Molecular Mechanisms Underlying Vibrio Tolerance in Ruditapes philippinarum Revealed by Comparative Transcriptome Profiling.

The clam Ruditapes philippinarum is an important species in the marine aquaculture industry in China. However, in recent years, the aquaculture of R. philippinarum has been negatively impacted by various bacterial pathogens. In this study, the transcriptome libraries of R. philippinarum showing different levels of resistance to challenge with Vibrio anguillarum were constructed and RNA-seq was performed using the Illumina sequencing platform. Host immune factors were identified that responded to V. anguillarum infection, including C-type lectin domain, glutathione S-transferase 9, lysozyme, methyltransferase FkbM domain, heat shock 70 kDa protein, Ras-like GTP-binding protein RHO, C1q, F-box and BTB/POZ domain protein zf-C2H2. Ten genes were selected and verified by RT-qPCR, and nine of the gene expression results were consistent with those of RNA-seq. The lectin gene in the phagosome pathway was expressed at a significantly higher level after V. anguillarum infection, which might indicate the role of lectin in the immune response to V. anguillarum. Comparing the results from R. philippinarum resistant and nonresistant to V. anguillarum increases our understanding of the resistant genes and key pathways related to Vibrio challenge in this species. The results obtained here provide a reference for future immunological research focusing on the response of R. philippinarum to V. anguillarum infection.

The ECSIT Mediated Toll3-Dorsal-ALFs Pathway Inhibits Bacterial Amplification in Kuruma Shrimp.

The Toll signaling pathway plays an important role in animal innate immunity. However, its activation and signal transmission greatly differ across species and need to be investigated. Shrimp farming is a worldwide economic activity affected by bacterial disease from the 1990s, which promoted research on shrimp immunity. In this study, we first proved that, among the three identified Toll receptors in Marsupenaeus japonicus kuruma shrimp, Toll 3 plays a pivotal role in initiating the antibacterial response in vivo, especially upon anti-Staphylococcus aureus infection. Further research showed that this result was due to the activation of the Dorsal transcription factor, which induced the expression of two anti-lipopolysaccharide factors (Alfs). Moreover, the evolutionarily conserved signaling intermediate in Toll pathways, ECSIT, was proved to be needed for signal transmission from Toll 3 to Dorsal and the expression of anti-lipopolysaccharide factors. Finally, the mortality assay showed that a Toll3-ECSIT-Dorsal-Alf axis was functional in the anti-S.aureus immunity of M. japonicus shrimp. The results provide new insights into the function and signal transduction of the Toll pathway in aquatic species and offer basic knowledge for shrimp disease control and genetic breeding.

Exon-Intron Circular RNA circRNF217 Promotes Innate Immunity and Antibacterial Activity in Teleost Fish by Reducing miR-130-3p Function.

Circular RNA (circRNA) is produced by splicing head to tail and is widely distributed in multicellular organisms, and circRNA reportedly can participate in various cell biological processes. In this study, we discovered a novel exon-intron circRNA derived from probable E3 ubiquitin-protein ligase RNF217 (RNF217) gene, namely, circRNF217, which was related to the antibacterial responses in teleost fish. Results indicated that circRNF217 played essential roles in host antibacterial immunity and inhibited the Vibrio anguillarum invasion into cells. Our study also found a microRNA miR-130-3p, which could inhibit antibacterial immune response and promote V. anguillarum invasion into cells by targeting NOD1. Moreover, we also found that the antibacterial effect inhibited by miR-130-3p could be reversed with circRNF217. In mechanism, our data revealed that circRNF217 was a competing endogenous RNA of NOD1 by sponging miR-130-3p, leading to activation of the NF-κB pathway and then enhancing the innate antibacterial responses. In addition, we also found that circRNF217 can promote the antiviral response caused by Siniperca chuatsi rhabdovirus through targeting NOD1. Our study provides new insights for understanding the impact of circRNA on host-pathogen interactions and formulating fish disease prevention to resist the severely harmful V. anguillarum infection.

IL-17/IL-17 Receptor Pathway-Mediated Inflammatory Response in Apostichopus japonicus Supports the Conserved Functions of Cytokines in Invertebrates.

