Functional characterization of irf3 against viral hemorrhagic septicemia virus infection using a CRISPR/Cas9-mediated zebrafish knockout model.

Interferon regulatory factors (IRFs) are transcription factors involved in immune responses, such as pathogen response regulation, immune cell growth, and differentiation. IRFs are necessary for the synthesis of type I interferons through a signaling cascade when pathogen recognition receptors identify viral DNA or RNA. We discovered that irf3 is expressed in the early embryonic stages and in all immune organs of adult zebrafish. We demonstrated the antiviral immune mechanism of Irf3 against viral hemorrhagic septicemia virus (VHSV) using CRISPR/Cas9-mediated knockout zebrafish (irf3-KO). In this study, we used a truncated Irf3 protein, encoded by irf3 with a 10 bp deletion, for further investigation. Upon VHSV injection, irf3-KO zebrafish showed dose-dependent high and early mortality compared with zebrafish with the wild-type Irf3 protein (WT), confirming the antiviral activity of Irf3. Based on the results of expression analysis of downstream genes upon VHSV challenge, we inferred that Irf3 deficiency substantially affects the expression of ifnphi1 and ifnphi2. However, after 5 days post infection (dpi), ifnphi3 expression was not significantly altered in irf3-KO compared to that in WT, and irf7 transcription showed a considerable increase in irf3-KO after 5 dpi, indicating irf7's control over ifnphi3 expression. The significantly reduced expression of isg15, viperin, mxa, and mxb at 3 dpi also supported the effect of Irf3 deficiency on the antiviral activity in the early stage of infection. The higher mortality in irf3-KO zebrafish than in WT might be due to an increased inflammation and tissue damage that occurs in irf3-KO because of delayed immune response. Our results suggest that Irf3 plays a role in antiviral immunity of zebrafish by modulating critical immune signaling molecules and regulating antiviral immune genes.

CRISPR/Cas9-mediated knockout of tnf-α1 in zebrafish reduces disease resistance after Edwardsiella piscicida bacterial infection.

Tumor necrosis factor (TNF) is an important cytokine involved in immune responses to bacterial infections in vertebrates, including fish. Although Tnf-α is a well-studied cytokine, there are contradictory findings about Tnf-α function following bacterial infection. In this study, we analyzed the expression and function of the Tnf-α-type I isoform (Tnf-α1) in zebrafish by knockout experiments using the CRISPR/Cas9 gene-editing tool. The open reading frame of tnf-α1 encodes a 25.82 kDa protein with 234 amino acids (aa). The expression of tnf-α1 in the early stages of zebrafish was observed from the 2-cell stage. Adult zebrafish spleens showed the highest expression of tnf-α1. To evaluate the function of Tnf-α1, an 8 bp deletion in the target region, resulting in a short truncated protein of 55 aa, was used to create the tnf-α1 knockout mutant. The pattern of downstream gene expression in 7-day larvae in wild-type (WT) and tnf-α1 knockout fish was examined. We also verified the fish mortality rate after Edwardsiella piscicida challenge and found that it was much higher in tnf-α1 knockout fish than in WT fish. Additionally, downstream gene expression analyses after E. piscicida exposure revealed a distinct expression pattern in tnf-α1 knockout fish compared to that in WT fish. Overall, our study using tnf-α1 deletion in zebrafish confirmed that Tnf-α1 is critical for immune regulation during bacterial infection.

CRISPR/Cas9-Induced Knockout of Sting Increases Susceptibility of Zebrafish to Bacterial Infection.

Stimulator of interferon genes (STING) is an adapter protein that is activated when cyclic dinucleotides (CDNs) are present. CDNs originate from the cytosolic DNA of both pathogens and hosts. STING activation promotes efficient immune responses against viral infections; however, its impact in bacterial infections is unclear. In this study, we investigated the role of Sting in bacterial infections by successfully creating a sting-deficient (sting(-/-) with a 4-bp deletion) knockout zebrafish model using CRISPR/Cas9. The transcriptional modulation of genes downstream of cGAS (cyclic GMP-AMP synthase)-Sting pathway-related genes was analyzed in seven-day-old wild-type (WT) and sting(-/-) embryos, as well as in four-day-old LPS-stimulated embryos. The expression of downstream genes was higher in sting(-/-) than in healthy WT fish. The late response was observed in sting(-/-) larvae following LPS treatment, demonstrating the importance of Sting-induced immunity during bacterial infection by activating the cGAS-STING pathway. Furthermore, adult sting(-/-) fish had a high mortality rate and significantly downregulated cGAS-STING pathway-related genes during Edwardsiella piscicida (E. piscicida) infection. In addition, we assessed NF-κB pathway genes following E. piscicida infection. Our results show fluctuating patterns of interleukin-6 (il6) and tumor necrosis factor-α (tnfα) expression, which is likely due to the influence of other NF-κB pathway-related immune genes. In summary, this study demonstrates the important role of Sting against bacterial infection.

