Jun-ichi Hikima

The cytosolic sensor, DDX41, activates antiviral and inflammatory immunity in response to stimulation with double-stranded DNA adherent cells of the olive flounder, Paralichthys olivaceus.

DDX41, a receptor belonging to the DExD family, functions as a DNA sensor in the mammalian cytoplasm and mediates the antiviral response in host cells. Here, the olive flounder DDX41 was found to have 2267-bp long and encodes a putative protein of 614 amino acid residues. The olive flounder DDX41 mRNA was presented in all tested tissues, and was distinctly expressed in fish naturally infected with LCDV. High expression levels were observed in the heart, liver, kidney and stomach. Furthermore, the olive flounder DDX41 mRNA expression increased significantly in adherent (monocyte-like) cells following stimulation with a DNA virus. Reporter assays showed that the transcriptional activity of the IFN-I promoter was enhanced in DDX41-overexpressing HINAE cells treated with C-di-GMP (dinucleotides). Overexpression of DDX41 also induced the antiviral and inflammatory cytokine gene expression through cytoplasmic C-di-GMP treatment. These results suggest that DDX41 functions as a cytosolic DNA sensor that is capable of inducing antiviral activity and inflammatory responses in the olive flounder.

Presence of two tumor necrosis factor (tnf)-α homologs on different chromosomes of zebrafish (Danio rerio) and medaka (Oryzias latipes).

Two or more isoforms of several cytokines including tumor necrosis factors (tnfs) have been reported from teleost fish. Although zebrafish (Danio rerio) and medaka (Oryzias latipes) possess two tnf-α genes, their genomic location and existence are yet to be described and confirmed. Therefore, we conducted in silico identification, synteny analysis of tnf-α and tnf-n from both the fish with that of human TNF/lymphotoxin loci and their expression analysis in zebrafish. We identified two homologs of tnf-α (named as tnf-α1 and tnf-α2) and a tnf-n gene from zebrafish and medaka. Genomic location of these genes was found to be as: tnf-α1, and tnf-n and tnf-α2 genes on zebrafish chromosome 19 and 15 and medaka chromosome 11 and 16, respectively. Several features such as existence of TNF family signature, conservation of genes in TNF loci with human chromosome, phylogenetic clustering and amino acid similarity with other teleost TNFs confirmed their identity as tnf-α and tnf-n. There were a constitutive expression of all three genes in different tissues, and an increased expression of tnf-α1 and -α2 and a varied expression of tnf-n ligand in zebrafish head kidney cells induced with 20 μg mL(-1) LPS in vitro. Our results suggest the presence of two tnf-α homologs on different chromosomes of zebrafish and medaka and correlate this incidence arising from the fish whole genome duplication event.

Inflammatory responses in the Japanese pufferfish (Takifugu rubripes) head kidney cells stimulated with an inflammasome-inducing agent, nigericin

A cytosolic receptor complex called inflammasome is responsible for mounting inflammatory response by releasing pro-inflammatory cytokines, interleukin (IL)-1β and IL-18. However, inflammatory cascades mediated by the inflammasome are unknown in a lower vertebrate like fish. Therefore, in an in vitro experiment, in order to obtain a preliminary information, we conducted transcriptomic analysis of 18 cytokines including pro-inflammatory cytokines in the Japanese pufferfish (Takifugu rubripes) head kidney (HK) cells stimulated with an inflammasome-inducing agent, nigericin, and a combination of nigericin and LPS by a multiplex RT-PCR assay (GenomeLab Genetic Analysis System, GeXPS; Beckman Coulter Inc.). Furthermore, expression of IL-1β, IL-6, IL-18, nuclear factor (NF)-κB, nucleotide-binding oligomerization domain 2 (NOD2) and NOD-like receptor X1 (NLRX1) genes was examined in HK cells by a quantitative real-time PCR. Additionally, to confirm functionality of activated inflammatory immunity, we also assessed phagocytic activity, superoxide anion production (NBT assay) and lysozyme activity in the nigericin-stimulated HK cells. An increased gene expression of pro-inflammatory cytokines (IL-1β and IL-18), NF-κB and NOD2 was recorded in nigericin and combined nigericin+LPS- stimulated HK cells. Enhanced cellular (phagocytic activity and NBT assay) and humoral (lysozyme activity) immune parameters in the stimulated cells confirmed induction of inflammatory response. Results suggested probable activation of inflammasome components for processing of the inflammatory cytokines in the Japanese pufferfish.