Inflammation participates in host defenses against infectious agents and contributes to the pathophysiology of many diseases. IL-17 is a well-known proinflammatory cytokine that contributes to various aspects of inflammation in vertebrates. However, the functional role of invertebrate IL-17 in inflammatory regulation is not well understood. In this study, we first established an inflammatory model in the Vibrio splendidus-challenged sea cucumber Apostichopus japonicus (Echinodermata). Typical inflammatory symptoms, such as increased coelomocyte infiltration, tissue vacuoles, and tissue fractures, were observed in the V. splendidus-infected and diseased tissue of the body wall. Interestingly, A. japonicus IL-17 (AjIL-17) expression in the body wall and coelomocytes was positively correlated with the development of inflammation. The administration of purified recombinant AjIL-17 protein also directly promoted inflammation in A. japonicus Through genome searches and ZDOCK prediction, a novel IL-17R counterpart containing FNIII and hypothetical TIR domains was identified in the sea cucumber genome. Coimmunoprecipitation, far-Western blotting, and laser confocal microscopy confirmed that AjIL-17R could bind AjIL-17. A subsequent cross-linking assay revealed that the AjIL-17 dimer mediates the inflammatory response by the specific binding of dimeric AjIL-17R upon pathogen infection. Moreover, silencing AjIL-17R significantly attenuated the LPS- or exogenous AjIL-17-mediated inflammatory response. Functional analysis revealed that AjIL-17/AjIL-17R modulated inflammatory responses by promoting A. japonicus TRAF6 ubiquitination and p65 nuclear translocation and evenly mediated coelomocyte proliferation and migration. Taken together, our results provide functional evidence that IL-17 is a conserved cytokine in invertebrates and vertebrates associated with inflammatory regulation via the IL-17-IL-17R-TRAF6 axis.

A unique NLRC4 receptor from echinoderms mediates Vibrio phagocytosis via rearrangement of the cytoskeleton and polymerization of F-actin.

Many members of the nucleotide-binding and oligomerization domain (NACHT)- and leucine-rich-repeat-containing protein (NLR) family play crucial roles in pathogen recognition and innate immune response regulation. In our previous work, a unique and Vibrio splendidus-inducible NLRC4 receptor comprising Ig and NACHT domains was identified from the sea cucumber Apostichopus japonicus, and this receptor lacked the CARD and LRR domains that are typical of common cytoplasmic NLRs. To better understand the functional role of AjNLRC4, we confirmed that AjNLRC4 was a bona fide membrane PRR with two transmembrane structures. AjNLRC4 was able to directly bind microbes and polysaccharides via its extracellular Ig domain and agglutinate a variety of microbes in a Ca2+-dependent manner. Knockdown of AjNLRC4 by RNA interference and blockade of AjNLRC4 by antibodies in coelomocytes both could significantly inhibit the phagocytic activity and elimination of V. splendidus. Conversely, overexpression of AjNLRC4 enhanced the phagocytic activity of V. splendidus, and this effect could be specifically blocked by treatment with the actin-mediated endocytosis inhibitor cytochalasin D but not other endocytosis inhibitors. Moreover, AjNLRC4-mediated phagocytic activity was dependent on the interaction between the intracellular domain of AjNLRC4 and the ß-actin protein and further regulated the Arp2/3 complex to mediate the rearrangement of the cytoskeleton and the polymerization of F-actin. V. splendidus was found to be colocalized with lysosomes in coelomocytes, and the bacterial quantities were increased after injection of chloroquine, a lysosome inhibitor. Collectively, these results suggested that AjNLRC4 served as a novel membrane PRR in mediating coelomocyte phagocytosis and further clearing intracellular Vibrio through the AjNLRC4-ß-actin-Arp2/3 complex-lysosome pathway.

Nitric Oxide Synthase Regulates Gut Microbiota Homeostasis by ERK-NF-κB Pathway in Shrimp.

