Chamilani Nikapitiya

Molecular evidence for the existence of lipopolysaccharide-induced TNF-α factor (LITAF) and Rel/NF-kB pathways in disk abalone (Haliotis discus discus)

The lipopolysaccharide-induced TNF-alpha factor (LITAF) and Rel family nuclear factor kappaB (Rel/NF-kB) are two important transcription factors which play major roles in the regulating inflammatory cytokine, apoptosis and immune related genes. Here, we report the discovery of disk abalone LITAF (AbLITAF) and Rel/NF-kB (AbRel/NF-kB) homologues and their immune responses. Full-length cDNA of AbLITAF consists of 441 bp open reading frame (ORF) that translates into putative peptide of 147 aa. Analysis of AbLITAF sequence showed it has characteristic LITAF (Zn(+2)) binding domain with two CXXC motifs. Phylogenetic analysis results further revealed that AbLITAF is a member of LITAF family. AbRel/NF-kB is 584 aa protein that contains several characteristic motifs including Rel homology domain (RHD), Rel protein signature, DNA binding motif, nuclear localization signal (NLS) and transcription factor immunoglobulin - like fold (TIG) similar to their invertebrate and vertebrate counterparts. Tissue specific analysis results showed that both AbLITAF and AbRel/NF-kB mRNA was expressed ubiquitously in all selected tissues in constitutive manner. However, constitutive expression of AbLITAF was higher than AbRel/NF-kB in all tissues except mantle. Upon immune challenge by bacteria (Vibrio alginolyticus, Vibrio parahemolyticus and Lysteria monocytogenes) and viral hemoragic septicemia virus (VHSV), AbLITAF showed the significant up-regulation in gills while AbRel/NF-kB transcription was not change significantly. Based on transcriptional response against immune challenge, we could suggest that regulation of TNF-alpha expression may have occurred mainly by LITAF activation rather than NF-kB in disk abalone. The cumulative data from other molluscs and our data with reference to TNF-alpha, LITAF and Rel/NF-kB from disk abalone provide strong evidence that LITAF and NF-kB are independent pathways likely to occur throughout the Phylum mollusca.

Allograft inflammatory factor-1 in disk abalone (Haliotis discus discus): molecular cloning, transcriptional regulation against immune challenge and tissue injury.

Here, we report the identification and characterization of allograft inflammatory factor-1 (AIF-1) from disk abalone Haliotis discus discus that was denoted as AbAIF-1. The full-length cDNA of AbAIF-1 consists of a coding region (453 bp) for 151 amino acids with a 17 kDa molecular mass. Analysis of AbAIF-1 sequence showed that it shares characteristic two EF hand Ca(+2)-binding motifs. Results from phylogenetic analysis further confirm that AbAIF-1 is a member of the AIF-1 family similar to invertebrate and vertebrate counterparts suggesting it has high evolutional conservation. Tissue-specific expression and transcriptional regulation of AbAIF-1 were analyzed after bacteria (Vibrio alginolyticus, Vibrio parahemolyticus and Lysteria monocytogenes), viral hemorrhagic septicemia virus (VHSV) immune challenge and during tissue injury by quantitative real-time PCR. It is shown that the expression of AbAIF-1 mRNA was expressed ubiquitously in all selected tissues in constitutive manner showing the highest level in hemocytes. Upon bacteria and VHSV challenge, AbAIF-1 showed the significant up-regulation in hemocytes than gills. After the tissue injury in shell and mantle, AbAIF-1 and antioxidant selenium-dependant glutathione peroxidase (SeGPx) transcripts were significantly upregulated in abalone hemocytes. Taken together, these findings suggest that AIF-1 could response against the pathogenic challenge or tissue injury in abalone like mollusks. Also, AbAIF-1 may involve in wound healing and shell repair after the tissue injury of abalone.

