• The Plant Pathology Journal
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• v.30 no.3
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• pp.245-253
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• 2014

Antimicrobial peptides (AMPs) are small but effective cationic peptides with variable length. In previous study, four hexapeptides were identified that showed antimicrobial activities against various phytopathogenic bacteria. KCM21, the most effective antimicrobial peptide, was selected for further analysis to understand its modes of action by monitoring inhibitory effects of various cations, time-dependent antimicrobial kinetics, and observing cell disruption by electron microscopy. The effects of KCM21 on Gram-negative strain, Pseudomonas syringae pv. tomato DC3000 and Gram-positive strain, Clavibacter michiganensis subsp. michiganensis were compared. Treatment with divalent cations such as $Ca^{2+}$ and $Mg^{2+}$ inhibited the bactericidal activities of KCM21 significantly against P. syringae pv. tomato DC3000. The bactericidal kinetic study showed that KCM21 killed both bacteria rapidly and the process was faster against C. michiganensis subsp. michiganensis. The electron microscopic analysis revealed that KCM21 induced the formation of micelles and blebs on the surface of P. syringae pv. tomato DC3000 cells, while it caused cell rupture against C. michiganensis subsp. michiganensis cells. The outer membrane alteration and higher sensitivity to $Ca^{2+}$ suggest that KCM21 interact with the outer membrane of P. syringae pv. tomato DC3000 cells during the process of killing, but not with C. michiganensis subsp. michiganensis cells that lack outer membrane. Considering that both strains had similar sensitivity to KCM21 in LB medium, outer membrane could not be the main target of KCM21, instead common compartments such as cytoplasmic membrane or internal macromolecules might be a possible target(s) of KCM21.

• Journal of Life Science
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• v.30 no.1
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• pp.64-69
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• 2020

The European honeybee (Apis mellifera L.) has multiple anti-microbial peptides, but many were unknown and demands for their characterization have increased. This study therefore focused on identifying novel anti-microbial peptides (AMPs) from A. mellifera L. To obtain high-throughput transcriptome data of the honeybee, we implemented next-generation sequencing (NGS), isolating novel AMPs from total RNA, and generated 15,314 peptide sequences, including 44 known, using Illumina HiSeq 2500 technology. The uncharacterized peptides were identified based on specific features of possible AMPs predicted in the sequencing analysis. AMP5, one such uncharacterized peptide, was expressed in the epidermis, body fat, and venom gland of the honeybee. We chemically synthesized this peptide and tested its anti-bacterial activity against Gram-negative Escherichia coli (KACC 10005) and Gram-positive Bacillus thuringiensis (KACC 10168) by anti-microbial assay. AMP5 exhibited anti-bacterial activity against E. coli (MIC50=22.04±0.66 μM) but not against B. thuringiensis. When worker bees were injected with E. coli, AMP5 was up-regulated in the body fat. This study therefore identified AMP5 in adult European honeybees and confirmed its anti-bacterial activity against Gram-negative E. coli.

• Journal of Microbiology and Biotechnology
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• v.19 no.8
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• pp.792-802
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• 2009

Antimicrobial peptides (AMPs) are considered to be a promising alternative to conventional antibiotics for future generations. We identified four novel hexapeptides with antimicrobial activity: KCM11 (TWWRWW-$NH_2$), KCM12 (KWRWlW-$NH_2$), KCM21 (KWWWRW-$NH_2$), and KRS22 (WRWFIH-$NH_2$), through positional scanning of a synthetic peptide combinatorial library (PS-SCL). The ability of these peptides to inhibit the growth of a variety of bacteria and unicellular fungi was evaluated. KCM11 and KRS22 preferentially inhibited the normal growth of fungal strains, whereas KCM12 and KCM21 were more active against bacterial strains. Bactericidal activity was addressed in a clear zone assay against phytopathogenic bacteria, including Pectobacterium spp., Xanthomonas spp., Pseudomonas spp., etc. KCM21 showed the highest activity and was effective against a wide range of target organisms. Application of KCM21 with inoculation of Pectobacterium carotovorum subsp. carotovorum on detached cabbage leaves resulted in an immune phenotype or a significant reduction in symptom development, depending on the peptide concentration. Cytotoxicity of the four hexapeptides was evaluated in mouse and human epithelial cell lines using an MTT test. The results revealed a lack of cytotoxic effects.