The gut microbiota is a complex group of microorganisms that is not only closely related to intestinal immunity but also affects the whole immune system of the body. Antimicrobial peptides and reactive oxygen species participate in the regulation of gut microbiota homeostasis in invertebrates. However, it is unclear whether nitric oxide, as a key mediator of immunity that plays important roles in antipathogen activity and immune regulation, participates in the regulation of gut microbiota homeostasis. In this study, we identified a nitric oxide synthase responsible for NO production in the shrimp Marsupenaeus japonicus. The expression of Nos and the NO concentration in the gastrointestinal tract were increased significantly in shrimp orally infected with Vibrio anguillarum. After RNA interference of Nos or treatment with an inhibitor of NOS, L-NMMA, NO production decreased and the gut bacterial load increased significantly in shrimp. Treatment with the NO donor, sodium nitroprusside, increased the NO level and reduced the bacterial load significantly in the shrimp gastrointestinal tract. Mechanistically, V. anguillarum infection increased NO level via upregulation of NOS and induced phosphorylation of ERK. The activated ERK phosphorylated the NF-κB-like transcription factor, dorsal, and caused nuclear translocation of dorsal to increase expression of antimicrobial peptides (AMPs) responsible for bacterial clearance. In summary, as a signaling molecule, NOS-produced NO regulates intestinal microbiota homeostasis by promoting AMP expression against infected pathogens via the ERK-dorsal pathway in shrimp.

The Role of Flagellin B in Vibrio anguillarum-Induced Intestinal Immunity and Functional Domain Identification.

Vibrio anguillarum, an opportunistic pathogen of aquatic animals, moves using a filament comprised of polymerised flagellin proteins. Flagellins are essential virulence factors for V. anguillarum infection. Herein, we investigated the effects of flagellins (flaA, flaB, flaC, flaD and flaE) on cell apoptosis, TLR5 expression, and production of IL-8 and TNF-α. FlaB exhibited the strongest immunostimulation effects. To explore the functions of flaB in infection, we constructed a flaB deletion mutant using a two-step recombination method, and in vitro experiments showed a significant decrease in the expression of TLR5 and inflammatory cytokines compared with wild-type cells. However in the in vivo study, expression of inflammatory cytokines and intestinal mucosal structure showed no significant differences between groups. Additionally, flaB induced a significant increase in TLR5 expression based on microscopy analysis of fluorescently labelled TLR5, indicating interactions between the two proteins, which was confirmed by native PAGE and yeast two-hybrid assay. Molecular simulation of interactions between flaB and TLR5 was performed to identify the residues involved in binding, revealing two binding sites. Then, based on molecular dynamics simulations, we carried out thirteen site-directed mutations occurring at the amino acid sites of Q57, N83, N87, R91, D94, E122, D152, N312, R313, N320, L97, H316, I324 in binding regions of flaB protein by TLR5, respectively. Surface plasmon resonance (SPR) was employed to compare the affinities of flaB mutants for TLR5, and D152, D94, I324, N87, R313, N320 and H316 were found to mediate interactions between flaB and TLR5. Our comprehensive and systematic analysis of V. anguillarum flagellins establishes the groundwork for future design of flagellin-based vaccines.

CircRNA75 and CircRNA72 Function as the Sponge of MicroRNA-200 to Suppress Coelomocyte Apoptosis Via Targeting Tollip in Apostichopus japonicus.

Circular RNAs (circRNAs) act as essential regulators in many biological processes, especially in mammalian immune response. Nonetheless, the functions and mechanisms of circRNAs in the invertebrate immune system are largely unclarified. In our previous work, 261 differentially expressed circRNAs potentially related to the development of Apostichopus japonicus skin ulceration syndrome (SUS), which is a major problem restricting the sea cucumber breeding industry, were identified by genome-wide screening. In this study, via miRanda analysis, both circRNA75 and circrRNA72 were shown to share the miR-200 binding site, a key microRNA in the SUS. The two circRNAs were verified to be increased significantly in LPS-exposed primary coelomocytes, similar to the results of circRNA-seq in sea cucumber under Vibrio splendidus-challenged conditions. A dual-luciferase assay indicated that both circRNA75 and circRNA72 could bind miR-200 in vivo, in which circRNA75 had four binding sites of miR-200 and only one for circRNA72. Furthermore, we found that miR-200 could bind the 3'-UTR of Toll interacting protein (Tollip) to negatively mediate the expression of Tollip. Silencing Tollip increased primary coelomocyte apoptosis. Consistently, inference of circRNA75 and circRNA72 could also downregulate Tollip expression, thereby increasing the apoptosis of primary coelomocytes, which could be blocked by miR-200 inhibitor treatment. Moreover, the rate of si-circRNA75-downregulated Tollip expression was higher than that of si-circRNA72 under an equivalent amount. CircRNA75 and circRNA72 suppressed coelomocyte apoptosis by sponging miR-200 to promote Tollip expression. The ability of circRNA to adsorb miRNA might be positively related to the number of binding sites for miRNA.

