G.I. Godahewa

Two carboxypeptidase counterparts from rock bream (Oplegnathus fasciatus): molecular characterization, genomic arrangement and immune responses upon pathogenic stresses.

Carboxypeptidases (CPs) are proteases that hydrolyze C-terminal peptide bonds. They are involved in regulating the complement system of the immune system. Here, we report the molecular characterization and immune response of two carboxypeptidases, named carboxypeptidase A (Rb-CPA) and carboxypeptidase N1 (Rb-CPN1), from rock bream. The genomic sequence of Rb-CPA contains 12 exons interrupted by 11 introns, while the genomic sequence of Rb-CPN1 has 9 exons and 8 introns. The cDNA sequence of Rb-CPA encodes a 421-amino-acid (AA) polypeptide (48kDa), and the cDNA of Rb-CPN1 encodes a 448-AA polypeptide (51kDa). The amino acid sequences of Rb-CPA and Rb-CPN1 were found to harbor two characteristic Zn-binding signature domains and a peptidase-M14 Zn carboxypeptidase site. Pairwise analysis revealed that Rb-CPA and Rb-CPN1 had the highest identity with the corresponding proteins from Anoplopoma fimbria (87.6%) and Dicentrarchus labrax (96.9%), respectively. qPCR results indicated that Rb-CPA and Rb-CPN1 were constitutively expressed mainly in the kidney, heart, liver, and head kidney. Both genes were transcriptionally regulated in the liver upon challenge with pathogenic bacteria (Streptococcus iniae, Edwardsiella tarda), rock bream iridovirus (RBIV), and the immune modulators polyinosinic:polycytidylic acid and lipopolysaccharide. Taken together, our findings suggest that Rb-CPA and Rb-CPN1 have immune-related functions in rock bream.

Mitochondrial peroxiredoxin 3 (Prx3) from rock bream (Oplegnathus fasciatus): immune responses and role of recombinant Prx3 in protecting cells from hydrogen peroxide induced oxidative stress.

Pathogenic infections and environmental factors cause a variety of stresses in fish including oxidative stress by rapid elevation of reactive oxygen species (ROS) and reactive nitrogen species (RNS). Transcriptional activation and expression of antioxidant enzymes are essential for reducing the oxidative stress. In this study, we present the molecular characterization, immune responses and ROS scavenging activity of mitochondrial peroxiredoxin 3 from Oplegnathus fasciatus (RbPrx3). Coding sequence (CDS) of RbPrx3 contains 248 amino acids polypeptide which consists of highly conserved peroxiredoxin super family domain and two cysteine residues. Pairwise sequence comparison revealed that RbPrx3 has the greatest identity (94.8%) to Sparus aurata Prx3. Transcriptional analysis of RbPrx3 indicated the ubiquitously expressed mRNA in wide array of organs showing the highest expression in the liver of rock bream. Upon immune challenge of Edwardsiella tarda, Streptococcus iniae, rock bream iridovirus (RBIV) and lipopolysaccharide (LPS), RbPrx3 mRNA level was up-regulated in immunocompetent liver tissues compared to unchallenged fish. Purified recombinant RbPrx3 treated THP-1 cells showed higher survival rate against H(2)O(2) induced oxidative stress and significantly reduced the level of intracellular ROS. Overall results from our study suggest that RbPrx3 may be involved in broader functions such as regulating oxidative stresses by scavenging ROS and activating immune responses in rock bream.

Characterization of rock bream (Oplegnathus fasciatus) complement components C1r and C1s in terms of molecular aspects, genomic modulation, and immune responsive transcriptional profiles following bacterial and viral pathogen exposure

