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.

Molecular characterization and immune response of suppressor of cytokine signaling 5b from redlip mullet (Planiliza haematocheilus): Disclosing its anti-viral potential and effect on cell proliferation.

The suppressor of cytokine signaling (SOCS) proteins family comprising eight proteins (SOCS1-7 and cytokine-inducible SH2-containing (CIS)) are classical feedback inhibitors of cytokine signaling. Although the biological role of CIS and SOCS1-3 have been extensively studied, the biological functions of SOCS4-7 remain unclear. Here, we elucidated the molecular characteristics, expression profile, immune response, anti-viral potential, and effect on cell proliferation of Phsocs5b, a member of the SOCS protein family from redlip mullet (Planiliza haematocheilus); phsocs5b comprised 1695 nucleotides. It was 564 amino acids long with a molecular weight of 62.3 kDa and a theoretical isoelectric point of 8.95. Like SOCS4-7 proteins, Phsocs5b comprised an SH2 domain, SOCS box domain, and a long N-terminal. SH2 domain is highly identical to its orthologs in other vertebrates. Phsocs5b, highly expressed in the brain tissue, was localized in the cytoplasm. Temporal changes in phsocs5b expression were observed following immune stimulation with polyinosinic: polycytidylic acid, lipopolysaccharide, and Lactococcus garvieae. In FHM cells, Phsocs5b overexpression suppressed the viral hemorrhagic septicemia virus (VHSV) infection and epidermal growth factor receptor (egfr) expression but increased the mRNA levels of pi3k, akt, pro-inflammatory cytokines (il1ß and il8), and anti-viral genes (isg15 and ifn). Overall, our findings suggest that Phsocs5b attenuates VHSV infection, either by hindering the cell entry via degradation of Egfr, enhancing pro-inflammatory cytokines and anti-viral factor production, or both. The results also indicated that Phsocs5b could directly activate Pi3k/Akt pathway by itself, thus enhancing the proliferation and migration of cells. Taken together, Phsocs5b may be considered a potential therapeutic target to enhance immune responses while positively regulating the proliferation and migration of cells.

Molecular delineation, expression profiling, immune response, and anti-apoptotic function of a novel clusterin homolog from big-belly seahorse (Hippocampus abdominalis).

Clusterin (CLU) is a glycoprotein that contains α- and ß-chains. CLU exerts multifunctional activities and plays a role in different cell signaling pathways that are associated with various diseases such as proteotoxic and oxidative stress, as well as cell death and survival. However, its role in marine teleost fish remains unclear. Therefore, the present study was carried out to characterize and investigate the immune responses and anti-apoptotic effects of CLU of the big-belly seahorse (Hippocampus abdominalis) (HaCLU) on oxidative stress-induced cell death. The HaCLU open reading frame was 1389 bp long and encoded a protein with 462 amino acids, a molecular weight of 51.28 kDa and an isoelectric point of 5.41. In-silico results demonstrated that HaCLU has a signal peptide in the 1-29 amino acid region, while the α- and ß-chains were in the 34-227 and 228-455 amino acid regions, respectively. Multiple sequence alignment clarified the low homology of the α-chain with other orthologs. The highest HaCLU mRNA expression level was observed in the liver, followed by the heart, spleen, and brain tissues of healthy big-belly seahorses. Further, HaCLU mRNA expression level was elevated in the liver in response to different stimuli, including lipopolysaccharides, polyinosinic:polycytidylic acid, Edwardsiella tarda, and Streptococcus iniae. HaCLU potentiates cell viability and weakens chromatin condensation in the nucleus of FHM cells following H2O2-induced oxidative stress and subsequent cell death. HaCLU overexpression resulted in a reduced Bax/Bcl-2 mRNA expression ratio. This study revealed the role of HaCLU in immune regulation against pathogenic infections and its anti-apoptotic effects on oxidative stress-induced cell death.

Cytosolic ß-catenin is involved in macrophage M2 activation and antiviral defense in teleosts: Delineation through molecular characterization of ß-catenin homolog from redlip mullet (Planiliza haematocheila).