A novel C-type lectin activates the complement cascade in the primitive oyster Crassostrea gigas.

The ancient origin of the lectin pathway of the complement system can be traced back to protochordates (such as amphioxus and tunicates) by the presence of components such as ficolin, glucose-binding lectin, mannose-binding lectin-associated serine protease (MASP), and C3. Evidence for a more primitive origin is offered in the present study on the Pacific oyster Crassostrea gigas. C3 protein in C. gigas (CgC3) was found to be cleaved after stimulation with the bacteria Vibrio splendidus. In addition, we identified a novel C-type lectin (defined as CgCLec) with a complement control protein (CCP) domain, which recognized various pathogen-associated molecular patterns (PAMPs) and bacteria. This protein was involved in the activation of the complement system by binding CgMASPL-1 to promote cleavage of CgC3. The production of cytokines and antibacterial peptides, as well as the phagocytotic ratio of haemocytes in CgCLec-CCP-, CgMASPL-1-, or CgC3-knockdown oysters, decreased significantly after V. splendidus stimulation. Moreover, this activated CgC3 participated in perforation of bacterial envelopes and inhibiting survival of the infecting bacteria. These results collectively suggest that there existed an ancient lectin pathway in molluscs, which was activated by a complement cascade to regulate the production of immune effectors, phagocytosis, and bacterial lysis.

Evaluation of the efficacy of a novel Vibrio vulnificus vaccine based on antibacterial peptide inactivation in turbot, Scophthalmus maximus.

Tongue sole tissue factor pathway inhibitor 2 (TFPI-2) C-terminus derived peptide, TC38, has previously been shown to kill Vibrio vulnificus cells without lysing the cell membrane; thus, the remaining bacterial shell has potential application as an inactivated vaccine. Therefore, this study aimed to evaluate the immune response induced by the novel V. vulnificus vaccine. The protective potential of TC38-killed V. vulnificus cells (TKC) was examined in a turbot model. Fish were intramuscularly vaccinated with TKC or FKC (formalin-killed V. vulnificus cells) and challenged with a lethal-dose of V. vulnificus. The results showed that compared with FKC, TKC was effective in protecting fish against V. vulnificus infection, with relative percent of survival (RPS) rates of 53.29% and 63.64%, respectively. The immunological analysis revealed that compared with the FKC and control groups, the TKC group exhibited: 1) significantly higher respiratory burst ability and bactericidal activity of macrophages at 7 d post-vaccination; 2) increased alkaline phosphatase, acid phosphatase, lysozyme, and total superoxide dismutase levels post-vaccination; 3) higher serum agglutinating antibody titer with corresponding higher serum bactericidal ability, and a more potent serum agglutination effect, as well as an increased IgM expression level; 4) higher expression of immune relevant genes, which were involved in both innate and adaptive immunity. Taken together, this is the first study to develop a novel V. vulnificus inactivated vaccine based on AMP inactivation, and TKC is an effective vaccine against V. vulnificus infection for aquaculture.

The Evolution and Diversity of Interleukin-17 Highlight an Expansion in Marine Invertebrates and Its Conserved Role in Mucosal Immunity.