The complement components C1r and C1s play a crucial role in innate immunity via activation of the classical complement cascade system. As initiators of the pathogen-induced signaling cascade, C1r and C1s modulate innate immunity. In order to understand the immune responses of teleost C1r and C1s, Oplegnathus fasciatus C1r and C1s genes (OfC1r and OfC1s) were identified and characterized. The genomic sequence of OfC1r was enclosed with thirteen exons that represented a putative peptide with 704 amino acids (aa), whereas eleven exons of OfC1s represented a 691 aa polypeptide. In addition, genomic analysis revealed that both OfC1r and OfC1s were located on a single chromosome. These putative polypeptides were composed of two CUB domains, an EGF domain, two CCP domains, and a catalytically active serine protease domain. Phylogenetic analysis of C1r and C1s showed that OfC1r and OfC1s were evolutionary close to the orthologs of Pundamilia nyererei (identity = 73.4%) and Oryzias latipes (identity = 58.0%), respectively. Based on the results of quantitative real-time qPCR analysis, OfC1r and OfC1s transcripts were detected in all the eleven different tissues, with higher levels of OfC1r in blood and OfC1s in liver. The putative roles of OfC1r and OfC1s in response to pathogenic bacteria (Edwardsiella tarda and Streptococcus iniae) and virus (rock bream iridovirus, RBIV) were investigated in liver and head kidney tissues. The transcription of OfC1r and OfC1s was found to be significantly upregulated in response to pathogenic bacterial and viral infections. Overall findings of the present study demonstrate the potential immune responses of OfC1r and OfC1s against invading microbial pathogens and the activation of classical signaling cascade in rock bream.

Molecular characterization and comparative expression analysis of two teleostean pro-inflammatory cytokines, IL-1β and IL-8, from Sebastes schlegeli.

Interleukin 1β (IL-1β) and interleukin 8 (IL-8) are two major pro-inflammatory cytokines which play a central role in initiation of inflammatory responses against bacterial- and viral-infections. IL-1β is a member of the interleukin 1 family proteins and IL-8 is classified as a CXC-chemokine. In the current study, putative IL-1β and IL-8 counterparts were identified from a black rockfish transcriptomic database and designated as RfIL-1β and RfIL-8. The RfIL-1β cDNA sequence consists of 1140 nucleotides with a 759bp open reading frame (ORF) which encodes a 252 amino acid (aa) protein, whereas the RfIL-8 cDNA sequence (898bp) harbors a 300bp ORF encoding a 99 aa protein. Furthermore, the RfIL-1β aa sequence contains an IL-1 super family-like domain and an N-terminal IL-1 super family propeptide, while the amino acid sequence of RfIL-8 consists of a typical chemokine-CXC domain. Analysis of sequenced BAC clones containing RfIL-1β and RfIL-8 showed each gene to contain 4 exons interrupted by 3 introns. Pairwise comparison and phylogeny analysis of these cytokine sequences clearly revealed their closer relationship with other corresponding members of teleosts compared to birds and mammals. Constitutive differences in RfIL-1β and RfIL-8 mRNA expression were detected in a tissue-specific manner with the highest expression of each mRNA in spleen tissue. Two immune challenge experiments were conducted with Streptococcus iniae and polyinosinic:polycytidylic acid (poly I:C; a viral double stranded RNA mimic), and transcripts were quantified in spleen and peripheral blood cells. Significantly increased RfIL-1β and RfIL8 transcript levels were detected with almost similar profile patterns, further suggesting a putative involvement of these pro-inflammatory cytokines in the rockfish immunity.

Molecular characterization and expression analysis of B cell activating factor from rock bream (Oplegnathus fasciatus)

B cell activating factor (BAFF) is a member of the tumor necrosis factor (TNF) ligand family. BAFF has been shown to induce survival and proliferation of lymphocytes. We characterized the gene encoding BAFF (RbBAFF) in rock bream (Oplegnathus fasciatus), and attempted to determine its biological functions upon immune responses. In silico analysis of RbBAFF demonstrated the presence of common TNF ligand family features, including a TNF domain, a D-E loop, and three cysteine residues that are crucial for trimer formation. Amino acid sequence alignment confirmed that RbBAFF and its homologs were conserved at secondary and tertiary levels. Transcriptional analysis indicated that RbBAFF mRNAs were ubiquitously expressed in wide array of tissues. The higher levels of constitutive expression were observed in the kidney, head kidney and spleen, suggesting an important physiological relationship with lymphocytes. Under pathological conditions, RbBAFF mRNA levels were significantly elevated. The role of RbBAFF in lymphocyte survival and proliferation was confirmed by MTT assays and flow cytometry. Recombinant RbBAFF protein (10 μg/mL) was able to prolong the survival and/or enhance the proliferation of rock bream lymphocytes by approximately 30%. Transcription of IL-10 and NFκB-1 was significantly stimulated by RbBAFF. Our findings provide further information regarding fish BAFF gene and its role in adaptive immunity.

