Jung-Kil Seo

Antimicrobial function of SHβAP, a novel hemoglobin β chain-related antimicrobial peptide, isolated from the liver of skipjack tuna, Katsuwonus pelamis

A 2.3xa0kDa of antimicrobial peptide was purified from an acidified liver extract of skipjack tuna, Katsuwonus pelamis, by preparative acid-urea-polyacrylamide gel electrophoresis and C18 reversed-phase HPLC. A comparison of the amino acid sequence of the purified peptide with those of other known polypeptides revealed high homology with the C-terminus of hemoglobin β-chain; thus, this peptide was designated as the Skipjack Hemoglobin β chain-related Antimicrobial Peptide (SHβAP). SHβAP showed potent antimicrobial activity against Gram-positive bacteria, such as Bacillus subtilis, Staphylococcus aureus, and Streptococcus iniae (minimal effective concentrations [MECs], 6.5-57.0xa0μg/mL), Gram-negative bacteria, such as Escherichia coli D31, Pseudomonas aeruginosa, Salmonella enterica, Shigella sonnei, and two Vibrio parahaemolyticus species (MECs, 2.0-19.0xa0μg/mL), and against Candida albicans (MEC; 12.0xa0μg/mL) without significant hemolytic activity. Antimicrobial activity of this peptide was heatstable and pH resistant but is sensitive to proteases and salt. SHβAP did not show membrane permeabilization and killing ability. The secondary structural prediction and the homology modeling expected that this peptide formed an amphipathic α-helical structure. This is the first report the purification of a novel antimicrobial peptide related to the C-terminus of hemoglobin β-chain from marine fish.

Antimicrobial function of the GAPDH-related antimicrobial peptide in the skin of skipjack tuna, Katsuwonus pelamis

A 3.4xa0kDa of antimicrobial peptide was purified from an acidified skin extract of skipjack tuna, Katsuwonus pelamis, by preparative acid-urea-polyacrylamide gel electrophoresis and C18 reversed-phase HPLC. A comparison of the N-terminal amino acid sequence of the purified peptide with that of other known polypeptides revealed high sequence homology with the YFGAP (Yellowfin tuna Glyceraldehyde-3-phosphate dehydrogenase-related Antimicrobial Peptide); thus, this peptide was identified as the skipjack tuna GAPDH-related antimicrobial peptide (SJGAP). SJGAP showed potent antimicrobial activity against Gram-positive bacteria, such as Bacillus subtilis, Micrococcus luteus, Staphylococcus aureus, and Streptococcus iniae (minimal effective concentrations [MECs], 1.2-17.0xa0μg/mL), Gram-negative bacteria, such as Aeromonas hydrophila, Escherichia coli D31, and Vibrio parahaemolyticus (MECs, 3.1-12.0xa0μg/mL), and against Candida albicans (MEC, 16.0xa0μg/mL) without significant hemolytic activity. Antimicrobial activity of this peptide is heat-stable but salt-sensitive. According to the secondary structural prediction and the homology modeling, this peptide consists of three secondary structural motifs, including one α-helix and two parallel β-strands, and forms an amphipathic structure. This peptide showed neither membrane permeabilization ability nor killing ability, but did display a small degree of leakage ability. These results suggest that SJGAP acts through a bacteriostatic process rather than bactericidal one. SJGAP is another GAPDH-related antimicrobial peptide isolated from skipjack tuna and likely plays an important role for GAPDH in the innate immune defense of tuna fish.

Antimicrobial peptide, hdMolluscidin, purified from the gill of the abalone, Haliotis discus.

A 4.7 kDa antimicrobial peptide was purified from the acidified gill extract of the Abalone, Haliotis discus, by cation-exchange and C18 reversed-phase high performance liquid chromatography (HPLC). Comparison of the amino acid sequences and molecular weight of this peptide with those of other known antimicrobial peptides revealed that this antimicrobial peptide have high sequence homology with that of cgMolluscidin and was designated hdMolluscidin. hdMolluscidin is composed of 46 amino acid residues containing several dibasic residue repeats like KK or K-R. hdMolluscidin showed potent antimicrobial activity against both Gram-positive bacteria including Bacillus subtilis and Staphylococcus aureus (minimal effective concentrations [MECs]; 0.8-19.0 μg/mL) and Gram-negative bacteria including Aeromonas hydrophila, Escherichia coli, Pseudomonas aeruginosa, Salmonella enterica, Shigella flexneri, and Vibrio parahemolyticus ([MECs]; 1.0-4.0 μg/mL) without hemolytic activity. However, hdMolluscidin did not show any significant activity against Candida albicans. The secondary structural prediction suggested that hdMolluscidin might not form an ordered or an amphipathic structure. hdMolluscidin did not show membrane permeabilization or leakage ability. The full-length hdMolluscidin cDNA contained 566-bp, including a 5-untranslated region (UTR) of 63-bp, a 3-UTR of 359-bp, and an open reading frame of 144-bp encoding 47 amino acids (containing Met). cDNA study of hdMolluscidin suggests that it is expressed as a mature peptide. Our results indicate that hdMolluscidin could relate to the innate immune defenses in abalone and it may not act directly on bacterial membrane.