The interleukin-17 (IL-17) family consists of proinflammatory cytokines conserved during evolution. A comparative genomics approach was applied to examine IL-17 throughout evolution from poriferans to higher vertebrates. Cnidaria was highlighted as the most ancient diverged phylum, and several evolutionary patterns were revealed. Large expansions of the IL-17 repertoire were observed in marine molluscs and echinoderm species. We further studied this expansion in filter-fed Mytilus galloprovincialis, which is a bivalve with a highly effective innate immune system supported by a variable pangenome. We recovered 379 unique IL-17 sequences and 96 receptors from individual genomes that were classified into 23 and 6 isoforms after phylogenetic analyses. Mussel IL-17 isoforms were conserved among individuals and shared between closely related Mytilidae species. Certain isoforms were specifically implicated in the response to a waterborne infection with Vibrio splendidus in mussel gills. The involvement of IL-17 in mucosal immune responses could be conserved in higher vertebrates from these ancestral lineages.

Characterization and functional study of a chimera galectin from yellow drum Nibea albiflora.

Galectins are protein that participates in a variety of immune responses in the process of pathogenic infections. In the present study, a chimera galectin gene was screened from the transcriptome database of Nibea albiflora, which was named as YdGal-3. The results of qRT-PCR showed that the mRNA transcripts of YdGal-3 were ubiquitously distributed in all the detected tissues. After infection with Vibrio harveyi, the expression of YdGal-3 in liver, spleen, and head kidney increased significantly. Immunohistochemistry showed that YdGal-3 protein was widely expressed in the head kidney. The purified YdGal-3 protein by prokaryotic expression agglutinated red blood cells. Sugar inhibition assay showed that the agglutinating activity of YdGal-3 protein was inhibited by different sugars including lactose, D-galactose, and lipopolysaccharide. In addition, we mutated YdGal-3 His 294 into proline (P), alanine (A), glycine (G), and aspartic acid (D), it was further proved that the residue plays a key role in agglutination. YdGal-3 agglutinated some gram-negative bacteria including Pseudomonas plecoglossicida, Vibrio parahemolyticus, V. harveyi, and Aeromonas hydrophila, and exhibited antibacterial activity. These results suggested that YdGal-3 protein played an important role in the innate immunity of N. albiflora.

Hepatopancreas-Specific Lectin Participates in the Antibacterial Immune Response by Regulating the Expression of Antibacterial Proteins.

The hepatopancreas is an important digestive and immune organ in crustacean. There were low but stable numbers of microbes living in the hemolymph of crustacean, whereas the organs (including hepatopancreas) of crustacean were immersed in the hemolymph. It is very important to study the immune mechanism of the hepatopancreas against bacteria. In this study, a novel CTL (HepCL) with two CRDs, which was mainly expressed in the hepatopancreas, was identified in red swamp crayfish (Procambarus clarkii). HepCL binds to bacteria in vitro and could enhance bacterial clearance in vivo. Compared with the C-terminal CRD of HepCL (HepCL-C), the N-terminal CRD (HepCL-N) showed weaker bacterial binding ability in vitro and stronger bacterial clearance activity in vivo. The expression of some antimicrobial proteins, such as FLP, ALF1 and ALF5, was downregulated under knockdown of HepCL or blocked with Anti-HepCL after challenge with Vibrio in crayfish. These results demonstrated that HepCL might be involved in the antibacterial immune response by regulating the expression of antimicrobial proteins.

Identification and functional analysis of the perforin-1 like gene in disease resistance in half smooth tongue sole (Cynoglossus semilaevis).

The pore-forming protein perforin is one of the effectors of cell-mediated killing via the granule exocytosis pathway. In this study, a genome-wide association study was conducted in Vibrio harveyi disease-resistant and disease-susceptible families of half smooth tongue sole (Cynoglossus semilaevis) to determine the genes accounting for host resistance, and a perforin homologue was identified, designated perforin-1 like (CsPRF1l). The full-length cDNA of CsPRF1l is 1835 bp, and encodes 514 amino acids. The CsPRF1l gene consists of 10 exons and 9 introns, spanning approximately 7 kb. The amino acid sequence of CsPRF1l shows 60.35, 54.03, 41.92, and 34.17% identities to Morone saxatilis PRF1l, Oryzias melastigma PRF1l, Danio rerio PRF1.5 and Homo sapiens PRF, respectively. Sequence analysis revealed the presence of membrane attack complex/perforin (MACPF) and C2 domains in CsPRF1l. Quantitative real-time PCR showed that CsPRF1l presented a higher intestinal expression level in disease-resistant families than in susceptible families. Tissue expression pattern analysis showed that CsPRF1l is present in most of the tested tissues and highly expressed in the intestine, brain, stomach and gills. After challenge with V. harveyi, CsPRF1l mRNA was markedly upregulated in the liver, spleen, kidney, intestine, gills and skin. In addition, the recombinant CsPRF1l protein exhibited obvious antimicrobial activity against V. harveyi in vitro and in a zebrafish model. Collectively, these data indicate that CsPRF1l modulates host immune defense against V. harveyi invasion and provide clues about the efficacy of rCsPRF1l in fish that will give rise to useful therapeutic applications for V. harveyi infection in C. semilaevis.