Inflammatory immune response by lipopolysaccharide-responsive nucleotide binding oligomerization domain (NOD)-like receptors in the Japanese pufferfish (Takifugu rubripes).

Some of NOD-like receptors (NLRs), the cytosolic pattern recognition receptors form a multi-protein complex, inflammasome consisting of one or more NLRs, the adaptor protein ASC and inflammatory caspase to generate mature inflammatory cytokines, interleukin (IL)-1β and IL-18. However, inflammasome-mediated inflammatory cascade involving any NLR member is unknown in a lower vertebrate like fish. Also, inflammatory cytokine induction pathway in response to a specific ligand, namely bacterial lipopolysaccharide (LPS) has not yet been clarified. Therefore, 13 predicted NLR sequences of the Japanese pufferfish, Fugu (Takifugu rubripes) were retrieved in silico and categorized as NLR-C1∼13. Expression analysis of these genes in Fugu head kidney (HK) cells stimulated with a heat-killed Lactobacillus paracasei spp. paracasei (Lpp), LPS, nigericin and a combination of nigericin + LPS showed consistent up-regulations of NLR-C1, 5, 7, 10 and 12 genes in both Lpp and LPS stimulations and NLR-C9 gene in LPS stimulation only. However, nigericin and nigericin + LPS caused an increased expression of NLR-C10 and 12 in HK cells and leukocytes. Fugu treated with Lpp and LPS (in vivo), and infected with Vibrio harveyi had an elevated expression of NLR-C10 and 12. Increased transcription of caspase-1, ASC, IL-1β and IL-18 was recorded in nigericin-stimulated HK cells and leukocytes. Results suggested activation of probable inflammasome-mediated inflammatory cytokine response in Fugu. Moreover, LPS may be a key ligand that induces some of the Fugu NLR-Cs (NLR-C9, 10 and 12). Further characterization and functional analysis of Fugu NLR-C10 and 12 for ligand sensing, and processing of pro-inflammatory cytokine, IL-1β would elucidate the inflammasome evolution in fish.

Inductive immune responses in the Japanese pufferfish (Takifugu rubripes) treated with recombinant IFN-γ, IFN-γrel, IL-4/13A and IL-4/13B

Immune regulation in response to recombinant cytokines (Th1- and Th2-types) is poorly understood in fish. Therefore, we synthesized and purified the Japanese pufferfish, (Takifugu rubripes) recombinant cytokines, namely interferon-gamma (IFN-γ), IFN-γrel, interleukin (IL)-4/13A and IL-4/13B, and then administered by injection to examine the immune regulatory effects on phagocytic activity, superoxide anion production and lysozyme activity, and cytokine gene expressions in treated fish head kidney (HK) at 1, 3 and 5 days post injection (dpi). Pufferfish that received rIFN-γ injection had an elevated phagocytic activity at 1 and 3 dpi, whereas rIFN-γrel and rIL-4/13A treated fish showed an increased phagocytic activity only at 1 and 3 dpi, respectively. Superoxide anion production increased only at 1 dpi in rIFN-γ, rIFN-γrel and rIL-4/13A injected pufferfish. Lysozyme showed a high activity at 1 dpi in rIFN-γ injected fish, at 5 dpi in rIL-4/13A injected fish and all through the time course in rIL-4/13B injected fish. rIFN-γ stimulation caused an elevated IL-1β and tumor necrosis factor-alpha (TNF-α) transcriptions, whereas stimulation with rIFN-γrel resulted in IFN-γ gene induction and a long term increase in IL-6 and IL-12p40 gene expressions. Injection of rIL-4/13A induced transcription of IL-6, IL-12p40 and TNF-α, while rIL-4/13B treatment enhanced IL-6, IL-12p35 and IL-12p40 gene expressions. Our results suggest that each recombinant cytokine has a role in activation of immune cells. More precisely, rIFN-γ may be potentially the most effective immune inducer among the tested cytokines. Therefore, use of recombinant Th1 and Th2 cytokines as immune enhancers could be an efficient technique for disease prevention in fish.