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.

Hemerythrin-related antimicrobial peptide, msHemerycin, purified from the body of the Lugworm, Marphysa sanguinea.

A ∼1.7xa0kDa antimicrobial peptide was purified from the acidified body extract of the Lugworm, Marphysa sanguinea, by preparative acid-urea-polyacrylamide gel electrophoresis and C18 reversed-phase high performance liquid chromatography (HPLC). The identified peptide is composed of 14 amino acids with the N-terminal acetylation. Comparison of the identified amino acid sequences and molecular weight of this peptide with those of other known proteins or peptides revealed that this peptide had high identity to the N-terminus of hemerythrin of marine invertebrates and named the msHemerycin. The full-length hemerythrin cDNA of Lugworm was contained 1027-bp, including a 5-untranslated region (UTR) of 60-bp, a 3-UTR of 595-bp, and an open reading frame of 372-bp encoding 123 amino acids including the msHemerycin at the N-terminus. Tissue distribution of the msHemerycin mRNA suggests that it is constitutively expressed as a non-tissue-specific manner, however, a relatively higher expression level was observed in muscle (6.8-fold) and brain (6.3-fold), and the lowest level in digestive gland. The secondary structural prediction and homology modeling studies indicate that the msHemerycin might form an unordered structure and might act via unconventional mechanism. Our results suggest that the msHemerycin might be an innate immune component related to the host defenses in the Lugworm. This is the first report on the antimicrobial function of the peptide derived from the N-terminus of hemerythrin in the Lugworm, Marphysa sanguinea.

Antimicrobial effect of the 60S ribosomal protein L29 (cgRPL29), purified from the gill of pacific oyster, Crassostrea gigas

Abstract We purified an ˜6.4‐kDa antimicrobial peptide from an acidified gill extract of the Pacific oyster, Crassostrea gigas, by cation‐exchange and C18 reversed‐phase high performance liquid chromatography (HPLC). The identified peptide was composed of 54 amino acids and had a molecular weight of 6484.6 Da. Comparison of the amino acid sequence and molecular weight with those of other known proteins or peptides revealed that the peptide had high identity with the 60S ribosomal protein L29, and so was named cgRPL29. The full‐length cgRPL29 cDNA of the Pacific oyster comprised 325‐bp, including a 5’‐untranslated region (UTR) of 100‐bp, a 3′‐UTR of 57‐bp, and an open reading frame of 168‐bp encoding 55 amino acids, with a Met residue at the N‐terminus. The cgRPL29 mRNA tissue distribution suggested that it is constitutively expressed in a non‐tissue‐specific manner. Secondary structural prediction and homology modeling indicated cgRPL29 have an unordered structure containing two partial &agr;‐helical regions. This is to our knowledge the first report of the antimicrobial effect of the 60S ribosomal protein L29 from marine invertebrates. HighlightsAntimicrobial effect of 60S ribosomal protein L29 (cgRPL29) was firstly identified from the Pacific oyster, crassostrea gigas.cgRPL29 consists of 54 amino acids with 6484.6 Da and showed strong antimicrobial activity against bacteria.cgRPL29 was constitutively expressed and might form unordered/non‐amphipathic conformation.cgRPL29 might contribute to innate defense function in the Pacific oyster, crassostrea gigas.

Evidence for the presence of cell-surface-bound and intracellular bactericidal toxins in the dinoflagellate Heterocapsa circularisquama

Heterocapsa circularisquama, a harmful dinoflagellate, has multiple haemolytic toxins that are considered to be involved in the toxic mechanism against shellfish and certain species of zooplankton. To evaluate the further nature of the toxins of H. circularisquama, we investigated its effects on several species of bacteria. By colony formation assay, we found that H. circularisquama had antibacterial activity toward the marine bacterium Vibrio alginolyticus in a cell density-dependent manner. When the inoculated bacterial cells were co-cultured with H. circularisquama under dinoflagellate cell culture conditions, the bacterial growth was significantly suppressed, whereas the number of live bacterial cells increased when cultured in the medium alone. Since the cell-free culture supernatant and the ruptured dinoflagellate cell suspension showed no toxic effects on V. alginolyticus, it is speculated that direct cell-to-cell contact mediated by the live dinoflagellate cells may be the major toxic mechanism. The decrease in bactericidal activity of theca-removed dinoflagellate cells may further support this speculation. H. circularisquama also showed bactericidal activities towards Escherichia coli and Staphylococcus aureus. In the dinoflagellate/bacteria co-culture system, the number of live bacterial cells declined with increasing incubation time. Light-dependent antibacterial activity of the ruptured dinoflagellate cells against S. aureus was observed, whereas no such activity was detected against E. coli. These results suggest that intracellular photosensitising bactericidal toxins, which were previously found to be porphyrin derivatives, may have specificity towards gram-positive bacteria. Based on these results together with previous studies, it is obvious that H. circularisquama possesses antibacterial activity, which may be mediated through toxins located on its cell surface. It is likely that such toxins play a role in the defence mechanism against predators and infectious bacteria. Although the exact biological significance of intracellular photosensitising toxins is still unclear, such toxins may have potential to be developed as novel photo-controllable 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.