Transcriptional Profiles of Genes Related to Stress and Immune Response in Rainbow Trout (Oncorhynchus mykiss) Symptomatically or Asymptomatically Infected With Vibrio anguillarum.

Rainbow trout (Oncorhynchus mykiss) is one of the most common aquaculture fish species worldwide. Vibriosis disease outbreaks cause significant setbacks to aquaculture. The stress and immune responses are bidirectionally modulated in response to the health challenges. Therefore, an investigation into the regulatory mechanisms of the stress and immune responses in trout is invaluable for identifying potential vibriosis treatments. We investigated the transcriptional profiles of genes associated with stress and trout immune functions after Vibrio anguillarum infection. We compared the control trout (CT, 0.9% saline injection), asymptomatic trout (AT, surviving trout with minor or no symptoms after bacteria injection), and symptomatic trout (ST, moribund trout with severe symptoms after bacteria injection). Our results showed activated immunomodulatory genes in the cytokine network and downregulated glucocorticoid and mineralocorticoid receptors in both AT and ST, indicating activation of the proinflammatory cytokine cascade as a common response in AT and ST. Moreover, the AT specifically activated the complement- and TNF-associated immune defenses in response to V. anguillarum infection. However, the complement and coagulation cascades, as well as steroid hormone homeostasis in ST, were disturbed by V. anguillarum. Our studies provide new insights toward understanding regulatory mechanisms in stress and immune functions in response to diseases.

Barbel steed (Hemibarbus labeo) NK-lysin protects against Aeromonas hydrophila infection via immunomodulatory activity.

NK-lysins (NKLs) are a family of multifunctional antimicrobial peptides that have activity against various microorganisms. However, the immunomodulatory activity of NKL in fish remains unclear. In this study, the cDNA sequence of barbel steed (Hemibarbus labeo) NKL gene was cloned. Barbel steed NKL amino acid sequence comprised a signal peptide and a mature peptide. The saposin B domain in the mature peptide has six conserved cysteines that form three disulfide bonds. Phylogenetic analysis showed that the barbel steed NKL was most closely related to that of the common carp (Cyprinus carpio) NKL. Differential expression analysis showed that the barbel steed NKL gene was expressed in all tested tissues, with the highest expression in the spleen. In response to Aeromonas hydrophila infection, NKL was significantly upregulated in the liver, spleen, head kidney, and gill. The barbel steed NKL showed strong antibacterial activity against Vibrio parahaemolyticus, V. alginolyticus, V. vulnificus, and Listeria monocytogenes. However, NKL had no antibacterial activity against the pathogenic bacteria A. hydrophila. Lactate dehydrogenase release assays showed that NKL damaged the V. parahaemolyticus cell membrane. NKL significantly increased barbel steed survival rate after A. hydrophila infection and upregulated IL-1ß and TNF-α expression in the spleen and head kidney. NKL induced monocyte/macrophage chemotaxis and enhanced the respiratory burst and proinflammatory cytokine expression. Our study shows that fish NKL exhibits immunomodulatory effects and protects the host from pathogenic infections independent of direct bacterial clearance.

Feeding olive flounder (Paralichthys olivaceus) with Lactococcus lactis BFE920 expressing the fusion antigen of Vibrio OmpK and FlaB provides protection against multiple Vibrio pathogens: A universal vaccine effect.

