Molecular characterization, cytoprotective, DNA protective, and immunological assessment of peroxiredoxin-1 (Prdx1) from yellowtail clownfish (Amphiprion clarkii).

Peroxiredoxin-1 (Prdx1) is a thiol-specific antioxidant enzyme that detoxifies reactive oxygen species (ROS) and regulates the redox status of cells. In this study, the Prdx1 cDNA sequence was isolated from the pre-established Amphiprion clarkii (A. clarkii) (AcPrdx1) transcriptome database and characterized structurally and functionally. The AcPrdx1 coding sequence comprises 597 bp and encodes 198 amino acids with a molecular weight of 22.1 kDa and a predicted theoretical isoelectric point of 6.3. AcPrdx1 is localized and functionally available in the cytoplasm and nucleus of cells. The TXN domain of AcPrdx1 comprises two peroxiredoxin signature VCP motifs, which contain catalytic peroxidatic (Cp-C52) and resolving cysteine (CR-C173) residues. The constructed phylogenetic tree and sequence alignment revealed that AcPrdx1 is evolutionarily conserved, and its most closely related counterpart is Amphiprion ocellaris. Under normal physiological conditions, AcPrdx1 was ubiquitously detected in all tissues examined, with the most robust expression in the spleen. Furthermore, AcPrdx1 transcripts were significantly upregulated in the spleen, head kidney, and blood after immune stimulation by polyinosinic:polycytidylic acid (poly (I:C)), lipopolysaccharide (LPS), and Vibrio harveyi injection. Recombinant AcPrdx1 (rAcPrdx1) demonstrated antioxidant and DNA protective properties in a concentration-dependent manner, as evidenced by insulin disulfide reduction, peroxidase activity, and metal-catalyzed oxidation (MCO) assays, whereas cells transfected with pcDNA3.1(+)/AcPrdx1 showed significant cytoprotective function under oxidative and nitrosative stress. Overexpression of AcPrdx1 in fathead minnow (FHM) cells led to a lower viral copy number following viral hemorrhagic septicemia virus (VHSV) infection, along with upregulation of several antiviral genes. Collectively, this study provides insights into the function of AcPrdx1 in defense against oxidative stressors and its role in the immune response against pathogenic infections in A. clarkii.

Involvement of tumor necrosis factor receptor-associated factor 6 (TRAF6) in NF-κB activation and antiviral immunity: Molecular and functional characterization of TRAF6 in red-spotted grouper (Epinephelus akaara).

Tumor necrosis factor receptor-associated factor 6 (TRAF6) is a member of the TRAF family of adaptor proteins involved in the signal transduction pathways of both TNF receptor and interleukin-1 receptor/Toll-like receptor superfamilies. In this study, red-spotted grouper (Epinephelus akaara) TRAF6 (EaTraf6) was identified and characterized. The open reading frame of EaTraf6, 1713 bp in length, encodes a putative protein of 570 amino acids and has a predicted molecular weight and theoretical isoelectric point of 64.11 kDa and 6.07, respectively. EaTraf6 protein contains an N-terminal RING-type zinc finger domain, two TRAF-type zinc finger domains, a coiled-coil region (zf-TRAF), and a conserved C-terminal meprin and TRAF homology (MATH) domain. EaTraf6 shared the highest amino acid sequence identity with its ortholog from Epinephelus coioides, and phylogenetic analysis showed all fish TRAF6s clustered together and apart from other species. qRT-PCR results revealed that EaTraf6 was ubiquitously expressed in all examined tissues, with the highest level detected in the blood. In the immune challenge, EaTraf6 exhibited modulated mRNA expression levels in the blood and spleen. The subcellular localization analysis revealed that the EaTraf6 protein was predominantly present in the cytoplasm; however, it could translocate into the nucleus following poly (I:C) stimulation. The antiviral function of EaTraf6 was confirmed by analyzing the expression of host antiviral genes and viral genomic RNA during viral hemorrhagic septicemia virus infection. Additionally, luciferase reporter assay results indicated that EaTraf6 is involved in the activation of the NF-κB signaling pathway upon poly (I:C) stimulation. Finally, the effect of EaTraf6 on cytokine gene expression and its role in regulating macrophage M1 polarization were demonstrated. Collectively, these findings suggest that EaTraf6 is a crucial immune-related gene that significantly contributes to antiviral functions and regulation of NF-κB activity in the red-spotted grouper.

