| Summary: | The increase and dissemination of antimicrobial resistance is a global public health issue. To address this, new antimicrobial agents have been developed. Antimicrobial peptides (AMPs) exhibit a wide range of antimicrobial activities against pathogens, including bacteria and fungi. Lycosin-II, isolated from the venom of the spider <i>Lycosa singoriensis</i>, has shown antibacterial activity by disrupting membranes. However, the mode of action of Lycosin-II and its antifungal activity have not been clearly described. Therefore, we confirmed that Lycosin-II showed antifungal activity against <i>Candida albicans</i> (<i>C. albicans</i>). To investigate the mode of action, membrane-related assays were performed, including an evaluation of <i>C. albicans</i> membrane depolarization and membrane integrity after exposure to Lycosin-II. Our results indicated that Lycosin-II damaged the <i>C. albicans</i> membrane. Additionally, Lycosin-II induced oxidative stress through the generation of reactive oxygen species (ROS) in <i>C. albicans</i>. Moreover, Lycosin-II exhibited an inhibitory effect on dual-species biofilm formation by <i>C. albicans</i> and <i>Staphylococcus aureus</i> (<i>S. aureus</i>)<i>,</i> which are the most co-isolated fungi and bacteria. These results revealed that Lycosin-II can be utilized against <i>C. albicans</i> and dual-species strain infections.
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