Dual-defense functionality of temporin-1LS from dark-spotted frogs (Pelophylax nigromaculatus): antimicrobial activity and immunomodulatory effects via macrophage activation

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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.

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