Amphiprion clarkii DDX41 modulates fish immune responses: Characterization by expression profiling, antiviral assay, and macrophage polarization analysis.

DDX41, a member of the DEAD-box helicase family, serves as a vital cytosolic DNA sensor and plays a pivotal role in controlling the activation of type I interferon responses in mammals. However, the functional aspects of fish DDX41 remain relatively unexplored. In this study, we identified and characterized the DDX41 gene in Amphiprion clarkii transcriptomes and designated the gene as AcDDX41. The complete open reading frame of AcDDX41 encoded a putative protein comprising 617 amino acids. Notably, the predicted AcDDX41 protein shared several structural features that are conserved in DDX41, including DEXDc, HELICc, and zinc finger domains, as well as conserved sequence "Asp-Glu-Ala-Asp (D-E-A-D)." AcDDX41 exhibited the highest sequence homology (99.68 % similarity) with DDX41 from Acanthochromis polyacanthus. Phylogenetic analysis revealed that DDX41s from fish formed a branch distinct from that in other animals. All investigated tissues were shown to express AcDDX41 constitutively, with blood showing the highest expression levels, followed by the brain. Furthermore, AcDDX41 expression was significantly induced upon stimulation with poly I:C, lipopolysaccharide, and Vibrio harveyi, indicating its responsiveness to immune stimuli. We confirmed the antiviral function of AcDDX41 by analyzing gene expression and viral replication during viral hemorrhagic septicemia virus infection. Additionally, using a luciferase reporter assay, we validated the ability of AcDDX41 to activate the NF-κB signaling pathway upon stimulation with poly I:C. Finally, AcDDX41 influenced cytokine gene expression and played a regulatory role in macrophage M1 polarization in RAW 264.7 cells. Collectively, these results highlight the significance of AcDDX41 as an immune-related gene that contributes substantially to antiviral defense and regulation of NF-κB activity.

Molecular characterization, immune expression, and functional delineation of peroxiredoxin 1 in Epinephelus akaara.

Peroxiredoxin 1 is a member of the typical 2-Cys peroxiredoxin family, which serves diverse functions in gene expression, immune and inflammatory responses, and tumor progression. In this study, we aimed to analyze the structural, functional, and immunomodulatory properties of peroxiredoxin 1 from Epinephelus akaara (EaPrx1). The open reading frame of EaPrx1 is 597 base pairs in length, encoding 198 amino acids, with a molecular weight of approximately 22 kDa. The in silico analysis revealed that EaPrx1 shares a conserved thioredoxin fold and signature motifs that are critical for its catalytic activity and oligomerization. Further, EaPrx1 is closely related to Epinephelus lanceolatus Prx1 and clustered in the Fishes group of the vertebrate clade, revealing that EaPrx1 was conserved throughout evolution. In terms of tissue distribution, a high level of EaPrx1 expression was observed in the spleen, brain, and blood tissues. Likewise, in immune challenge experiments, significant transcriptional modulations of EaPrx1 upon lipopolysaccharide, polyinosinic:polycytidylic acid, and nervous necrosis virus injections were noted at different time points, indicating the immunological role of EaPrx1 against pathogenic infections. In the functional analysis, rEaPrx1 exhibited substantial DNA protection, insulin disulfide reduction, and tissue repair activities, which were concentration-dependent. EaPrx1/pcDNA™ 3.1 (+)-transfected fathead minnow cells revealed high cell viability upon arsenic toxicity, indicating the heavy metal detoxification activity of EaPrx1. Taken together, the transcriptional and functional studies imply critical roles of EaPrx1 in innate immunity, redox regulation, apoptosis, and tissue-repair processes in E. akaara.

Molecular characterization and expression analysis of B-cell lymphoma-2 protein in Amphiprion clarkii and its role in virus infections.

