Antifungal Effect of Vitamin D(3) against Cryptococcus neoformans Coincides with Reduced Biofilm Formation, Compromised Cell Wall Integrity, and Increased Generation of Reactive Oxygen Species

Cryptococcus neoformans 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 D(3) (VD(3)) against C. neoformans was found to be 0.4 mg/mL by broth microdilution assay. The antifungal activities of VD(3) were further verified by solid dilution assays and “time-kill” curves. The results showed that VD(3) 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 VD(3) compromised cell integrity. Reverse transcription quantitative PCR demonstrated that VD(3) treatment upregulated the expression of fungal genes related to cell wall synthesis (i.e., CDA3, CHS3, FKS1, and AGS1). Moreover, VD(3) enhanced cell membrane permeability and caused the accumulation of intracellular reactive oxygen species. Finally, VD(3) significantly reduced the tissue fungal burden and prolonged the survival of Galleria mellonella larvae infected with C. neoformans. These results showed that VD(3) could exert significant antifungal activities both in vitro and in vivo, demonstrating its potential application in the treatment of cryptococcal infections.