• Molecules and Cells
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• v.20 no.3
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• pp.409-415
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• 2005

Infection of Drosophila melanogaster adults with 6 Vibrio species revealed that V. cholerae was lethal (100% mortality) within 20 h as a result of systemic infection. Avirulent infection by V. vulnificus restricted the subsequent virulent infection by V. cholerae. The immediate transcription of antimicrobial peptides (AMPs), most notably Attacin A, was delayed in V. cholerae infection compared to V. vulnificus infection. Ectopic expression of Attacin A and Metchnikowin enhanced the survival of D. melanogaster upon V. cholerae infection. These results suggest that AMPs are important in the response to infections by Vibrio species and that the signaling pathways governing their expression may be targeted by V. cholerae virulence factors to elude the innate immunity of Drosophila.

• Journal of Marine Bioscience and Biotechnology
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• v.16 no.1
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• pp.55-62
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• 2024

Various antimicrobial peptides (AMPs) from microalgae have shown antibacterial, antiviral, antifungal, anticancer, and antioxidant effects, and play crucial roles in medical applications, aquaculture-related disease management, and the food industry. Magainin 2 (MAG2), an AMP, exhibits high antibacterial and antitumor activity, necessitating an efficient recombinant expression system for low-cost, large-scale production. To enhance MAG2 secretion efficiency in Chlorella, we constructed the SS:MAG2:His vector using the known Chlamydomonas reinhardtii CA1 signal sequence (SS) and obtained a stable transformant via an Agrobacterium-mediated transformation method and RT-qPCR. ELISA results revealed that the MAG2 content secreted into the medium by the SS:MAG2:His transformants increased proportionally with mRNA expression. These findings offer a strategy for high MAG2 secretion in the Chlorella vulgaris platform, potentially minimizing downstream processing costs.

• Journal of Microbiology and Biotechnology
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• v.30 no.9
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• pp.1305-1309
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• 2020

Insects possess biological defense systems that can effectively combat the invasion of external microorganisms and viruses, thereby supporting their survival in diverse environments. Antimicrobial peptides (AMPs) represent a fast-acting weapon against invading pathogens, including various bacterial or fungal strains. A 37-residue antimicrobial peptide, papiliocin, derived from the swallowtail butterfly Papilio xuthus larvae, showed significant antimicrobial activities against several human pathogenic bacterial and fungal strains. Jelleines, isolated as novel antibacterial peptides from the Royal Jelly (RJ) of bees, exhibit broad-spectrum protection against microbial infections. In this study, we developed a novel antimicrobial peptide, PAJE (RWKIFKKPFKISIHL-NH₂), which is a hybrid peptide prepared by combining 1-7 amino acid residues (RWKIFKK-NH₂) of papiliocin and 1-8 amino acid residues (PFKISIHL-NH₂) of Jelleine-1 to alter length, charge distribution, net charge, volume, amphipaticity, and improve bacterial membrane interactions. This novel peptide exhibited increased hydrophobicity and net positive charge for binding effectively to the negatively charged membrane. PAJE demonstrated antimicrobial activity against both gram-negative and gram-positive bacteria, with very low toxicity to eukaryotic cells and an inexpensive process of synthesis. Collectively, these findings suggest that this novel peptide possesses great potential as an antimicrobial agent.

• Food Science of Animal Resources
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• v.36 no.4
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• pp.547-557
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• 2016

This review discusses the status, antimicrobial mechanisms, application, and regulation of natural preservatives in livestock food systems. Conventional preservatives are synthetic chemical substances including nitrates/nitrites, sulfites, sodium benzoate, propyl gallate, and potassium sorbate. The use of artificial preservatives is being reconsidered because of concerns relating to headache, allergies, and cancer. As the demand for biopreservation in food systems has increased, new natural antimicrobial compounds of various origins are being developed, including plant-derived products (polyphenolics, essential oils, plant antimicrobial peptides (pAMPs)), animal-derived products (lysozymes, lactoperoxidase, lactoferrin, ovotransferrin, antimicrobial peptide (AMP), chitosan and others), and microbial metabolites (nisin, natamycin, pullulan, ε-polylysine, organic acid, and others). These natural preservatives act by inhibiting microbial cell walls/membranes, DNA/RNA replication and transcription, protein synthesis, and metabolism. Natural preservatives have been recognized for their safety; however, these substances can influence color, smell, and toxicity in large amounts while being effective as a food preservative. Therefore, to evaluate the safety and toxicity of natural preservatives, various trials including combinations of other substances or different food preservation systems, and capsulation have been performed. Natamycin and nisin are currently the only natural preservatives being regulated, and other natural preservatives will have to be legally regulated before their widespread use.