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.

Expression profile and molecular function of beclin-1 in Epinephelus akaara in response to immune stimuli and oxidative stress.

Beclin-1, the mammalian ortholog of the yeast autophagy-related gene 6 (Atg 6), is a key regulator of autophagy. A variety of health and disease conditions in mammals are intricately related to the broad spectrum of beclin-1 functions. Nevertheless, few studies have investigated the role of beclin-1 in fish. In this study, we identified and cloned the beclin-1 cDNA (EaBECN-1) of Epinephelus akaara (red-spotted grouper) and carried out in silico analysis, tissue-specific expression analysis, immune challenge experiment, and in vitro analysis of its roles against viral infection and oxidative stress. The open reading frame was 1344 bp long and encoded 447 amino acids with a molecular weight of 51.2 kDa. Beclin-1 consisted of a conserved N-terminal BH3 and APG6 domains, and shared more than 88% identity with other vertebrates, according to a pairwise sequence alignment. EaBECN-1 expression profile analysis in E. akaara revealed that it is mostly expressed in the blood. Moreover, transcriptional modulation of EaBECN-1 was observed following stimulation with lipopolysaccharide (LPS), polyinosinic-polycytidylic acid (poly (I:C)), and nervous necrosis virus. During the viral hemorrhagic septicemia virus challenge, increased viral gene expression was observed at 12 h post-infection in FHM cells ectopically expressing EaBECN-1, and decreased thereafter at 24 h post-infection compared to control cells. However, increased antiviral gene expression at 12 and 24 h confirmed the antiviral function of EaBECN-1. Furthermore, EaBECN-1 overexpression protected the cells against H2O2-mediated apoptosis, as evidenced by the MTT assay, analysis of mRNA expression levels of apoptotic genes, and AO-EtBr staining. Overall, our study demonstrated the protective role of EaBECN-1 against viral pathogenesis and oxidative stress through autophagy, increasing our understanding of the role of beclin-1 in fish.

Insight into the molecular structure and function of peptidoglycan recognition protein SC2 (PGRP-SC2) from Amphiprion clarkii: Investigating the role in innate immunity.

Peptidoglycan recognition proteins (PGRPs) belong to the pattern recognition receptor (PRR) family and are conserved from insects to mammals. PGRPs show specific binding abilities to peptidoglycans (PGNs) in various microbes. In this study, molecular and functional analyses of PGRP-SC2 from Amphiprion clarkii (AcPGRP-SC2) were conducted. The 492 bp ORF of AcPGRP-SC2 encoded a protein of 164 amino acids with a molecular weight of 17.58 kDa and pI of 8.9. The PGRP superfamily domain was identified from the protein sequence of AcPGRP-SC2 and sequence similarities were observed with homologous proteins. Quantitative polymerase chain reaction (qPCR) analysis revealed that AcPGRP-SC2 transcripts were ubiquitously expressed in all tested tissues, with high levels in the skin, and transcript expression was significantly modulated by immune stimulation with lipopolysaccharide (LPS), Polyinosinic:polycytidylic acid (poly I:C), and Vibrio harveyi post-immune challenge. Recombinant AcPGRP-SC2 with the maltose-binding protein fusion (rAcPGRP-SC2) was used to evaluate LPS-, PGN-, and bacterial-binding activities and to conduct bacterial agglutination assays, and the results demonstrated that AcPGRP-SC2 exhibited bacterial recognition, binding, and colonization abilities to a range of Gram-positive and Gram-negative bacterial strains. Moreover, rAcPGRP-SC2-pre-treated Fat Head Minnow (FHM) cells exhibited significant upregulation in NF-ĸB1, NF-ĸB2, and stat3 expression upon treatment with killed bacteria. Taken together, our findings suggest that AcPGRP-SC2 plays an important role in the immune response against microbial pathogens in A. clarkii.

Generation of cd63-deficient zebrafish to analyze the role of cd63 in viral infection.

