Antifungal Effect of Vitamin D3 against Cryptococcus neoformans Coincides with Reduced Biofilm Formation, Compromised Cell Wall Integrity, and Increased Generation of Reactive Oxygen Species

Abstract

<i>Cryptococcus neoformans</i> is an invasive fungus that causes both acute and chronic infections, especially in immunocompromised patients. Owing to the increase in the prevalence of drug-resistant pathogenic fungi and the limitations of current treatment strategies, drug repositioning has become a feasible strategy to accelerate the development of new drugs. In this study, the minimum inhibitory concentration of vitamin D₃ (VD₃) against <i>C. neoformans</i> was found to be 0.4 mg/mL by broth microdilution assay. The antifungal activities of VD₃ were further verified by solid dilution assays and "time-kill" curves. The results showed that VD₃ reduced fungal cell adhesion and hydrophobicity and inhibited biofilm formation at various developmental stages, as confirmed by crystal violet staining and the 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide assay. Fluorescence staining of cellular components and a stress susceptibility assay indicated that VD₃ compromised cell integrity. Reverse transcription quantitative PCR demonstrated that VD₃ treatment upregulated the expression of fungal genes related to cell wall synthesis (i.e., <i>CDA3</i>, <i>CHS3</i>, <i>FKS1</i>, and <i>AGS1</i>). Moreover, VD₃ enhanced cell membrane permeability and caused the accumulation of intracellular reactive oxygen species. Finally, VD₃ significantly reduced the tissue fungal burden and prolonged the survival of <i>Galleria mellonella</i> larvae infected with <i>C. neoformans</i>. These results showed that VD₃ could exert significant antifungal activities both in vitro and in vivo, demonstrating its potential application in the treatment of cryptococcal infections.