• Journal of Life Science
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• v.26 no.12
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• pp.1409-1414
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• 2016

In the larvae of the black soldier fly, Hermetia illucens, innate immunity mechanisms are activated in response to various pathogens and stimulants, resulting in the expression of antimicrobial peptides (AMPs). To induce the mass production of AMPs, H. illucens fifth instar larvae were immunized with five different kinds of bacteria. We isolated from the hemolymph of the H. illucens larvae after bacterial challenge, and their antimicrobial activities against Gram-positive bacteria (Staphylococcus aureus) and Gram-negative bacteria (Escherichia coli) were measured using the inhibition zone assay. Among these five different kinds of bacteria, the hemolymph of Bacillus subtilis-challenged H. illucens larvae showed the strongest antimicrobial activity against both Gram-positive bacteria and Gram-negative bacteria. The antimicrobial activity of the hemolymph of $1{\times}10^9cfu/ml$ B. subtilis-challenged H. illucens peaks at 24 hr at 48 hr post-infection and gradually declines with time. Moreover, the immunized hemolymph also showed strong antimicrobial activity against various poultry pathogens such as S. enteritidis, S. typhimurium, and S. pullorum. These results suggest that the expression of AMP genes in B. subtilis-challenged H. illucens is up-regulated by innate immune responses, and that B. subtilis-challenged H. illucens overexpressing AMPs may be useful as a feed additive in livestock diets to reduce the need for antibiotics.

• Journal of Life Science
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• v.32 no.12
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• pp.956-961
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• 2022

Plant Chloroplast have several advantages as an expression platform of biopharmaceuticals over conventional expression platforms such as mammalian cells, yeast and bacteria. First, plants do not serve as a host for mammalian infectious virus and have endotoxin like bacteria which can cause anaphylactic shock. In addition, high copy number of chloroplast genome allows for chloroplast transformants to reach the high level of expression of heterologous genes. Moreover, the integration of transgenes into specific region of chloroplast genomes makes chloroplast transformants unaffected by positional effect which can be frequently observed from nuclear transformants, resulting in loss of transgene expressions. Antimicrobial peptides (AMPs) are a kind of innate immunity which is found from bacteria to humans. Unlike conventional antibiotics, very less dosage of AMPs can have catastrophic effect on bacterial survival. Further, the repeated use of AMPs does not trigger the development of bacterial resistance. Moricin, one of the AMPs, was isolated from Bombyx mori, a silkworm moth. The C-terminal of moricin consists largely of basic amino acids, and the N-terminal has an α-helix structure. Moricin was chosen and expressed in a SUMO/SUMOase without leaving any unwanted amino acids which could potentially affect the anti-bacterial activity of the moricin. The transformation vector used in this study has already been created in this lab for the expression in both prokaryotic systems such as E. coli and chloroplast. The expressed moricin was purified using Ni columns and SUMOase, and the antibacterial activity of the purified moricin was confirmed using an agar diffusion assay.

• Journal of Veterinary Science
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• v.24 no.2
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• pp.20.1-20.13
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• 2023

Background: As Actinobacillus pleuropneumonniae (APP) infection causes considerable losses in the pig industry, there is a growing need to develop effective therapeutic interventions that leverage host immune defense mechanisms to combat these pathogens. Objectives: To demonstrate the role of microRNA (miR)-127 in controlling bacterial infection against APP. Moreover, to investigate a signaling pathway in macrophages that controls the production of anti-microbial peptides. Methods: Firstly, we evaluated the effect of miR-127 on APP-infected pigs by cell count/enzyme-linked immunosorbent assay (ELISA). Then the impact of miR-127 on immune cells was detected. The cytokines tumor necrosis factor (TNF)-α and interleukin (IL)-6 were evaluated by ELISA. The expression of cytokines (anti-microbial peptides [AMPs]) was assessed using quantitative polymerase chain reaction. The expression level of IL-6, TNF-α and p-P65 were analyzed by western blot. The expression of p65 in the immune cells was investigated by immunofluorescence. Results: miR-127 showed a protective effect on APP-infected macrophage. Moreover, the protective effect might depend on its regulation of macrophage bactericidal activity and the generation of IL-22, IL-17 and AMPs by targeting sphingosine-1-phosphate receptor3 (SIPR3), the element involved in the Toll-like receptor (TLR) cascades. Conclusions: Together, we identify that miR-127 is a regulator of S1PR3 and then regulates TLR/nuclear factor-κB signaling in macrophages with anti-bacterial acticity, and it might be a potential target for treating inflammatory diseases caused by APP.