Vibriosis, an illness caused by the Vibrio bacteria species, results in significant economic loss in olive flounder farms. Here we present a novel anti-Vibrio feed vaccine protecting multiple strains of Vibrio pathogens, a universal vaccine effect. The vaccine was generated by engineering Lactococcus lactis BFE920 to express the fusion antigens of Vibrio outer membrane protein K (OmpK) and flagellin B subunit (FlaB). These antigen genes are highly conserved among Vibrio species. Olive flounder (7.1 ± 0.8 g and 140 ± 10 g) were fed the vaccine adsorbed to a regular feed (1 × 107 CFU/g) for one week with a 1-week interval, repeating three times (a triple boost). The vaccinated fish increased the significant levels of antigen-specific antibodies, T cell numbers (CD4-1, CD4-2, and CD8α), cytokine production (T-bet and IFN-γ), and innate immune responses (TLR5M, IL-1ß, and IL-12p40). Also, the survival rates of adult and juvenile fish fed the vaccine were significantly elevated when challenged with V. anguillarum, V. alginolyticus, and V. harveyi. In addition, weight gain rate and feed conversion ratio were improved in vaccinated fish. The feed vaccine protected multiple Vibrio pathogens, a universal vaccine effect, by activating innate and adaptive immune responses. This oral vaccine may be developed as an anti-Vibrio vaccine to protect against a broad spectrum of Vibrio pathogens.

A haemocyte-expressed Methyltransf_FA domain containing protein (MFCP) exhibiting microbe binding activity in oyster Crassostrea gigas.

The Methyltransf_FA domain is well-known as a key protein domain of enzyme synthesizing juvenile hormone, and Methyltransf_FA domain containing proteins (MFCPs) are widely existed in vertebrates and invertebrates. In the present study, a CgMFCP with a single Methyltransf_FA domain was screened from oyster Crassostrea gigas, and its open reading frame of CgMFCP was of 1128 bp, encoding a polypeptide of 376 amino acids with a signal peptide, a Methyltransf_FA domain and a transmembrane region. CgMFCP was clustered with FAMeTs from insecta and crustacea of arthropod. The mRNA transcripts of CgMFCP were detected in different tissues, with the extremely high expression level in haemocytes, which was 131.36-fold (p < 0.05) of that in gills. The expression level of CgMFCP protein was verified to be highly expressed in haemocytes. The expression level of CgMFCP mRNA in primarily cultured haemocytes significantly up-regulated at 3 h, 24 h and 48 h post LPS stimulation, which was 3.25-fold (p < 0.01), 2.04-fold (p < 0.05) and 3.59-fold (p < 0.01) compared to that in blank group. After the oysters were stimulated with Vibrio splendidus in vivo, the expression level of CgMFCP mRNA in haemocytes was also significantly up-regulated at 3 h, 12 h, and 24 h, which was 4.22-fold (p < 0.05), 4.39-fold (p < 0.05) and 6.35-fold (p < 0.01) of that in control group, respectively. By flow cytometry analysis, anti-rCgMFCP can label 95% of oyster haemocytes. And by fluorescence microscope analysis, CgMFCP was mainly distributed in cytomembrane of haemocytes. The recombinant CgMFCP (rCgMFCP) exhibited higher affinity towards MAN and LPS in a dose-dependent manner, while relatively lower affinity to PGN and poly (I:C). rCgMFCP also displayed binding activities towards Gram-negative bacteria (Vibrio anguillarum and V. splendidus), Gram-positive bacteria (Staphylococcu aureu) and fungi (Pichia pastoris). These results collectively indicated that CgMFCP specifically expressed in haemocytes and functioned as a pattern recognition receptor by binding to various microbes in oyster C. gigas, which provided insight into the function of Methyltransf_FA domain containing proteins.

Upregulation of polymeric immunoglobulin receptor expression in flounder (Paralichthys olivaceus) gill cells by cytokine tumor necrosis factor-α via activating PI3K and NF-κB signaling pathways.