Chitosan Silver Nano Composites (Cagncs) as Potential Antibacterial Agent to Control Vibrio tapetis

Nanocomposites exhibit high performance on antimicrobial activities and have the potential to be developed alternative antibiotics. In this study, antibacterial effects of chitosan silver nano composites (CAgNCs) were investigated using pathogenic Vibrio tapetis as a bacterial model. Agar disc diffusion and turbidimetric assay results showed that CAgNCs could inhibit the growth of V. tapetis in concentration depended manner. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of CAgNCs against V. tapetis were 50 μg/mL and 100 μg/mL, respectively. The field emission scanning electron microscopy (FE-SEM) images analysis of V. tapetis showed severe structural damage to cells after treating CAgNCs at 75 μg/mL compared to un-treated bacteria. Moreover, CAgNCs induce the intracellular ROS level, leakage of nucleic materials (RNA and DNA), decrease the cell viability and protein level in V. tapetis cells. Herein, we demonstrate that CAgNCs as effective antibacterial agent with capability to disrupt cell membrane, de-stabilize the membrane permeability, induce the oxidative stress and inhibit the expression of protein or synthesis of macromolecules. Overall results from this study suggest that mode of action of CAgNCs may be associated with excessive generation of ROS, loss in membrane integrity and inhibiting protein synthesis that cause the bacterial cell death.

Chitosan silver nanocomposite (CAgNC) as an antifungal agent against Candida albicans

Abstract Due to limited numbers of antifungal drugs and emergence of drug resistance have directed to develop nonconventional therapeutic agents against fungal pathogen Candida albicans. In this study, anticandidal activity of chitosan silver nanocomposite (CAgNC) was tested against C. albicans. Minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) of CAgNC were determined as 25 and 100 μg/ml, respectively. Electron microscopic image results confirmed the ultrastructural cell wall deformities and injuries caused by CAgNC. Propidium iodide (PI) penetration into the CAgNC treated cells could be considered as an evidence for loss of cell membrane integrity and cell death at MFC. Level of intracellular reactive oxygen species (ROS) was increased, while cell viability was decreased with the increased of CAgNC concentrations. In our protein profile results, several induced proteins were observed under CAgNC treatment, and they could be related to multidrug and stress resistant proteins such as CDR1 (55 kDa) and CaHSP70 based on the protein band size. CAgNC mediated cell wall damage, loss of cell membrane integrity, elevated ROS level, and associated oxidative stress have been identified as the main causative factors for the anticandidal activity. Overall results from our study indicated that CAgNC could affect negatively on physiological and biochemical functions of C. albicans suggesting CAgNC as a potential alternative for antifungal chemotherapy.

A homolog of Kunitz-type serine protease inhibitor from rock bream, Oplegnathus fasciatus: Molecular insights and transcriptional modulation in response to microbial and PAMP stimulation, and tissue injury.

Serine proteases and their inhibitors play vital roles in diverse biological processes. In this study, we identified and characterized cDNA coding for a Kunitz-type serine protease inhibitor (SPI), which we designated as RbKSPI, in a commercially important species, rock bream. The full-length cDNA sequence of RbKSPI consisted of 2452 bp with an open reading frame (ORF) of 1521 bp encoding a polypeptide of 507 amino acid (aa) residues. In the RbKSPI protein, MANEC, PKD, LDLa, and two Kunitz domains responsible for various functions were identified as characteristic features. Homology analysis revealed that RbKSPI shared the highest identity with the Kunitz homolog in Takifugu rubripes (77.6%). Phylogenetic analysis indicated that RbKSPI clusters with other teleostean KSPIs. In tissue-specific expression analysis, RbKSPI transcripts were detected in all the tested tissues, with the highest expression in gill tissue, followed by kidney and intestine. The mRNA expression of RbKSPI significantly increased in blood cells upon stimulation with two strains of bacteria (Edwardsiella tarda and Streptococcus iniae) and two pathogen-associated molecular patterns (PAMPs; LPS and poly I:C). Meanwhile, down-regulated expression of RbKSPI was observed in response to tissue injury. Collectively, these results suggest that the RbKSPI may be involved in essential immune defense against microbial pathogens and in the wound-healing process.