ß-catenin is a structural protein that makes the cell-cell connection in adherence junctions. Besides the structural functions, it also plays a role as a central transducer of the canonical Wnt signaling cascade, regulating nearly four hundred genes related to various cellular processes. Recently the immune functions of ß-catenin during pathogenic invasion have gained more attention. In the present study, we elucidated the immune function of fish ß-catenin by identifying and characterizing the ß-catenin homolog (PhCatß) from redlip mullet, Planiliza haematocheila. The complete open reading frame of PhCatß consists of 2352 bp, which encodes a putative ß-catenin homolog (molecular weight: 85.7 kDa). Multiple sequence alignment analysis revealed that ß-catenin is highly conserved in vertebrates. Phylogenetic reconstruction demonstrated the close evolutionary relationship between PhCatß and other fish ß-catenin counterparts. The tissue distribution analysis showed the highest mRNA expression of PhCatß in heart tissues of the redlip mullet under normal physiological conditions. While in response to pathogenic stress, the PhCatß transcription level was dramatically increased in the spleen and gill tissues. The overexpression of PhCatß stimulated M2 polarization and cell proliferation of murine RAW 264.7 macrophage. In fish cells, the overexpression of PhCatß resulted in a significant upregulation of antiviral gene transcription and vice versa. Moreover, the overexpression of PhCatß could inhibit the replication of VHSV in FHM cells. Our results strongly suggest that PhCatß plays a role in macrophage activation and antiviral immune response in redlip mullet.

Identification, expression profiling and functional characterization of interleukin 11a ortholog from redlip mullet Liza haematocheila: Insight into its roles in the inflammation and apoptosis regulation.

Interleukin 11 (IL-11) is a secretory cytokine with pleotropic properties, including anti-inflammatory and anti-apoptotic functions. This study aimed to functionally characterize a teleostean IL-11a ortholog from redlip mullet (LhIL-11a) through bioinformatic analysis, transcriptional expression profiling and protein function assays. The deduced LhIL-11a protein sequence is 200 amino acids long, with a predicted molecular weight of 23.168 kDa. Multiple sequence alignment indicates that LhIL-11a has a typical four-bundle architecture of α-helixes as observed in other IL-11s. The identity-similarity matrix show a higher identity between LhIL-11a and other fish IL-11a sequences. Phylogenetic analysis demonstrated that LhIL-11a falls within a clade including other fish counterparts. In the tissue distribution analysis, the highest constitutive expression of LhIL-11a mRNA was observed in the mullet gastrointestinal tract and brain tissues. Following the challenges with LPS, poly I:C and Lactococcus garvie, the transcription levels of LhIL-11a were significantly upregulated in both PBCs and liver. In the biological functional assay, recombinant LhIL-11a protein showed strong activities of suppressing pro-inflammatory cytokines and apoptotic gene expression in mullet kidney cells and reducing LPS stimulated NO production in murine macrophage cells. Overall, the findings in this study provide the experimental clues to understanding the functional roles of fish IL-11a in inflammation and apoptosis regulation during host defense against invading microbial pathogens.

Molecular, transcriptional and functional delineation of Galectin-8 from black rockfish (Sebastes schlegelii) and its potential immunological role.

