Anandamide Targets Membrane Integrity in Non-Albicans Candida: A Novel Antifungal Approach

Fungal infections remain a major threat to human health, with non-albicans Candida (NAC) species causing more than half of all clinical cases and many strains gaining resistance to current treatments rapidly. Previously, N-arachidonoyl ethanolamine (anandamide, AEA) has been studied and shown to possess antibacterial and antifungal properties against various bacteria and Candida albicans. Given the previous findings, we aim here to expand the current preliminary research on AEA to investigate its antifungal activities against clinically relevant NAC species in vitro: Candida glabrata, Candida parapsilosis, and Candidaozyma auris. The minimum inhibitory concentration (MIC) and growth curve analysis determined planktonic inhibition. MTT metabolic assay and ATP production via BacTiter-Glo luminescence assay evaluated biofilm formation. Membrane fluidity, polarization and efflux pump activity were examined using fluorescence probes Laurdan, DiS-C3(3), and Rhodamine 6G, respectively. Reactive oxygen species (ROS) were assessed using DCFH-DA. Biofilm architecture and cell viability were analyzed by spinning disk confocal microscopy (SDCM). AEA reduced MIC values and slowed planktonic growth, while MTT and ATP assays demonstrated a pronounced dose-dependent reduction in biofilm metabolic activity. Membrane-targeted effects revealed increased fluidity and permeability at 125 µg/mL. Notably, AEA rapidly impaired efflux pump activity and induced intracellular ROS production. This effect was accompanied by reduced cell viability, increased proportions of PI-positive cells, and enhanced intracellular dye retention, as confirmed by SDCM. Together, these findings demonstrate that AEA exerts antifungal activity by disrupting membrane integrity and associated cellular functions and provide the first comparative characterization of species-specific membrane and oxidative stress responses to AEA across three major clinically relevant multidrug-resistant NAC species.