Min Jeong Lee

cgMolluscidin, a novel dibasic residue repeat rich antimicrobial peptide, purified from the gill of the Pacific oyster, Crassostrea gigas

A 5.5 kDa antimicrobial peptide consisting of 55 amino acids, cgMolluscidin, was purified from the acidified gill extract of the Pacific oyster, Crassostrea gigas, by ion-exchange and C18 reversed-phase high performance liquid chromatography. By comparing the N-terminal amino acid sequences and the molecular weight of this peptide with those of other known antimicrobial peptides, it has been revealed that this peptide had no homology with any known peptides. cgMolluscidin showed potent antimicrobial activity against both Gram-positive bacteria, including Bacillus subtilis, Micrococcus luteus, and Staphylococcus aureus (minimal effective concentrations [MECs]; 1.3-31.3 μg/mL), and Gram-negative bacteria, including Escherichia coli, Salmonella enterica, and Vibrio parahaemolyticus ([MECs]; 0.4-2.3 μg/mL), without hemolytic activity. However, cgMolluscidin did not show any significant activity against Candida albicans. The deduced amino acid sequence of the cgMolluscidin showed no hit in public protein databases, while the nucleotide sequence had a 99% homology (E value = 0) with only the unknown ESTs sequenced by C. gigas EST project. Tissue distribution of the cgMolluscidin mRNA suggests that it is constitutively expressed as a mature form in a non-tissue-specific manner. The cgMolluscidin mRNA expression level was significantly up-regulated at 12 h (2.8-fold) post injection with Vibrio sp. This peptide is highly basic and contains several dibasic residue repeats including Lysine-Lysine or Lysine-Arginine in the sequence, but may not form an ordered structure. These results suggest that cgMolluscidin might be an oyster-specific novel antimicrobial peptide.

Purification and antimicrobial function of ubiquitin isolated from the gill of Pacific oyster, Crassostrea gigas

An antimicrobial polypeptide was purified from an acidified gill extract of Pacific oyster (Crassostrea gigas) by C(18) reversed-phase HPLC. The purified polypeptide had a molecular weight of 8471Da containing 74 amino acid residues. Comparison of the obtained N-terminal sequences with those of others revealed that it was identical to ubiquitin reported from other species and named cgUbiquitin. cgUbiquitin showed broad potent antimicrobial activity against Gram-positive and -negative bacteria including Streptococcus iniae and Vibrio parahemolyticus (minimal effective concentrations, 7.8 and 9.8μg/mL), respectively, without hemolytic activity. The cgUbiquitin cDNA was identified from an expressed sequence tag (EST) library of oyster gill as a precursor form, encoding ubiquitin consisting of 76 amino acids fused to ribosomal protein of S27. Although the cgUbiquitin precursor mRNA was expressed at the intermediate level in the gill, the mRNA was significantly up-regulated at 48h post injection with Vibrio sp. Analysis of the cgUbiquitin C-terminus by carboxypeptidase B treatment and comparison of the retention times revealed that cgUbiquitin lacks the terminal Gly-Gly doublet and ends in an C-terminal Arg residue which might be related to antimicrobial activity. Study of the kinetics of killing and membrane permeabilization showed that this peptide was not membrane permeable and acted through a bacteriostatic process. According to the homology modeling, this peptide is composed of three secondary structural motifs including three α-helices and four β-strands separated by 7 loops regions. Our results indicate that cgUbiquitin might be related to the innate immune defenses in the Pacific oyster and this is the first report for antimicrobial function of ubiquitin isolated from any oyster species.

Functional analysis of Pacific oyster (Crassostrea gigas) β-thymosin: Focus on antimicrobial activity

An antimicrobial peptide, ∼5 kDa in size, was isolated and purified in its active form from the mantle of the Pacific oyster Crassostrea gigas by C18 reversed-phase high-performance liquid chromatography. Matrix-assisted laser desorption ionisation time-of-flight analysis revealed 4656.4 Da of the purified and unreduced peptide. A comparison of the N-terminal amino acid sequence of oyster antimicrobial peptide with deduced amino acid sequences in our local expressed sequence tag (EST) database of C. gigas (unpublished data) revealed that the oyster antimicrobial peptide sequence entirely matched the deduced amino acid sequence of an EST clone (HM-8_A04), which was highly homologous with the β-thymosin of other species. The cDNA possessed a 126-bp open reading frame that encoded a protein of 41 amino acids. To confirm the antimicrobial activity of C. gigas β-thymosin, we overexpressed a recombinant β-thymosin (rcgTβ) using a pET22 expression plasmid in an Escherichia coli system. The antimicrobial activity of rcgTβ was evaluated and demonstrated using a bacterial growth inhibition test in both liquid and solid cultures.