Galectins are ß-galactoside-binding lectins, which are involved in pattern recognition, cell adhesion, and stimulation of the host innate immune responses against microbial pathogens. In spite of several functional studies on different galectins isolated from vertebrates and invertebrates, this is the first report to present functional studies for galectin-8 from the marine teleost tissues. In the present study, we characterized galectin-8 homolog from black rockfish (Sebastes schlegelii), in molecular and functional aspects. Rockfish galectin-8 (SsGal8) was found to consist of a 969 bp long open reading frame (ORF), encoding a protein of 322 amino acids and the predicted molecular weight was 35.82 kDa. In silico analysis of SsGal8 revealed the presence of two carbohydrate binding domains (CRDs), at both N and C-termini and a linker peptide of 40 amino acids, in between the two domains. As expected, the phylogenetic tree categorized SsGal8 as a tandem-repeat galectin, and ultimately positioned it in the sub-clade of fish galectin-8. rSsGal8 was able to strongly agglutinate fish erythrocytes and the inhibition of agglutination was successfully exhibited by lactose and d-galactose. Bacterial agglutination assay resulted in agglutination of both Gram (+) and Gram (-) bacteria, including Escherichia coli, Vibrio harveyi, Vibrio parahaemolyticus, Streptococcus parauberis, Lactococcus garvieae, Streptococcus iniae and Vibrio tapetis. The tissue distribution analysis based on qPCR assays, revealed a ubiquitous tissue expression of SsGal8 for the examined rockfish tissues, with the most pronounced expression in blood, followed by brain, intestine, head kidney and kidney. Furthermore, the mRNA transcription level of SsGal8 was significantly up-regulated in spleen, liver and head kidney, upon immune challenges with Streptococcus iniae, LPS and poly I:C, in a time dependent manner. Taken together, these findings strongly suggest the contribution of SsGal8 in regulating innate immune responses to protect the rockfish from bacterial infections.

Modulation of fish immune response by interferon regulatory factor 4 in redlip mullet (Liza haematocheilus): Delineation through expression profiling, antiviral assay, and macrophage polarization analysis.

Interferon regulatory factor 4 (IRF4) is a crucial member of IRF family, which acts as an imperative transcription factor in the development and maturation of multiple lineages of blood cells and also plays a pivotal role in host defense against microbial infections. In the present study, we aimed to investigate the detailed structural and functional aspects of a redlip mullet IRF4 homolog (LhIRF4). The LhIRF4 open reading frame consists of 1347 base pairs encoding 449 amino acids, with the DNA-binding domain sharing significant homology with that of other vertebrate IRF4 homologs. The highest transcription levels of LhIRF4 were observed in the mullet intestine and spleen under normal physiological conditions. Furthermore, a time-dependent upregulation of LhIRF4 transcription was observed in the spleen and head kidney tissues upon pathogenic challenges. When overexpressed in mullet cells, LhIRF4 was localized to the nucleus and significantly stimulated the transcription of several host antiviral genes. Moreover, the overexpression of LhIRF4 strongly inhibited the replication of viral hemorrhagic septicemia virus (VHSV) in vitro. The function of LhIRF4 in regulation of macrophage M2 polarization has also been evidently demonstrated in RAW 264.7 cells. Taken together, our findings indicate the profound role of LhIRF4 in modulating immune responses against microbial infections in redlip mullet.

Molecular and functional characterization of a mitochondrial glutathione reductase homolog from redlip mullet (Liza haematocheila): Disclosing its antioxidant properties in the fish immune response mechanism.

Glutathione reductase (GSHR) is a biologically important enzyme involved in the conversion of oxidized glutathione (GSSG) into its reduced form, reduced glutathione (GSH), with the catalytic activity of NADPH. Most animals and aquatic organisms, including fish, possess high levels of this enzyme system to neutralize oxidative stress in cells. The current study was conducted to broaden our knowledge of GSHR in fish by identifying a mitochondrial isoform of this enzyme (LhGSHRm) in redlip mullet, Liza haematocheila, and clarifying its structure and function. The complete open reading frame of LhGSHRm consists of 1527 base pairs, encoding 508 amino acids, with a predicted molecular weight of 55.43 kDa. Multiple sequence alignment revealed the conservation of important amino acids in this fish. Phylogenetic analysis demonstrated the closest evolutionary relationship between LhGSHRm and other fish GSHRm counterparts. In tissue distribution analysis, the highest mRNA expression of LhGSHRm was observed in the gill tissue under normal physiological conditions. Following pathogenic challenges, the LhGSHRm transcription level was upregulated in a time-dependent manner in the gill and liver tissues, which may modulate the immune reaction against pathogens. rLhGSHRm showed considerable glutathione reductase activity in an enzyme assay. Further, the biological activity of rLhGSHRm in balancing cellular oxidative stress was observed in both disk diffusion and DPPH assays. Collectively, these results support that LhGSHRm has profound effects on modulating the immune reaction in fish to sustain precise redox homeostasis.