Transcriptome of American Oysters, Crassostrea virginica, in Response to Bacterial Challenge: Insights into Potential Mechanisms of Disease Resistance

The American oyster Crassostrea virginica, an ecologically and economically important estuarine organism, can suffer high mortalities in areas in the Northeast United States due to Roseovarius Oyster Disease (ROD), caused by the gram-negative bacterial pathogen Roseovarius crassostreae. The goals of this research were to provide insights into: 1) the responses of American oysters to R. crassostreae, and 2) potential mechanisms of resistance or susceptibility to ROD. The responses of oysters to bacterial challenge were characterized by exposing oysters from ROD-resistant and susceptible families to R. crassostreae, followed by high-throughput sequencing of cDNA samples from various timepoints after disease challenge. Sequence data was assembled into a reference transcriptome and analyzed through differential gene expression and functional enrichment to uncover genes and processes potentially involved in responses to ROD in the American oyster. While susceptible oysters experienced constant levels of mortality when challenged with R. crassostreae, resistant oysters showed levels of mortality similar to non-challenged oysters. Oysters exposed to R. crassostreae showed differential expression of transcripts involved in immune recognition, signaling, protease inhibition, detoxification, and apoptosis. Transcripts involved in metabolism were enriched in susceptible oysters, suggesting that bacterial infection places a large metabolic demand on these oysters. Transcripts differentially expressed in resistant oysters in response to infection included the immune modulators IL-17 and arginase, as well as several genes involved in extracellular matrix remodeling. The identification of potential genes and processes responsible for defense against R. crassostreae in the American oyster provides insights into potential mechanisms of disease resistance.

Molecular characterization and expression analysis of Cathepsin B and L cysteine proteases from rock bream (Oplegnathus fasciatus).

Cathepsins are lysosomal cysteine proteases of the papain family that play an important role in intracellular protein degradation and turn over within the lysosomal system. In the present study, full-length sequences of cathepsin B (RbCathepsin B) and L (RbCathepsin L) were identified after transcriptome sequencing of rock bream Oplegnathus fasciatus mixed tissue cDNA. Cathepsin B was composed of 330 amino acid residues with 36 kDa predicted molecular mass. RbCathepsin L contained 336 amino acid residues encoding for a 38 kDa predicted molecular mass protein. The sequencing analysis results showed that both cathepsin B and L contain the characteristic papain family cysteine protease signature and active sites for the eukaryotic thiol proteases of cysteine, asparagine and histidine. In addition, RbCathepsin L contained EF hand Ca(2+) binding and cathepsin propeptide inhibitor domains. The rock bream cathepsin B and L showed the highest amino acid identity of 90 and 95% to Lutjanus argentimaculatus cathepsin B and Lates calcarifer cathepsin L, respectively. By phylogenetic analysis, cathepsin B and L exhibited a high degree of evolutionary relationship to respective cathepsin family members of the papain superfamily. Quantitative real-time RT-PCR analysis results confirmed that the expression of cathepsin B and L genes was constitutive in all examined tissues isolated from un-induced rock bream. Moreover, activation of RbCathepsin B and L mRNA was observed in both lipopolysaccharide (LPS) and Edwardsiella tarda challenged liver and blood cells, indicating a role of immune response in rock bream.

Abhisin: A potential antimicrobial peptide derived from histone H2A of disk abalone (Haliotis discus discus)