Purification and cDNA cloning of the antimicrobial peptide apMolluscidin from the pen shell, Atrina pectinata

&NA; A 5.6 kDa antimicrobial peptide (AMP) was purified from acidified gill extract of the pen shell, Atrina pectinata, by cation exchange and C18 reversed‐phase high performance liquid chromatography. Comparison of the amino acid sequences and molecular weight of this peptide with those of other known AMPs revealed that it had high sequence homology with that of cgMolluscidin or hdMolluscidin; it was designated apMolluscidin. apMolluscidin comprises 59 amino acid residues containing several dibasic residue repeats and sequence repeats such as Lys‐Lys and Lys‐Gly. apMolluscidin exhibited potent antimicrobial activity against both Gram‐positive bacteria including Bacillus subtilis (minimal effective concentration [MEC], 2.1 &mgr;g/mL), and Gram‐negative bacteria including E. coli D31 (MEC, 0.5 &mgr;g/mL), without hemolytic activity. However, it did not show any activity against fungi such as Candida albicans. Secondary structure prediction suggested that it might form two helical regions and have an amphipathic structure. Full‐length apMolluscidin cDNA contained 812 base pairs (bp), including a 5′‐untranslated region (UTR) of 82 bp, a 3′‐UTR of 547 bp, and a coding sequence of 183 bp encoding 60 amino acids (containing Met). Furthermore, qPCR analyses revealed that the mature peptide translated from apMolluscidin mRNA is expressed in a tissue‐specific manner in locations such as the gill and siphon. These results indicate that apMolluscidin might be related to the innate immune defense system of abalone and may not act directly on the bacterial membrane. This is the first report of an AMP from the pen shell with a fully identified amino acid sequence. HighlightsA novel antimicrobial peptide was purified from the pen shell, Atrina pectinata.This was named apMolluscidin because it showed a high sequence homology with cgMolluscidin or hdMolluscidin.We found that apMolluscidin had broad‐spectrum antibacterial activity and low hemolytic activity.Full‐length apMolluscidin cDNA was cloned and its mRNA expression in tissues was evaluated with qPCR.

Identification of a novel antimicrobial peptide from the sea star Patiria pectinifera

Abstract Antimicrobial peptides (AMPs) are components of innate immunity found in many forms of life. However, there have been no reports of AMPs in sea star (Phylum Echinodermata). Here we report the isolation and characterization of a novel antimicrobial peptide from the coelomic epithelium extract of the sea star Patiria pectinifera. The isolated peptide comprises 38 amino acid residues, is cationic (pI 9.2), has four cysteine residues that form two disulfide bonds (C1‐C3 and C2‐C4), is amidated at the C‐terminus, and is designated P. pectinifera cysteine‐rich antimicrobial peptide (PpCrAMP). Synthetic PpCrAMP identical to the native peptide exhibited the most potent antimicrobial activity compared to analogs with different disulfide bond configurations. Expression analysis of PpCrAMP precursor transcripts revealed constitutive expression in the coelomic epithelium and tube feet of P. pectinifera. Analysis of genomic DNA and cDNA encoding the PpCrAMP precursor protein revealed that an intron splits the coding region of the mature peptide into a positively charged N‐terminal domain and a C‐terminal domain harboring four cysteine residues and a glycine for C‐terminal amidation. No significant homology with other known AMPs was observed, while orthologs of PpCrAMP were found in other echinoderm species. These findings indicate that PpCrAMP is the prototype of a family a novel cysteine‐rich AMPs that participate in mechanisms of innate immunity in echinoderms. Furthermore, the discovery of PpCrAMP may lead to the identification of related AMPs in vertebrates and protostome invertebrates. HighlightsA novel cysteine‐rich AMP (PpCrAMP) is identified from the starfish Patiria pectinifera.PpCrAMP adopts two disulfide bonds with Cys16‐C27 and Cys25‐Cys38 pairings.PpCrAMP transcripts are highly expressed in the tube feet and the coelomic epithelium.PpCrAMP gene contains an intron.PpCrAMP exhibits antimicrobial activity to different bacteria.