Antifungal and Antivirulence Activity of Alizarin against <i>Candida dubliniensis</i>

Candida dubliniensis is a relatively common yet understudied fungal species present in the oral cavity of both healthy and immunocompromised individuals. Despite its increasing clinical relevance, research addressing its pathogenesis and therapeutic targeting remains limited. In this context, the present study investigated the antifungal potential of alizarin against C. dubliniensis. Alizarin exhibited notable antifungal activity, with a minimum inhibitory concentration (MIC) of 16 μg/mL. Checkerboard analysis further demonstrated a significant enhancement of fluconazole activity when used in combination with alizarin, indicating a synergistic interaction. In vitro antibiofilm assays demonstrated that alizarin, at its MIC level, significantly reduces both biofilm formation and preformed biofilms of C. dubliniensis. Notably, administration of alizarin improved the survival of Galleria mellonella by decreasing the fungal burden; however, its immunomodulatory potential remained lower than that of standard antifungal treatments. Furthermore, alizarin effectively inhibited filamentation, a key virulence factor, at 16 μg/mL within 2 h of exposure. After 24 h, only 2.12% of cells retained the ability to undergo hyphal transition (p < 0.0001). Proteomic analysis revealed significant modulation of the protein expression profile, including downregulation of proteins associated with cell wall synthesis and integrity. Overall, these findings demonstrate that alizarin exerts antifungal effects against C. dubliniensis, primarily by inhibiting morphological transition and modulating virulence-associated pathways. Further studies are warranted to elucidate the underlying molecular mechanisms and to explore its potential as a therapeutic agent.