Three novel clade B serine protease inhibitors from disk abalone, Haliotis discus discus: Molecular perspectives and responses to immune challenges and tissue injury.

Serine protease inhibitors (SERPINs) control cellular protease activity in order to maintain cellular homeostasis. The immune and inflammatory responses of invertebrate clade B SERPINs have not been widely reported. In the present study, three proteins with high similarity to clade B SERPINs, referred to as AbSERPIN-1, AbSERPIN-2 and AbSERPIN-3, were identified from disk abalone (Haliotis discus discus). While AbSERPIN-1 (399 aa) was of a typical size for this protein class, AbSERPIN-2 (506 aa) and AbSERPIN-3 (532 aa) were relatively larger. Bioinformatic analysis revealed the characteristic SERPIN domain in each AbSERPIN. In addition, the N-terminal region of both AbSERPIN-2 and AbSERPIN-3 contained a predicted low complexity region (LCR) and a signal peptide, suggesting that these proteins are secretory proteins and are, thus, novel peptides. Tertiary structural models of the AbSERPINs highlighted their structural and functional conservation. Ubiquitous expression of AbSERPIN transcripts was evaluated by quantitative real time PCR (qPCR) analysis in seven tissue types. AbSERPIN-1, AbSERPIN-2, and AbSERPIN-3 transcript levels were highest in mantle, hemocytes, and muscles, respectively. Temporal expression analysis revealed that AbSERPINs were significantly (P < 0.05) elevated in hemocytes during the early/middle stages following the injection of a bacterial pathogen (Vibrio parahaemolyticus or Listeria monocytogenes) or an immuno-stimulant (lipopolysaccharide). Moreover, mantle tissue injury led to significant changes in the temporal expression of AbSERPIN mRNA. Specifically, transcription of AbSERPIN-1 and AbSERPIN-3 was considerably up-regulated, while expression of AbSERPIN-2 was suppressed. These results suggest a potential role of AbSERPINs in response to pathogen invasion and tissue injury in disk abalone.

Manganese-superoxide dismutase (MnSOD), a role player in seahorse (Hippocampus abdominalis) antioxidant defense system and adaptive immune system

Abstract Manganese superoxide dismutase (MnSOD) is a metaloenzyme that catalyzes dismutation of the hazardous superoxide radicals into less hazardous H2O2 and H2O. Here, we identified a homolog of MnSOD from big belly seahorse (Hippocampus abdominalis; HaMnSOD) and characterized its structural and functional features. HaMnSOD transcript possessed an open reading frame (ORF) of 672 bp which codes for a peptide of 223 amino acids. Pairwise alignment showed that HaMnSOD shared highest identity with rock bream MnSOD. Results of the phylogenetic analysis of HaMnSOD revealed a close proximity with rock bream MnSOD which was consistent with the result of homology alignment. The intense expression of HaMnSOD was observed in the ovary, followed by the heart and the brain. Further, immune related responses of HaMnSOD towards pathogenic stimulation were observed through bacterial and viral challenges. Highest HaMnSOD expression in response to stimulants Edwardsiella tarda, Streptococcus iniae, lipopolysaccharide (LPS), and polyinosinic‐polycytidylic acid (Poly I:C) was observed in the late stage in the blood tissue. Xanthine/xanthine oxidase assay (XOD assay) indicated the ROS‐scavenging ability of purified recombinant HaMnSOD (rHaMnSOD). The optimum conditions for the SOD activity of rHaMnSOD were pH 9 and the 25 °C. Collectively, the results obtained through the expressional analysis profiles and the functional assays provide insights into potential immune related and antioxidant roles of HaMnSOD in the big belly seahorse. HighlightsMnSOD was identified from big belly seahorse (HaMnSOD).HaMnSOD was cloned and expressed to evaluate its distinct functional features.XOD (Xanthine oxidase) assay confirmed the superoxide scavenging ability of HaMnSOD.Transcriptional level of HaMnSOD was modulated by pathological stress.