DNA vaccine-mediated innate immune response triggered by PRRs in teleosts

Aquacultured fish are threatened by many pathogens, often with serious consequences. Vaccination is one of the most effective tools for enhancing host immunity and protecting fish from infections. Vaccination with DNA vaccine is based on the administration of the gene encoding a vaccine antigen. Several effective DNA vaccines that encode viral or bacterial antigenic proteins have already been shown to be effective for cultured fish. This review summarizes current knowledge on fish DNA vaccines, and the mechanism of interaction between the DNA vaccines and host immunity, especially focusing on the enhancement of innate immunity mediated through direct-recognition of DNA vaccine by pattern recognition receptors (PRRs). To date, numerous fish PRR genes have been identified, and the primordial functions of PRRs involved in the innate immune response to viral and bacterial nucleic acids have been increasingly clarified. The evolutionary conservations and divergences in the PRR mechanisms of teleosts and mammals are focused on their molecular features and the recognition of DNA vaccine mediated by TANK binding kinase 1. In addition, the mechanism of type I interferon production in teleosts, which is enhanced after the recognition of cytosolic nucleic acids and current topics on DNA sensing by PRRs are also introduced.

Comparative Genomic Characterization of Three Streptococcus parauberis Strains in Fish Pathogen, as Assessed by Wide-Genome Analyses

Streptococcus parauberis, which is the main causative agent of streptococcosis among olive flounder (Paralichthys olivaceus) in northeast Asia, can be distinctly divided into two groups (type I and type II) by an agglutination test. Here, the whole genome sequences of two Japanese strains (KRS-02083 and KRS-02109) were determined and compared with the previously determined genome of a Korean strain (KCTC 11537). The genomes of S. parauberis are intermediate in size and have lower GC contents than those of other streptococci. We annotated 2,236 and 2,048 genes in KRS-02083 and KRS-02109, respectively. Our results revealed that the three S. parauberis strains contain different genomic insertions and deletions. In particular, the genomes of Korean and Japanese strains encode different factors for sugar utilization; the former encodes the phosphotransferase system (PTS) for sorbose, whereas the latter encodes proteins for lactose hydrolysis, respectively. And the KRS-02109 strain, specifically, was the type II strain found to be able to resist phage infection through the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas system and which might contribute valuably to serologically distribution. Thus, our genome-wide association study shows that polymorphisms can affect pathogen responses, providing insight into biological/biochemical pathways and phylogenetic diversity.

Evolutionary evidence of tumor necrosis factor super family members in the Japanese pufferfish (Takifugu rubripes): Comprehensive genomic identification and expression analysis