Amphiprion clarkii is increasingly being used as a captive-bred ornamental fish in South Korea. However, its breeding has recently been greatly hindered by destructive diseases due to pathogens. B-cell lymphoma-2 (Bcl2), a mitochondrial apoptosis regulatory gene involved in immune responses, has not been investigated in anemonefish, including A. clarkii. Herein, we aimed to annotate Bcl2 in the A. clarkii transcriptome and examined its role against virus infections. Sequence analysis indicated that Bcl2 in A. clarkii (AcBcl2) contained all four Bcl-2 homology domains. The structure of AcBcl2 closely resembled those of previously analyzed anti-apoptotic Bcl2 proteins in mammals. Expression analysis showed that the highest level of AcBcl2 was expressed in blood. AcBcl2 expression in the blood was downregulated within 24 hpi when challenged with immune stimulants poly I:C and lipopolysaccharides. AcBcl2 reduced poly I:C-induced cell death. The propagation of viral hemorrhagic septicemia virus (VHSV) was higher in the presence of AcBcl2. Cell mortality was higher in AcBcl2 when transfected cells were infected with VHSV, and a higher viral transcript was observed compared to their respective controls. In conclusion, AcBcl2 is an anti-apoptotic protein, and its activity may facilitate the propagation of VHSV.

Molecular cloning, expression analysis of interleukin 17D (cysteine knot cytokine) from Amphiprion clarkii and their functional characterization and NFκB pathway activation using FHM cells.

Interleukin 17D (IL-17D), a pro-inflammatory cytokine, is a signature cytokine of T helper 17 (Th17) cells. However, studies characterizing the functions of IL-17D in teleost are scarce. Therefore, we aimed to characterize the properties of IL-17D in Amphiprion clarkii. We performed spatial and temporal expression, AcIL-17D-mediated antibacterial and inflammatory gene expression, NFκB pathway-related gene expression analyses, and bacterial colony counting and cell protection assays. We found that AcIL-17D contains a 630 bp coding sequence and encodes 210 amino acids. The spatial expression analysis of AcIL-17D in 12 tissues showed ubiquitous expression, with the highest expression in the brain, followed by blood and skin. Temporal expression analysis of AcIL-17D in blood showed upregulated expression at 6 and 24 h (polyinosinic: polycytidylic acid and lipopolysaccharide), 12 h (all stimulants), and 48 h (polyinosinic: polycytidylic acid and Vibrio harveyi). AcIL-17D expression in the blood gradually decreased at later hours in response to all the stimulants. After treatment of fathead minnow (FHM) cells with different recombinant AcIL-17D concentrations, the downstream gene expression analysis showed increased expression of antimicrobial genes in the FHM cells, namely [NK-Lysin (NKL), Hepcidin antimicrobial peptide-1 (HAMP-1), Defensin-ß (DEFB1)] and some inflammatory genes such as IL-1ß, TNF-α, IL-11, and STAT3. Further nuclear factor κB (NFκB) subunits (NFκB1, NFκB2, RelA, and Rel-B) showed upregulated gene expression at 12 and 24 h. The bacterial colony counting assay using FHM cells showed lower bacterial colony counts in rAcIL-17D-treated cells than in control. Furthermore, the Water-Soluble Tetrazolium Salt (WST -1) assay confirmed the ability of rAcIL-17D in the protection of FHM cells from bacterial infection and conducted the Hoechst 33342 staining upon treatment with rAcIL-17D and rMBP. Therefore, our findings provide important insights into the activation of IL-17D pathway genes in FHM cells, the protective role of AcIL-17D against bacterial infection, and host defense mechanisms in teleost.

PDI family thioredoxin from disk abalone (Haliotis discus discus): Responses to stimulants (PAMPs, bacteria, and viral) and functional characterization.

Thioredoxin, a highly conserved class of proteins involved in redox signaling, is found in a range of organisms from bacteria to higher-level eukaryotes. Thioredoxin acts as an active regulatory enzyme to eliminate excessive reactive oxygen species, thereby preventing cellular damage. In this study, the cDNA sequence of thioredoxin domain-containing 5 (AbTXNDC5) from the disk abalone transcriptomic database was characterized. An in silico analysis of AbTXNDC5 was performed, and its spatial and temporal expression patterns in hemocytes and gills in response to bacteria (Vibrio parahaemolyticus, Listeria monocytogenes), viral hemorrhagic septicemia virus, and pathogen-associated molecular pattern molecules were observed. Furthermore, AbTXNDC5 expression was examined in different developmental stages. Functional assays to explore insulin disulfide reduction, anti-apoptotic activity, and protection against hypoxic cell death of AbTXNDC5 were conducted through recombinant proteins or overexpression in cells. AbTXNDC5 contains a 1179-bp open reading frame coding for 392 amino acids. Conserved thiol-disulfide cysteine residues within two Cys-X-X-Cys motifs were found in AbTXNDC5. Quantitative real-time polymerase chain reaction indicated that healthy digestive tract and hemocyte tissues expressed high levels of AbTXNDC5 mRNA, which may protect the host from invading pathogens. Immune-challenged abalone hemocytes and gills exhibited upregulated expression of AbTXNDC5 at different time points. rAbTXNDC5 also exhibited a functional insulin disulfide reductase activity. AbTXNDC5 conferred protection to cultured cells from apoptosis and hypoxia-induced stress, compared to the pcDNA3.1(+) transfected control cells. Therefore, AbTXNDC5 can be considered an important gene in abalones in relation to the primary immune system and regulation of redox homeostasis and confers protection from stress.

