Alternariol from the endophytic fungus Alternaria sp. S4 acts as a membrane-targeting bactericidal agent against...

The endophytic fungus Alternaria sp. S4, derived from Ruta graveolens, shows significant antimicrobial potential, but its active components and mode of action require clarification. Through bioassay-guided fractionation, alternariol (1) and alternariol-5-O-methyl ether (2) were isolated from the ethyl acetate fraction of Alternaria sp. S4 and characterized spectroscopically. Compound 1 exhibited potent bactericidal activity against Staphylococcus aureus (MIC = 4 μg/mL, MBC = 16 μg/mL), whereas compound 2 was inactive. Time-growth assays confirmed concentration-dependent bacterial eradication, with 2× MIC of compound 1 achieving complete growth inhibition. Mechanistic investigations revealed that compound 1 targets the bacterial membrane, inducing depolarization, compromising permeability (evidenced by ion leakage and release of intracellular constituents), and causing ultrastructural damage visualized via SEM. Furthermore, compound 1 prevented biofilm formation via bacterial eradication and disrupted preformed biofilms. Checkerboard assays indicated additive effects when combined with conventional antibiotics. Notably, compound 1 displayed no hemolytic activity even at 1024 μg/mL, highlighting its membrane selectivity. This work identifies alternariol as a promising membrane-targeting bactericidal agent from an endophytic source, capable of preventing biofilm establishment, with potential for combinatorial therapy against S. aureus.