The tetraspanin superfamily proteins are transmembrane proteins identified in a diverse range of eukaryotic organisms. Tetraspanins are involved in a variety of essential biological functions, including cell differentiation, adhesion, migration, signal transduction, intracellular trafficking, and immune responses. For an infection to occur, viruses must interact with various cell surface components, including receptors and signaling molecules. Tetraspanin CD63 is involved in the organization of the cell membrane and trafficking of cellular transmembrane proteins that interact with many viruses. In this study, the cd63 gene was characterized by studying its expression and function in a zebrafish model. The functional domains and structural features of Cd63, such as the Cys-Cys-Gly (CCG) motif in the large extracellular loop and cysteine residues, are conserved in zebrafish. We confirmed that cd63 was expressed in immune system organs, such as the axial vein and pronephric duct, during the embryonic development of zebrafish. To better understand the role of cd63 in the zebrafish immune system, we established cd63-deficient zebrafish lines using the clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) system. A 19 bp insertion mutation was generated in single guide RNA (sgRNA) target sequence of exon 3 of the cd63 gene, to create a pre-mature stop codon. We then analyzed the expression of cd63-related genes cxcr4a and cxcr4b in wild type (WT) and cd63-deficient zebrafish. We believe our study provides an important model that could be used to investigate the roles of cd63 in viral infection in vivo.

Transcription profile, NF-ĸB promoter activation, and antiviral activity of Amphiprion clarkii Akirin-2.

Animal defense system constitutes a series of distinct mechanisms that specifically defend against microbial invasion. Understanding these complex biological mechanisms is of paramount importance for implementing disease prevention strategies. In this study, the transcription factor, Akirin-2 was identified from ornamental fish Amphiprion clarkii and its involvement in immune response was characterized. A. clarkii Akirin-2 (AcAkirin-2) was identified as a highly conserved protein with two nuclear localization signals. In-vitro localization analysis in fathead minnow cells revealed that AcAkirin-2 is strictly localized to the nucleus. With regard to tissue-specific expression without immune challenge, AcAkirin-2 expression was highest in the brain and lowest in the liver. Immune challenge experiments revealed that AcAkirin-2 expression was the strongest in response to poly I:C. Overexpression of AcAkirin-2 alone did not enhanced NF-ĸB activity significantly in HEK293T cells; however, it significantly enhanced NF-ĸB activity in the presence of poly I:C. AcAkirin-2-mediated expression of antiviral genes was analyzed using qPCR in mullet kidney cells and plaque assay was performed to decipher the involvement of AcAkirin-2 in antiviral immunity. AcAkirin-2 overexpression significantly enhanced the expression of Viperin but not of Mx. Plaque assays revealed the ability of AcAkirin-2 to enervate VHSV titers. Taken together, this study unveiled the involvement of AcAkirin-2 in NF-ĸB-mediated transcription of antiviral genes.

Glutathione-S-transferase alpha-4 in Hippocampus abdominalis (big-belly seahorse): Molecular characterization, antioxidant properties, and its potent immune response.

Glutathione-S-transferase (GST) is a key enzyme in the phase-II detoxification process and is a biomarker of oxidative stress. In this study, we analyzed the molecular, biochemical, and antioxidant properties of GST alpha-4 from Hippocampus abdominalis (HaGSTA-4). Also, the spatial and temporal expression of HaGSTA-4 upon immune challenge with abiotic and biotic stimulants were evaluated. The HaGSTA-4 ORF encodes 223 amino acids with a molecular weight of 25.7 kDa, and an estimated isoelectric point (pI) of 8.47. It consists of the GST_C superfamily and thioredoxin-like superfamily domain. The phylogenetic tree revealed that HaGSTA-4 is evolutionarily conserved with its GST alpha class counterparts. From pairwise alignment, the highest values of identity (78.5%) and similarity (85.7%) were with Parambassis ranga GSTA-4. Protein rHaGSTA-4 exhibited the highest conjugation activity towards 1-chloro-2,4-dinitrobenzene (CDNB) at pH 7 and 20 °C. A disk diffusion assay showed that rHaGSTA-4 significantly protects cells from the stress of exposure to ROS inducers such as CuSO4, CdCl2, and ZnCl2. Furthermore, overexpressed HaGSTA-4 defended cells against oxidative stress caused by H2O2; evidence of selenium-independent peroxidase activity. From qPCR, the tissue-specific expression profile demonstrates that HaGSTA-4 is most highly expressed in the kidney, followed by the intestine and stomach, among fourteen different tissues extracted from healthy seahorses. The mRNA expression profile of HaGSTA-4 upon immune challenge varied depending on the tissue and the time after challenge. Altogether, this study suggests that HaGSTA-4 may be involved in protection against oxidative stress, in immune defense regulation, and xenobiotic metabolism.

Identification, molecular characterization, expression analysis and wound-healing ability of multifunctional calreticulin from big-belly seahorse Hippocampus abdominalis.

