Antimicrobial Activity and Action Mechanisms of Arg-Rich Short Analog Peptides Designed from the C-Terminal Loop Region of American Oyster Defensin (AOD).

American oyster defensin (AOD) was previously purified from acidified gill extract of the American oyster, Crassostrea virginica. AOD is composed of 38 amino acids with three disulfide bonds and exhibits strong antimicrobial activity against Gram-positive bacteria as well as significant activity against Gram-negative bacteria. Here, to develop promising peptides into antibiotic candidates, we designed five arginine-rich analogs (A0, A1, A2, A3, and A4), predicted their loop and extended strand/random structures-including nine amino acids and a disulfide bond derived from the C-terminus of AOD-and described their antimicrobial and cytotoxic effects, as well as their modes of action. In our experimental results, the A3 and A4 analogs exhibited potent antimicrobial activity against all test organisms-including four Gram-positive bacteria, six Gram-negative bacteria, and Candida albicans-without cell toxicity. A sequence of experiments, including a membrane permeabilization assay, DNA binding study, and DNA polymerization inhibition test, indicated that the two analogs (A3 and A4) possibly did not act directly on the bacterial membrane but instead interacted with intracellular components such as DNA or DNA amplification reactions. AOD analogs also showed strong bacterial inhibition activity in the plasma environment. In addition, analog-treated microbial cells clearly exhibited membrane disruption, damage, and leakage of cytoplasmic contents. Collectively, our results suggest that two analogs, A3 and A4, have potent antimicrobial activity via DNA interaction and have the potential for development into novel antimicrobial agents.

Two metalloenzymes from rockfish (Sebastes schligellii): Deciphering their potential involvement in redox homeostasis against oxidative stress.

Disturbance in the balance between pro-oxidants and anti-oxidants result oxidative stress in aerobic organisms. However, oxidative stress can be inhibited by enzymatic and non-enzymatic defense mechanisms. Superoxide dismutases (SODs) are well-known scavengers of superoxide radicals, and they protect cells by detoxifying hazardous reactive oxygen species. Here, we have identified and characterized two different SODs, CuZnSOD and MnSOD, from black rockfish (RfCuZnSOD and RfMnSOD, respectively). In silico analysis revealed the well-conserved molecular structures comprising all essential properties of CuZnSOD and MnSOD. Phylogenetic analysis revealed that both RfCuZnSOD and RfMnSOD cladded with their fish counterparts. The recombinant RfSOD proteins demonstrated their potential superoxide scavenging abilities through a xanthine oxidase assay. The optimum temperature and pH conditions for both rRfSODs were 25 °C and pH 8, respectively. Moreover, the potential peroxidation function of rRfCuZnSOD was observed in the presence of HCO3-. The highest peroxidation activity was observed at 100 µg/mL of rRfCuZnSOD using the MTT cell viability assay and flow cytometry. The analogous tissue-specific expression profile indicated ubiquitous expression of both RfCuZnSOD and RfMnSOD in selected tissues of healthy juvenile rockfish. An immune challenge experiment illustrated the altered expression profiles of both RfCuZnSOD and RfMnSOD against lipopolysaccharide, Streptococcus iniae, and polyinosinic-polycytidylic acid (poly I:C). Collectively, these results strengthen the general understanding of the structural and functional characteristics of SODs within the host defense system.

A teleostan homolog of catalase from black rockfish (Sebastes schlegelii): insights into functional roles in host antioxidant defense and expressional responses to septic conditions.