Molecular characterization, expression profile, and antiviral activity of redlip mullet (Liza haematocheila) viperin.

Viperin is known to exhibit activity against RNA viral infection. Viral hemorrhagic septicemia virus (VHSV) is a negative-sense single-stranded RNA virus that causes severe loss in aquaculture species. Susceptible species include redlip mullets (Liza haematocheila), which has become an economically important euryhaline mugilid species in offshore aquaculture along the west coast of Korea. Although interferon-stimulated genes are suspected to act against VHSV, specific pathways or mechanisms of these antiviral actions in redlip mullets have not yet been established. In silico studies of the mullet viperin (Lhrsad2) revealed an S-adenosyl methionine binding conserved domain containing the 77CNYKCGFC84 sequence. In the tissue distribution, the highest levels of lhrsad2 expression were observed in the blood. When injected with poly(I:C), an approximately 17-fold upregulation (compared to the control) of viperin was detected in the blood after 24 h. Furthermore, non-viral immune stimuli, including Lactococcus garvieae (L. garvieae) and lipopolysaccharide (LPS), that were injected into redlip mullets were not found to induce considerable levels of viperin expression. Subcellular analysis revealed that Lhrsad2 localized to the endoplasmic reticulum (ER). To investigate Lhrsad2's antiviral effects against VHSV, cells overexpressing lhrsad2 were infected with VHSV, and then the viral titer and viral gene expression were analyzed. Both assays revealed the potential of Lhrsad2 to significantly reduce VHSV transcription and replication. In brief, the current study illustrates the remarkable ability of viperin to weaken VHSV in redlip mullet.

Molecular characterization, immune and xenobiotic responses of glutathione S-transferase omega 1 from the big-belly seahorse: Novel insights into antiviral defense.

Glutathione S-transferases (GSTs) are important enzymes involved in phase II detoxification and function by conjugating with the thiol group of glutathione. In this study, we isolated an omega class GST from the big-belly seahorse (Hippocampus abdominalis; HaGSTO1) to study the putative xenobiotic responses and defense ability against viral and bacterial infections in this animal. The isolated HaGSTO1 gene, with a cording sequence of 720 bp, encodes a peptide of 239 amino acids. The predicted molecular mass and theoretical isoelectric point of HaGSTO1 was 27.47 kDa and 8.13, respectively. In-silico analysis of HaGSTO1 revealed a characteristic N-terminal thioredoxin-like domain and a C-terminal domain. Unlike other GSTs, the C-terminal of HaGSTO1 reached up to the N-terminal, and the N-terminal functional group was cysteine rather than tyrosine or serine, as observed in other GSTs. Phylogenetic analysis showed the evolutionary proximity of HaGSTO1 with other identified vertebrate and invertebrate GST orthologs. For the first time, we demonstrated the viral defense capability of HaGSTO1 against viral hemorrhagic septicemia virus (VHSV) infection. All six nucleoproteins of VHSV were significantly downregulated in HaGSTO1-overexpressing FHM cells at 24 h after infection compared with those in the control. Moreover, arsenic toxicity was significantly reduced in HaGSTO1-overexpressing FHM cells, and cell viability increased. Real-time polymerase chain reaction analysis showed that HaGSTO1 transcripts were highly expressed in the pouch and gill when compared with those in other tissues. Blood HaGSTO1 transcripts were significantly upregulated after Edwardsiella tarda, Streptococcus iniae, lipopolysaccharide, and polyinosinic:polycytidylic acid challenge experiments. Collectively, these findings suggest the involvement of HaGSTO1 in the host defense mechanism of seahorses.