Calreticulin (CRT) is a multifunctional ubiquitous protein that is widely presented in all cells in eukaryotes except erythrocytes. CRT is well known for diverse cellular functions such as endoplasmic reticulum (ER)-specialized protein quality control during protein synthesis and folding, in-vivo Ca2+ homeostasis, antigen presentation, phagocytosis, wound-healing, proliferation, adhesion, and migration of cells. In the current study, we identified CRT from Hippocampus abdominalis (HaCRT) and analyzed expression profiles and functional properties. The cDNA sequence of HaCRT was identified with an open reading frame of 1226 bp. The molecular weight of HaCRT was estimated as 49 kDa. The in-silico study revealed conserved sequence arrangements such as two CRT signature motifs (5'-KHEQSIDCGGGYVKVF-3' and 5'-LMFGPDICG-3'), triplicate repeats (5'-IKDPEAKKPEDWD-3', 5'-IPDPDDTKPEDWD-3', 5'-IPDPDAKKPDDWD-3'), signal peptide and an ER-targeting 5'-KDEL-3' sequence of HaCRT. Close sequence similarity of HaCRT was observed with Hippocampus comes from phylogenetic analysis and pairwise sequence comparison. From quantitative polymerase chain reaction (qPCR) results, HaCRT was ubiquitously distributed in all tested tissues and expression levels of HaCRT were significantly modulated in blood, liver and gill tissues after stimulation with Streptococcus iniae, Edwardsiella tarda, polyinosinic:polycytidylic acid, and lipopolysaccharides. Bacterial- and pathogen-associated molecular patterns-binding activities were observed with recombinant HaCRT (rHaCRT). The treatment of murine macrophages with rHaCRT induced the expression of immune genes, such as tumor necrosis factor-α (TNF-α), interleukin 6 (IL-6), inducible nitric oxide synthase (iNOS), and interleukin-1ß (IL-1ß). Furthermore, rHaCRT exhibited wound-healing ability. Based on the results from the above study, we suggest that HaCRT play an indispensable role in the immunity of big-belly seahorses by recognition and elimination of pathogens as well as the tissue repairing process.

Molecular and functional insights into a novel teleost malectin from big-belly seahorse Hippocampus abdominalis.

Malectin is a carbohydrate-binding lectin protein found in the endoplasmic reticulum (ER). It selectivity binds to Glc2-N-glycan and is involved in a glycoprotein quality control mechanism. Even though malectin may play a role in immunity, its role in innate immunity is not fully known. In the present study, we identified and characterized the malectin gene from Hippocampus abdominalis (HaMLEC). We analyzed sequence features, spatial expression levels, temporal expression profiles upon immune responses, bacterial and carbohydrate binding abilities and anti-viral properties to investigate the potential role of HaMLEC in innate immunity. The molecular weight and isoelectric point (pI) were estimated to be 31.99 kDa and 5.17, respectively. The N-terminal signal peptide, malectin superfamily domain and C-terminal transmembrane region were identified from the amino acid sequence of HaMLEC. The close evolutionary relationship of HaMLEC with other teleosts was identified by phylogenetic analysis. According to quantitative PCR (qPCR) results, HaMLEC expression was observed in all the examined tissues and high expression was observed in the ovary and brain, compared to other tested tissues. Temporal expression of HaMLEC in liver and blood tissues were significant modulated upon exposure to immunogens Edwardasiella tarda, Streptococcus iniae, polyinosinic:polycytidylic and lipopolysaccharide. The presence of carbohydrate binding modules (CBMs) of bacterial glycosyl hydrolases were functionally confirmed by a bacterial binding assay. Anti-viral activity significantly reduced viral hemorrhagic septicemia virus (VHSV) replication in cells overexpressing HaMLEC. The observed results suggested that HaMLEC may have a significant role in innate immunity in Hippocampus abdominalis.

Characterization and expression analysis of rockfish (Sebastes schlegelii) myeloid differentiation factor-88 (SsMyD88) and evaluation of its ability to induce inflammatory cytokines through NF-ĸB.