Antimicrobial peptides (AMPs) play an important role in the immune defense against pathogenic microorganisms. In this study, a histone H2A full-length cDNA was cloned from disk abalone Haliotis discus discus. We identified a 40-amino acid AMP designated as abhisin from the N-terminus of the abalone histone H2A sequence. Abhisin shows the characteristic features of AMPs including net positive charge (+13), higher hydrophobic residues (27%) and 2.82 kcal/mol protein binding potential. Abhisin shares 80% amino acid identity with the buforin I peptide that is derived from Asian toad histone H2A. We synthesized the synthetic peptide of abhisin, and characterized its antimicrobial activities. Our results showed the growth inhibition of Gram positive (G+) Listeria monocytogenes, Gram negative (G-) Vibrio ichthyoenteri bacteria, and fungi (yeast) Pityrosporum ovale by abhisin treatment at 250 microg/mL. However, stronger activity was displayed against the G+ than G- bacteria. Additionally, scanning electron microscope (SEM) observation results confirmed that P. ovale cells were damaged by abhisin treatment. Interestingly, abhisin treatment (50 microg/mL) decreased the viability of THP-1 leukemia cancer cells approximately by 25% but there was no effect on the normal vero cells, suggesting that abhisin has cytotoxicity against cancer cells but not normal cells. Quantitative real time RT-PCR results revealed that histone H2A transcription was significantly induced at 3 h post-infection with bacteria in abalone gills and digestive tract. These results suggest that abhisin is a potential antimicrobial agent, and its precursor histone H2A may be involved in the innate immune defense system in abalone.

Molecular cloning, characterization and expression analysis of peroxiredoxin 6 from disk abalone Haliotis discus discus and the antioxidant activity of its recombinant protein

Peroxiredoxins (Prxs) play an important role against various oxidative stresses and intra-cellular signal transduction. Peroxiredoxin 6 (PrxVI) was identified from the disk abalone Haliotis discus discus cDNA library and named HdPrxVI. The full length cDNA of HdPrxVI was 1457 bp with a 654 bp open reading frame (ORF) encoding 218 amino acids. The predicted molecular mass and estimated isoelectric point (pI) of HdPrxVI were 24 kDa and 7.3, respectively. The deduced amino acid sequence demonstrated the greatest degree (72.4%) of identity with Crassostrea gigas PrxVI. The conserved peroxidase catalytic center (42PVCTTE47) with a conserved cysteine residue (Cys44) and a catalytic center for PLA2 activity (27GGSWA31) were observed in the sequence, indicating that it is a member of 1-Cys Prx. Real time PCR results revealed that HdPrxVI mRNA is constitutively expressed in all tissues in a tissue-specific manner. During exposure to haemorrhagic septicaemia virus (VHSV), HdPrxVI mRNA transcription was down-regulated in the gill, suggesting that the abalone responded to the viral infection quickly, and HdPrxVI played a physiological role against virus-induced oxidative stress. The purified recombinant HdPrxVI, together with dithiothreitol (DTT), was shown to scavenge H2O2 in human leukemia THP-1 cells and provided protection against H2O2-induced apoptosis.

A novel Fas ligand in mollusk abalone: Molecular characterization, immune responses and biological activity of the recombinant protein

Fas ligand is a member of the TNF superfamily that plays an important role by inducing apoptosis and homeostasis of immune responses. The gene encoding Fas ligand was isolated from a disk abalone (Haliotis discus discus) cDNA library, denoted as the AbFas ligand. It contains an 1832bp transcript with a 945bp open reading frame, encoding 315 amino acids. The AbFas ligand showed characteristic transmembrane and TNF family signature domains. The deduced amino acid comparison showed that the AbFas ligand exhibits 22.0, 16.1 and 14.5% identities to human Fas ligand, TNF-alpha, and lymphotoxin (LT-alpha), respectively. Phylogenetic analysis indicates that the AbFas ligand belongs to the invertebrate TNF family and it is closely related to vertebrate Fas ligand counterparts. Quantitative real-time PCR analysis results showed that the AbFas ligand transcripts were constitutively expressed in abalone hemocytes, gills, mantle, muscle, digestive tract and digestive gland in a tissue-specific manner. By immune stimulation, AbFas ligand mRNA was significantly (p<0.05) up-regulated after infection with a mixture of bacteria (Vibrio alginolyticus, Vibrio parahemolyticus, and Listeria monocytogenes), viral haemorrhagic septicaemia virus (VHSV), and lipopolysaccharide (LPS) in abalone gills. The recombinant AbFas ligand was over-expressed in Escherichia coli (E. coli) and purified using a pMAL protein fusion system. This recombinant AbFas ligand showed its biological activity by inducing both superoxide anion (O(2-) and H(2)O(2) in human THP-1 cells in concentration-dependant manner. Correlating the AbFas ligand transcriptional up-regulation against bacteria, virus and LPS with the biological activity of its recombinant protein, we could suggest that the abalone Fas ligand may control microbial infection by inducing O(2-), H(2)O(2) and other ROS.

