and characterized a teleost SRCR protein from red drum Sciaenops ocellatus. The protein was named S. ocellatus SRCR1 (SoSRCRP1). SoSRCRP1 is 410-residue in length and was predicted to be a transmembrane protein, with the extracellular region containing a collagen triple helix repeat and a SRCR domain. The SRCR domain has six conserved cysteines, of which, C338 and C399, C351 and C409, and C379 and C389 were predicted to form three disulfide bonds. SoSRCRP1 expression was detected mainly in immune-relevant tissues and upregulated by bacterial and viral infection. In head kidney leukocytes, bacterial infection stimulated the expression of SoSRCRP1, and the expressed SoSRCRP1 was localized on cell surface. Recombinant SoSRCRP1 (rSoSRCRP1) corresponding to the SRCR domain was purified from Escherichia coli and found to be able to bind Gram-negative and Gram-positive bacteria. To examine the structure-function relationship of SoSRCRP1, the mutant proteins SoSRCRP1M1, SoSRCRP1M2, SoSRCRP1M3, and SoSRCRP1M4 were created, which bear C351S and C409S, C338S, C379S, and R325A mutations respectively. Compared to rSoSRCRP1, all mutants were significantly reduced in the ability of bacterial interaction, with the highest reduction observed with SoSRCRP1M4. Taken together, these results indicate that SoSRCRP1 is a cell surface-localized SRCR protein that binds bacterial ligands in a manner that depends on the conserved structural features of the SRCR domain.

Selection of normalization factors for quantitative real time RT-PCR studies in Japanese flounder (Paralichthys olivaceus) and turbot (Scophthalmus maximus) under conditions of viral infection.

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.

Characterization of a c-type lysozyme of Scophthalmus maximus: expression, activity, and antibacterial effect.

Lysozyme is a key component of the innate immune system and plays an important role in antibacterial infection. In this study, we analyzed the expression and activity of a chicken-type (c-type) lysozyme (named SmLysC) from turbot (Scophthalmus maximus). SmLysC is composed of 143 residues and shares 67-90% overall sequence identities with the c-type lysozymes of a number of teleost fish. SmLysC possesses a typical c-type lysozyme domain, which contains the conserved residues E50 and D67 that form the putative catalytic site. SmLysC expression was detected, in increasing order, in head kidney, gill, heart, muscle, brain, spleen, blood, and liver. Bacterial infection caused significant inductions of SmLysC expression in head kidney, spleen, and liver in a time-dependent manner. Immunoblot analysis indicated that SmLysC has a subcellular localization in the extracellular milieu. Recombinant SmLysC (rSmLysC) was able to bind to bacterial cells and inhibit bacterial growth. Enzyme assay showed that the optimal temperature and pH of rSmLysC were 37 °C and pH 6.0 respectively. In contrast to rSmLysC, the mutant protein rSmLysCM1, which bears alanine substitutions at E50 and D67, displayed drastically reduced bacteriolytic activity. rSmLysC was able to inhibit the growth of several fish bacterial pathogens in a manner that depended on the dose of the protein; however, Gram-positive bacteria were in general more sensitive to rSmLysC than Gram-negative bacteria. Together these results indicate that SmLysC is a functional lysozyme that is likely to participate in innate immune defense against extracellular bacterial pathogens, in particular those of Gram-positive nature.

The galectin-3-binding protein of Cynoglossus semilaevis is a secreted protein of the innate immune system that binds a wide range of bacteria and is involved in host phagocytosis.

Galectin-3 binding protein (G3BP) is a secreted glycoprotein that binds galectin-3 and is involved in various pathological conditions including cancer and viral infection. In fish, G3BP-like sequences have been identified in very few species and their biological properties are entirely unknown. In this work, we reported for the first time the identification and analysis of a teleost G3BP, CsG3BP, from half-smooth tongue sole (Cynoglossus semilaevis). CsG3BP is composed of 565 amino acids and possesses a Scavenger Receptor Cysteine-rich (SRCR) domain, the latter containing six conserved cysteine residues that were predicted to form three intramolecular disulfide bridges. Expression of CsG3BP was detected in a wide range of tissues and upregulated by bacterial and megalocytivirus infection in a time-dependent manner. Immunoblot analysis detected CsG3BP in the culture medium of peripheral blood leukocytes (PBL) and in serum following bacterial stimulation. Purified recombinant CsG3BP (rCsG3BP) exhibited bacterial binding ability in a dose-dependent manner. In contrast, the mutant forms of CsG3BP that bear deletion of the SRCR domain or serine substitutions at three cysteine residues involved in disulfide bond formation lost the capacity of bacterial interaction. rCsG3BP displayed a certain substrate preference and bound more effectively to Gram-negative bacteria than to Gram-positive bacteria. Further study showed that when the CsG3BP produced by PBL was blocked by anti-rCsG3BP antibodies, the phagocytic activity of the cells was significantly reduced. Taken together, these results indicate that CsG3BP is a secreted protein that probably plays a role in innate immune defense by binding to bacterial cells via the SRCR domain and thereby facilitating host phagocytosis.

Comparative study of four flagellins of Vibrio anguillarum: vaccine potential and adjuvanticity.

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.

C312M: an attenuated Vibrio anguillarum strain that induces immunoprotection as an oral and immersion vaccine.

Vibrio anguillarum, a Gram-negative bacterial pathogen, is the causative agent of vibriosis that affects a wide range of aquatic animals. In this study, we obtained a mutant V. anguillarum, C312M, derived from the pathogenic V. anguillarum C312 by selection of rifampicin resistance. C312M was slower in growth than the wild type C312, particularly under conditions of iron depletion. Compared to C312, C312M was altered in protein production profile and exhibited a dramatically increased median lethal dose. Safety analysis showed that C312M was stable in virulence in the absence of selective pressure. To examine the potential of C312M as a live attenuated vaccine, Japanese flounder Paralichthys olivaceus were vaccinated with C312M via oral, immersion, and oral plus immersion routes. Microbiological analysis showed that C312M was recovered from the gut, liver, kidney, and spleen of the vaccinated fish in 1 to 14 d post-vaccination. When the fish were challenged with C312 at 1 mo post-vaccination, C312M-vaccinated fish exhibited relative percent survival rates of 60 to 84%. Comparable protection was observed when the fish were challenged with a heterologous V. anguillarum strain. Further analysis showed that C312M-vaccinated fish produced specific serum antibodies which enhanced serum bactericidal activity in a manner that is probably complement-dependent. These results indicate that C312M is highly attenuated in virulence but still retains residual infectivity, and that C312M is an effective vaccine when delivered alive via immersion and oral feeding.

Edwardsiella tarda sialidase: pathogenicity involvement and vaccine potential.

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.