Innate immunity is characterized by nonspecific, prompt reactions toward armada of antigens. Animals funnel down a repertoire of immune stimulants to activate non-selective defense mechanisms rapidly. This study was conducted to characterize the rockfish (Sebastes schlegelii) adaptor protein MyD88 (SsMyD88), which interacts with both toll-like receptors and interleukin receptors. The tissue expression of unchallenged SsMyD88 was evaluated by quantitative real time PCR (qPCR). Fish were intraperitoneally injected with immune stimulants including poly I:C, lipopolysaccharides, and Streptococcus iniae. Then, the temporal expression of SsMyD88 was analyzed. Finally, the inflammatory gene expression and downstream promoter activation were analyzed. Strongest expressions were reported in the liver, gills and spleen in unchallenged conditions. All diverse immune stimulants were found to be capable of significantly altering SsMyD88 transcription during the challenge experiment. Evaluation of downstream promoter biases by SsMyD88 found a predominant activation of NF-ĸB transcription factors when compared with the AP-1, revealing significant and substantial upregulation of major inflammatory mediators such as IL-1-ß, IL-6, iNOS, COX-2 and TNF-α. Fluorescent detection confirmed an intense production of NO and the predominant differentiation of macrophages into M1 lineage with the overexpression of SsMyD88 in vitro. These results further corroborate the role of SsMyD88 as a mediatory molecule that bridges distinct immune stimulants to induce drastic immune responses in fish.

Molecular and functional explication of thioredoxin mitochondrial-like protein (Trx-2) from big-belly seahorse (Hippocampus abdominalis) and expression upon immune provocation.

Thioredoxin (Trx) is a small ubiquitous multifunctional protein with a characteristic WCGPC thiol-disulfide active site that is conserved through evolution. Trx plays a crucial role in the antioxidant defense system. Further, it is involved in a variety of biological functions including gene expression, apoptosis, and growth regulation. Trx exists in several forms, with the cytosolic (Trx-1) and mitochondrial (Trx-2) forms being the most predominant. In this study, the mitochondrial Trx protein (HaTrx-2), from the big-belly seahorse (Hippocampus abdominalis) was characterized, and its molecular features and functional properties were investigated. The cDNA sequence of HaTrx-2 consists of a 519 bp ORF, and it encodes a polypeptide of 172 amino acids. This protein has a calculated molecular mass of 18.8 kDa and a calculated isoelectric point (pI) of 7.80. The highest values of identity (78.7%) and similarity (86.2%) were observed with Fundulus heteroclitus Trx-2 from the pairwise alignment results. The phylogenetic analysis revealed that HaTrx-2 is closely clustered with teleost fishes. The qPCR results showed that HaTrx-2 was prevalently expressed at various levels in all the tissues examined. The ovary showed the highest expression, followed by the brain and kidney. HaTrx-2 showed varying mRNA expression levels during the immune challenge experiment, depending on the type of tissue and the time interval. Our results confirmed the antioxidant property of HaTrx-2 by performing the MCO assay, DPPH radical scavenging activity, and cell viability assays. Further, an insulin disulfide reduction assay revealed the dithiol remove the enzymatic activity of HaTrx-2. Altogether these results indicate that HaTrx-2 plays indispensable roles in the regulation of oxidative stress and immune response in the seahorse.

Molecular cloning, characterization, and expression level analysis of a marine teleost homolog of catalase from big belly seahorse (Hippocampus abdominalis).

Organisms possess a cellular antioxidant defense system inclusive of ROS scavengers to maintain the homeostasis of antioxidant levels. Catalase is a major ROS scavenger enzyme that plays a significant role in the antioxidant defense mechanism of organisms by reducing toxic hydrogen peroxide molecules into a nontoxic form of oxygen and water with a high turnover rate. In the present study, we performed molecular and functional characterization of the catalase homolog from Hippocampus abdominalis (HaCat). The HaCat cDNA sequence was identified as a 1578 bp ORF (open reading frame) that encodes a polypeptide of 526 amino acids with 59.33 kDa molecular weight. Its estimated pI value is 7.7, and it does not have any signal sequences. HaCat shared a conserved domain arrangement including the catalase proximal active site signature and heme ligand signature domain with the previously identified catalase counterparts. Phylogenetic analysis displayed close evolutionary relationships between HaCat and catalases from other teleost fish. According to our qPCR results, ubiquitous expression of HaCat transcripts were observed in all the tested tissues with high expression in the kidney followed by liver. Significant modulations of HaCat transcription were observed in blood, liver, and kidney tissues post-challenge with Streptococcus iniae, Edwardsiella tarda, poly I:C, and LPS. Peroxidase activity of recombinant HaCat (rHaCat) was evaluated using an ABTS assay and the ROS removal effect was further confirmed by oxidative DNA damage protection and cell viability assays. The rHaCat showed more than 97% activity over a temperature and pH range of 10 °C-40 °C and 5 to 6, respectively. The above results suggest that HaCat plays an indispensable role in the oxidative homeostasis of the seahorse during pathogenic attack.