Synthesis and Biological Activities of Myomodulin E and its Analogs

Previous work has characterized myomodulin A (MMA, PMSMLRLamide) and myomodulin E (MME, GLQMLRLamide) purified from the central nervous systems of the sea hare, Aplysia Kurodai, using the anterior byssus retractor muscle (ABRM) of the mussel, Mytilus edulis. The amino acid sequences of MMA and MME were the same as those of the myomodulin family peptide found in other mollusks. In this study, we synthesized MME, its derivatives, and other neuropeptides to investigate the relationship between the structure and biological activity of MME. The primary structures of MME’s derivatives, Des[Gly¹]-MME, Des[Gly¹,Leu²]-MME, and Des[Gly¹,Leu²,Gln³]-MME, were LQMLRLamide, QMLRLamide, and MLRLamide, respectively. MMA and synthetic peptides were tested on ABRM in M. edulis as well as muscle preparations in Achatina fulica. MME displayed an inhibitory effect on phasic contraction of the ABRM at 1×10 -9 M or higher. MME also had a relaxing effect on the catch-tension of AMRM at 1×10 -8 M. Both MMA and its analogs stimulated a contractile response on the crop and relaxed the catch-relaxing response on the penial retractor muscle of A. fulica. These results suggest that MME and its analogs have modulatory effects on various muscles of mollusks. This study has also laid the groundwork for future neural and circuit modulation studies during animal behavioral changes.

Antibacterial Activity of Bacteria Isolated from Rocks on the Seashore

There is a great deal of research interest regarding substitutes for antibiotics because of various obstacles to the efficacy and use of antibiotics. We isolated and analyzed diversity of microbiota which exhibited antibacterial activity against 23 pathogenic bacteria, to develop alternative agent of antibiotics. By investigating the microbiota from rocks on the seashore, we characterized and obtained various antibacterial material-producing bacteria. Thirty-one isolates belong to four genera and seven species, according to 16S rDNA sequence analysis, showed antibacterial activities against 23 pathogenic bacteria. The Identity of 16S rDNA sequences indicated three species of Bacillus, one species of Paenibacillus, one species of Pseudomonas and two species of Enterobacter. Two isolates were similar to Bacillus aerophilus, four isolates were similar to Bacillus pumilus, seven isolates were similar to Bacillus safensis, 15 isolates were similar to Paenibacillus polymyxa, respectively. In addition, one isolate was similar with Pseudomonas poae, one isolate was similar to Enterobacter asburiae, and one isolate was similar to Enterobacter ludwigii, respectively. Variations of antibacterial activity and level among the same species were indicated the diverse strains of isolates. Vibrio vulnificus showed the highest degree of growth inhibition by 29 isolates. Further studies regarding antibacterial materials and bacteria suggest that development of probiotic strains or alternative agents to antibiotics.

Purification and characterization of an antimicrobial peptide mytichitin-chitin binding domain from the hard-shelled mussel, Mytilus coruscus

Abstract An antimicrobial peptide with 55 amino acid residues was purified by C18 reversed‐phase high‐performance liquid chromatography (HPLC) from foot extract of the hard‐shelled mussel, Mytilus coruscus. This peptide showed strong antimicrobial activity against Gram‐positive and Gram‐negative bacteria, as well as fungi. The purified peptide was determined to have a molecular mass of 6202 Da by matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrophotometry (MALDI‐TOF/MS). The identified 20‐amino acid sequence of the purified peak by Edman degradation shared 100% identity with the N‐terminal regions of mytichitin‐1, mytichitin‐2, mytichitin‐3, mytichitin‐4, mytichitin‐5, and chitinase‐like protein‐1, and so was named mytichitin‐CBD. The cDNA of mytichitin‐CBD was cloned and sequenced by rapid amplification of cDNA ends (RACE). The mRNA transcripts were mainly detected in foot tissue, and they were up‐regulated and peaked at 4 h after bacterial infection. We constructed and expressed recombinant mytichitin‐CBD protein which displayed antimicrobial activity against Gram‐negative bacteria Gram‐positive bacteria and the fungus as well as anti‐parasitic activity against scuticociliates. The results of this study demonstrate that the peptide isolated from M. coruscus is related to the innate immune system of this marine invertebrate and is a possible alternative to antibiotics. HighlightsAntimicrobial effect of mytichin‐chitin binding domain (mytichitin‐CBD) was identified from foot extract of the Korean hard‐shelled mussel, Mytilus coruscus.6.2 kDa of the mytichitin‐CBD peptide containing 55 amino acid residues exhibited antibacterial and anti‐parasite activity.Expression levels and patterns were dependent on the tissue type and phase of infection.Mytichitin‐CBD peptide might cooperate in the innate immune defense system of Korean hard‐shelled mussel, Mytilus coruscus.