Cloning and Functional Analysis of Skin Host Defense Peptides from Yakushima Tago’s Brown Frog (Rana tagoi yakushimensis) and Development of Serum Endotoxin Detection System

• Published in: Antibiotics (Basel) 2024-11, Vol.13 (12), p.1127
• Main Authors: Aono, Taichi, Tamura, Saki, Suzuki, Yua, Imanara, Taichi, Niwa, Ryosei, Yamane, Yoshie, Kobayashi, Tetsuya, Kikuyama, Sakae, Hasunuma, Itaru, Iwamuro, Shawichi
• Format: Article
• Language: English
• Subjects: Amino acids; Amphibians; amurin-9; Antibacterial activity; Antibiotics; Antiinfectives and antibacterials; Antimicrobial activity; Antimicrobial agents; Antioxidants; Binding; brevinin-1; Cloning; Coding; Cytotoxicity; Defense industry; Detectors; E coli; Endotoxins; Enzymes; Escherichia coli; frog skin; Frogs; Functional analysis; Glucan; host defense peptides; Lipopolysaccharides; Lipoteichoic acid; Microorganisms; Mitochondrial DNA; Organic compounds; Pathogens; Peptides; Pharmacology; Phylogenetics; Precursors; Proteins; Rana tagoi; Rana tagoi yakushimensis; Reptiles & amphibians; Sequences; Skin; Toxicity; yakushimin; β-Glucan
• ISSN: 2079-6382; 2079-6382
• DOI: 10.3390/antibiotics13121127

Background/Objective: Amphibian skin is a valuable source of host defense peptides (HDPs). This study aimed to identify HDPs with novel amino acid sequences from the skin of Rana tagoi yakushimensis and analyze their functions. Methods: cDNAs encoding HDP precursors were cloned and sequenced using RT-PCR and 3′-RACE. The novel HDPs were synthesized to evaluate their antimicrobial activity, antioxidant activity, and cytotoxicity. Antimicrobial activity was evaluated by way of broth microdilution and endotoxin- and β-glucan-binding capacity using an enzyme-linked endotoxin binding assay (ELEBA) and a modified ELEBA, respectively. Results: Nine cDNAs encoding precursors for various HDP families, including temporin, ranatuerin-2, brevinin-1, amurin-9, and a novel yakushimin peptide, were identified. Brevinin-1TYa exhibited antibacterial activity against Staphylococcus aureus, and brevinin-1TYa and amurin-9TYa induced morphological changes in Escherichia coli and S. aureus. Yakushimin-TYa, amurin-9TYa, and brevinin-1TYa showed concentration-dependent antibacterial effects against the plant pathogens Xanthomonas oryzae pv. oryzae and Clavibacter michiganensis subsp. michiganensis. Amurin-9TYa demonstrated strong binding affinity to lipopolysaccharide, lipoteichoic acid, and β-glucan, exhibited antioxidant activity, and lacked cytotoxicity, making it a promising therapeutic candidate. Moreover, brevinin-1TYa showed strong cytotoxicity, whereas yakushimin-TYa exhibited weak cytotoxicity. Conclusions: These findings highlight the potential of these peptides, particularly amurin-9TYa, for future applications as antimicrobial and therapeutic agents.