Molecular characterization, gene expression analysis and biochemical properties of α-amylase from the disk abalone, Haliotis discus discus.

The present study reports the molecular characterization, cloning, expression, and biochemical characterization of alpha-amylase identified from the disk abalone, Haliotis discus discus cDNA library. The full length of the alpha-amylase cDNA was 1650 bp, and it encoded a polypeptide of 511 amino acids. The predicted HdAmyI molecular mass of mature protein was 54 kDa and the estimated isoelectric point (pI) was 8.3. The alpha-amylase gene showed its characteristic motifs, catalytic sites, substrate binding sites and conserved regions with other known species of alpha-amylases. Purified recombinant HdAmyI exhibited a relatively low activity of 0.1 U/mg protein towards 1% starch. HdAmyI had an optimum temperature and pH of 50 degrees C and 6.5, respectively. It also demonstrated stability in a wide range of temperatures and pH. Tissue-specific mRNA expression results showed that HdAmyI is expressed only in the digestive tract and hepatopancreas, with the highest levels in the hepatopancreas. Over 8 weeks of starvation, alpha-amylase transcription was decreased significantly relative to basal levels. However, after starvation, mRNA transcription was increased and returned to normal level by the 2nd week of feeding, suggesting that the alpha-amylase mRNA expression changes according to variations in food availability at the transcriptional level in disk abalone.

Bioactive secondary metabolites from marine microbes for drug discovery.

The isolation and extraction of novel bioactive secondary metabolites from marine microorganisms have a biomedical potential for future drug discovery as the oceans cover 70% of the planets surface and life on earth originates from sea. Wide range of novel bioactive secondary metabolites exhibiting pharmacodynamic properties has been isolated from marine microorganisms and many to be discovered. The compounds isolated from marine organisms (macro and micro) are important in their natural form and also as templates for synthetic modifications for the treatments for variety of deadly to minor diseases. Many technical issues are yet to overcome before wide-scale bioprospecting of marine microorganisms becomes a reality. This chapter focuses on some novel secondary metabolites having antitumor, antivirus, enzyme inhibitor, and other bioactive properties identified and isolated from marine microorganisms including bacteria, actinomycetes, fungi, and cyanobacteria, which could serve as potentials for drug discovery after their clinical trials.

Molecular cloning, characterization and enzymatic properties of a novel βeta-agarase from a marine isolate Psudoalteromonas sp. AG52

An agar-degrading Pseudoalteromonas sp. AG52 bacterial strain was identified from the red seaweed Gelidium amansii collected from Jeju Island, Korea. A β-agarase gene which has 96.8% nucleotide identity to Aeromonas β-agarase was cloned from this strain, and was designated as agaA. The coding region is 870 bp, encoding 290 amino acids and possesses characteristic features of the glycoside hydrolase family (GHF)-16. The predicted molecular mass of the mature protein was 32 kDa. The recombinant β-agarase (rAgaA) was overexpressed in Escherichia coli and purified as a fusion protein. The optimal temperature and pH for activity were 55 °C and 5.5, respectively. The enzyme had a specific activity of 105.1 and 79.5 unit/mg toward agar and agarose, respectively. The pattern of agar hydrolysis demonstrated that the enzyme is an endo-type β-agarase, producing neoagarohexaose and neoagarotetraose as the final main products. Since, Pseudoalteromonas sp. AG52 encodes an agaA gene, which has greater identity to Aeromonas β-agarase, the enzyme could be considered as novel, with its unique bio chemical characteristics. Altogether, the purified rAgaA has potential for use in industrial applications such as development of cosmetics and pharmaceuticals.