Abstract
INTRODUCTION: The escalating crisis of antibiotic resistance highlights the urgent need for novel antimicrobial agents. This study aims to identify and characterize temporin-1LS, a temporin-family antimicrobial peptide from the dark-spotted frog (Pelophylax nigromaculatus), and to investigate its biological functions, including antimicrobial and immunomodulatory activities. METHODS: Structural and phylogenetic analyses were conducted to determine the peptide's conformation and evolutionary relationship. Expression levels of temporin-1LS in various tissues were assessed under basal and pathogen-challenged conditions using Aeromonas hydrophila. The antimicrobial activity of synthetic temporin-1LS was evaluated by determining minimum inhibitory concentrations (MICs) against bacterial strains. Immunomodulatory functions, including macrophage chemotaxis, phagocytosis, and respiratory burst, were examined in vitro. RESULT: Temporin-1LS possesses a conserved amphipathic α-helical structure and shows high phylogenetic homology with temporin-1GY. It was constitutively highly expressed in frog skin and significantly upregulated in the gut (47.4-fold) and liver (24.3-fold) following A. hydrophila challenge. The synthetic peptide exhibited potent and selective antibacterial activity against Staphylococcus warneri and Vibrio alginolyticus (MIC = 3.125 µg/mL). Temporin-1LS significantly increased bacterial LDH release at higher concentrations (50-100 μg/mL) relative to the BSA control, suggesting possible membrane damage under these conditions. Although it did not show measurable in vitro antibacterial activity against A. hydrophila, temporin-1LS administration significantly improved survival in a frog infection model. Furthermore, the peptide demonstrated strong immunomodulatory effects, stimulating macrophage chemotaxis (2.4-fold), enhancing phagocytosis (4.4-fold), and amplifying respiratory burst (1.6-fold) in a dose-dependent manner. DISCUSSION AND CONCLUSIONS: Temporin-1LS employs a dual-defense mechanism, combining direct antibacterial activity against susceptible pathogens with strategic enhancement of key macrophage functions to resolve infection. These findings position temporin-1LS as a promising scaffold for developing novel immunomodulatory anti-infectives.