The polymeric immunoglobulin receptor (pIgR) transports secretory immunoglobulins across mucosal epithelial cells into external secretions, playing critical roles in mucosal surface defenses, but the regulation mechanism of pIgR expression is not clarified in teleost fish. In this study, the dynamic changes of flounder (Paralichthys olivaceus) pIgR (fpIgR) and pro-inflammatory cytokine tumor necrosis factor-α (TNF-α) mRNA expression in mucosal tissues were first analyzed post inactivated Vibrio anguillarum immunization, and increased production of TNF-α was found to correlate with increased expression of fpIgR. To determine that cytokine TNF-α influenced fpIgR expression, following confirming that natural fpIgR expressed on flounder gill (FG) cells, FG cells were incubated with various concentrations of recombinant TNF-α for different time, the results showed that the expressions of fpIgR were significantly upregulated at gene and protein levels in a dose-dependent and time-dependent manner, and similar change trend was observed for free secretory component (SC) secreted by fpIgR into the culture supernatant. After FG cells were treated with TNF-α, specific phosphoinositide 3-kinase (PI3K) inhibitor wortmannin, nuclear factor kappa-B (NF-κB) inhibitor Bay11-7082, and the mixtures of TNF-α and wortmannin / Bay11-7082 respectively, the fpIgR protein and mRNA levels, together with SC secretion, obviously decreased in wortmannin- and Bay11-7082-treated cells compared with the untreated control, and cotreatment with wortmannin / Bay11-7082 plus TNF-α resulted in lower expression compared with that upon treatment with TNF-α alone, indicating that the inhibition of PI3K and NF-κB both blocked the ability of TNF-α to increase cellular fpIgR and SC levels. Furthermore, the gene expressions of PI3K and NF-κB were upregulated and present a tendency to increase first and then decrease after TNF-α treatment of FG cells; However, the expression of PI3K mRNA was inhibited significantly by wortmannin but not by Bay11-7082, and the expression of NF-κB mRNA was suppressed obviously by Bay11-7082 but not by wortmannin, suggesting that inhibition of PI3K or NF-κB had no influence on each other. All these results collectively revealed that TNF-α could transcriptionally upregulate fpIgR expression and SC production, and this TNF-α-induced pIgR expression was regulated by complex mechanisms that involved PI3K and NF-κB signaling pathways, which provided evidences for pro-inflammatory cytokine TNF-α acting as a regulator in pIgR expression and better understanding of regulation mechanism of pIgR expression in teleost fish.

A hexokinase from the oyster Crassostrea gigas is involved in immune recognition as a pattern recognition receptor.

Hexokinase (HK) is generally recognized as a crucial enzyme participating in glycolysis. In the present study, a HK (named CgHK) was identified as a potential pattern recognition receptor (PRR) from the Pacific oyster Crassostrea gigas. The open reading frame (ORF) of CgHK was of 1395 bp, encoding a peptide of 464 amino acids with one Hexokinase_1 domain and one Hexokinase_2 domain. The predicted amino acid sequence of CgHK shared 17%-29% similarities with that of other known HKs. The mRNA transcripts of CgHK were constitutively detected in all the examined tissues, with relative high expression level in labial palp and haemocytes. CgHK protein was mainly observed in the cytoplasm of oyster haemocytes. The mRNA expression level of CgHK in haemocytes was significantly up-regulated and peaked at 3 h after Vibrio splendidus (7.64-fold, p < 0.001) and lipopolysaccharide (LPS) (11.86-fold, p < 0.001) stimulations. The recombinant CgHK protein (rCgHK) exhibited Mg2+-dependent adenosine triphosphate (ATP) binding activity in vitro and activity to bind D-(+)-glucose (GLU) and various pathogen-associated molecular pattern (PAMPs) such as LPS and peptidoglycan (PGN) in the absence of Mg2+. It also displayed higher binding activity towards V. splendidus and relatively lower binding activity towards Staphylococcus aureus, Escherichia coli, and Micrococcus luteus. After the mRNA expression of CgHK in haemocytes was knocked down by dsRNA interference, the expression of CgIL17-5 mRNA in haemocytes was considerably down-regulated at 3 h after the stimulation with V. splendidus (0.33-fold, p < 0.001). These results collectively indicated that CgHK was able to recognize various PAMPs and pathogenic bacteria as a PRR apart from being the enzyme to exert ATP binding activity in glycolysis, and activate the anti-bacterial immune response by promoting the expression of pro-inflammatory cytokines CgIL17-5 in oyster haemocytes.