Antioxidative defense renders a significant protection against environmental stress in organisms and maintains the correct redox balance in cells, thereby supporting proper immune function. Catalase is an indispensable antioxidant in organisms that detoxifies hydrogen peroxides produced in cellular environments. In this study, we sought to molecularly characterize a homolog of catalase (RfCat), identified from black rockfish (Sebastes schlegelii). RfCat consists of a 1581 bp coding region for a protein of 527 amino acids, with a predicted molecular weight of 60 kD. The protein sequence of RfCat harbored similar domain architecture to known catalases, containing a proximal active site signature and proximal heme ligand signature, and further sharing prominent homology with its teleostan counterparts. As affirmed by multiple sequence alignments, most of the functionally important residues were well conserved in RfCat. Furthermore, our phylogenetic analysis indicates its common vertebrate ancestral origin and a close evolutionary relationship with teleostan catalases. Recombinantly expressed RfCat demonstrated prominent peroxidase activity that varied with different substrate and protein concentrations, and protected against DNA damage. RfCat mRNA was ubiquitously expressed among different tissues examined, as detected by qPCR. In addition, RfCat mRNA expression was modulated in response to pathogenic stress elicited by Streptococcus iniae and poly I:C in blood and spleen tissues. Collectively, our findings indicate that RfCat may play an indispensable role in host response to oxidative stress and maintain a correct redox balance after a pathogen invasion.

Gene cloning and catalytic characterization of cold-adapted lipase of Photobacterium sp. MA1-3 isolated from blood clam.

A lipase-producing Photobacterium strain (MA1-3) was isolated from the intestine of a blood clam caught at Namhae, Korea. The lipase gene was cloned by shotgun cloning and encoded 340 amino acids with a molecular mass of 38,015 Da. It had a very low sequence identity with other bacterial lipases, with the exception of that of Photobacterium lipolyticum M37 (83.2%). The MA1-3 lipase was produced in soluble form when Escherichia coli cells harboring the gene were cultured at 18°C. Its optimum temperature and pH were 45°C and pH 8.5, respectively. Its activation energy was calculated to be 2.69 kcal/mol, suggesting it to be a cold-adapted lipase. Its optimum temperature, temperature stability, and substrate specificity were quite different from those of M37 lipase, despite the considerable sequence similarities. Meanwhile, MA1-3 lipase performed a transesterification reaction using olive oil and various alcohols including methanol, ethanol, 1-propanol, and 1-butanol. In the presence of t-butanol as a co-solvent, this lipase produced biodiesel using methanol and plant or waste oils. The highest biodiesel conversion yield (73%) was achieved using waste soybean oil and methanol at a molar ratio of 1:5 after 12 h using 5 units of lipase.

Identification and molecular characterization of two hepcidin genes from black rockfish (Sebastes schlegelii).

Antimicrobial peptides (AMPs) are important mediators of the immune response against bacteria and cysteine-rich peptide hepcidin is a 20-26 residues member with functions in iron regulation and antimicrobial activity. Here, we have identified two different types of hepcidin cDNA from the Black rockfish, Sebastes schlegelii, by EST analysis. Both hepcidin genes (hepcidin I and II) consist of two introns and three exons that encode a prepropeptide (88 amino acids). A TATA box and several consensus-binding motifs for transcription factors were found in the upstream of the transcriptional start site. Semi-quantitative RT-PCR analysis suggested that hepcidin I transcripts were detected in various tissues, while hepcidin II was only expressed in the liver. During the bacterial challenge with the fish pathogen, Streptococcus iniae, two hepcidin genes were differentially expressed. Hepcidin I and II dramatically increased at 24 h post-injection, then gradually declined at 3 days in hepcidin II, while hepcidin I expression continued at 3 days after challenge.

Molecular cloning and expression analysis of two hepcidin genes from olive flounder Paralichthys olivaceus.

Hepcidin is a cysteine-rich cationic antimicrobial peptide central to iron metabolism. We report a comparative analysis of the sequences, gene organization and expression of two hepcidin genes from olive flounder Paralichthys olivaceus. Both consist of two introns and three exons that encode a prepropeptide (81 amino acids for hepcidin I and 89 amino acids for hepcidin II). A TATA box and several consensus-binding motifs for transcription factors were found upstream of the transcriptional starting site. Hepcidin II was predominantly expressed in the liver and highly inducible under the effect of lipopolysaccharide (LPS), while a large amount of hepcidin I transcripts was detected in various tissues but did not appear to have a significant effect during LPS-stimulation.