Tumor necrosis factor (TNF) and its superfamily (TNFSF) members are important inflammatory cytokines. Although fish have fourteen TNFSF genes, their genomic location and existence are yet to be described and confirmed in the Japanese pufferfish (Fugu) (Takifugu rubripes). Therefore, we conducted in silico identification, synteny analysis of TNFSF genes from Fugu with that of zebrafish and human TNFSF loci and their expression analysis in various tissues. We identified ten novel TNFSF genes, viz. TNFSF5 (CD40L), TNFSF6 (FasL), three TNFSF10 (TRAIL) (-1, 2 and 3), TNFSF11 (RANKlg), TNFSF12 (TWEAK), two TNFSF13B (BAFF) (1 and 2) and TNFSF14 (LIGHT) belonging to seven TNFSFs in Fugu. Several features such as existence of TNF family signature, conservation of genes in TNF loci with human and zebrafish chromosomes and phylogenetic clustering with other teleost TNFSF orthologs confirmed their identity. Fugu TNFSF genes were constitutively expressed in all eight different tissues with most of them expressed highly in liver. Fugu TNFSF10 gene has three homologs present on chromosomes 10 (TNFSF10-1), 8 (TNFSF10-2) and 2 (TNFSF10-3). Moreover, a phylogenetic analysis containing all available vertebrate (mammals, birds, reptiles, amphibians and fish) TNFSF10 orthologs showed that Fugu TNFSF10-1 and 10-3 are present in all vertebrates, whereas TNFSF10-2 was not related to any mammalian and avian orthologs. Viral double-stranded RNA mimic poly (I:C) caused an elevated expression of three Fugu TNFSF10 genes in head kidney cells at 4h indicating probable role of these genes to induce apoptosis in virus-infected cells. In conclusion, Fugu possesses genes belonging to nine TNFSFs including the newly identified seven and previously reported two, TNFSF New (TNF-N) and TNFSF2 (TNF-α). Our findings would add up information to TNFSF evolution among vertebrates.

Complete genome sequence of Photobacterium damselae subsp. piscicida strain OT-51443 isolated from yellowtail (Seriola quinqueradiata) in Japan

ABSTRACT Pseudotuberculosis caused by infection of Photobacterium damselae subsp. piscicida has caused serious economic damages to aquaculture farms worldwide. Here, the whole-genome sequence of P. damselae subsp. piscicida strain OT-51443, isolated in Japan, was determined and suggests that this genome consists of two chromosomes and five plasmids.

Transcriptome analysis of immune response against Vibrio harveyi infection in orange-spotted grouper (Epinephelus coioides)

ABSTRACT Vibrio harveyi is a gram‐negative bacterium reported as found in many aquaculture species. To increase knowledge of the immune response against V. harveyi, in this study we performed transcriptome analysis of head kidney and spleen in orange‐spotted grouper (Epinephelus coioides) at 1 and 2 days post‐infection (dpi), using the Illumina sequencing platform. After de novo assembly, a total of 79,128 unigenes was detected with an N50 of 2511 bp. After alignments with sequences recorded in the major databases (NT, NR, Swiss‐Prot COG, KEGG, Interpro and GO), based on sequence similarity, 61,208 (77.4%) of the unigene total could be annotated using at least one database. Comparison of gene expression levels between V. harveyi and a control group at each time point revealed differentially expressed genes (DEGs) (P < 0.05): a total of 7918 (5536 upregulated and 2282 downregulated genes) from head kidney at 1 day post infection (dpi), 4260 (1444 upregulated and 2816 downregulated genes) from head kidney at 2 dpi, 7887 (4892 upregulated and 2995 downregulated genes) from spleen at 1 dpi, and 8952 (7388 upregulated and 1564 downregulated genes) from spleen at 2 dpi. The DEGs were mainly annotated into signal transduction and immune system categories, based on the KEGG database. The DEGs were enriched in immune‐related pathway functions, NOD‐like receptor signaling pathways, Toll‐like receptor signaling pathways, NF‐&kgr;B signaling pathways, and Jak‐STAT signaling pathways. Additionally, we selected several DEGs and validated their expression level by RT‐qPCR. The data generated in this study may provide a valuable resource for further immune response research and offer improved strategies against V. harveyi infection in teleost fishes. HighlightsTranscriptome analysis of head kidney and spleen at 1 and 2 days post‐infection of Vibrio harveyi was performed in orange‐spotted grouper (Epinephelus coioides).A total of 79,128 unigenes were obtained after de novo assembly.A number of differentially expressed genes were annotated in immune system.The increase of RANKL, IL‐l&bgr;, INF‐&ggr; genes and these target genes expression indicated the activation of immune response against Vibrio harveyi infection.