Juglone suppresses morphogenesis and early biofilm formation in <i>Candida albicans</i> through disruption of oxidative homeostasis

Candida albicans is an important opportunistic pathogen causing superficial to life-threatening infections, especially in immunocompromised individuals. Its pathogenicity is largely driven by biofilm formation and phenotypic switching, especially the yeast-to-hypha (germ tube) transition, which plays a crucial role in antifungal tolerance and clinical progression. Despite growing interest in natural antifungals, the role of juglone (5-hydroxy-1,4-naphthoquinone, a natural naphthoquinone derived from Juglans species) in germ tube formation and redox-dependent virulence remains underexplored. This study evaluated juglone's antifungal and anti-virulence effects and whether these involve disruption of oxidative homeostasis. Antifungal activity and virulence-related effects were assessed using MIC/MFC determination, well diffusion, germ tube and biofilm assays, and oxidative stress measurements. Juglone exhibited dose-dependent inhibition of C. albicans growth (MIC 15.63 µg/mL, MFC = MIC) and an additive interaction with amphotericin B (MIC 0.39 µg/mL; FICI = 0.5625). Inhibition zones increased in combination, supporting an additive effect. Although effects on mature biofilms, phospholipase, and hemolysin activities were limited, juglone markedly inhibited key virulence factors, reducing germ tube formation to 9% (60% for amphotericin B; 93% control), and suppressing early biofilm formation and extracellular polymeric substance production. Juglone caused only a slight reduction in lactate dehydrogenase release, suggesting membranes remained largely intact, but significantly modulated stress responses by decreasing superoxide dismutase activity and total antioxidant capacity, while malondialdehyde levels remained unchanged, consistent with a redox-based antifungal mechanism. Overall, juglone shows potent antifungal and anti-virulence effects against C. albicans, likely in part by modulating oxidative balance and weakening virulence mechanisms, while enhancing conventional antifungal treatment.