Riboflavin inhibits growth and reduces virulence of <i>Cryptococcus neoformans in vitro</i> by membrane disruption and excessive accumulation of reactive oxygen species and exhibits efficacy against pulmonary cryptococcosis and meningitis

The incidences of pulmonary cryptococcosis and meningitis cause significant morbidity and mortality. Effective and affordable drugs for treatment of cryptococcal meningitis are urgently needed. Drug reuse is an effective strategy for the development of new antifungals against <i>Cryptococcus neoformans</i> infection. In this study, riboflavin (RF) significantly inhibited growth of <i>C. neoformans</i> as determined by the broth microdilution and spot dilution methods. Moreover, RF significantly inhibited biofilm formation and reduced virulence (capsule, melanin, and urease). In addition, RF caused cell membrane damage, compromised cell wall integrity, and promoted accumulation of intracellular reactive oxygen species (ROS). RT-qPCR analysis confirmed that RF treatment up-regulated expression genes related to cell wall biosynthesis (<i>CHS3</i>, <i>CDA1</i>, and <i>FKS1</i>), the cell wall damage repair pathway (<i>Pkc1</i> and <i>Mpk1</i>), and virulence (<i>CAP59</i>, <i>Lac1</i>, <i>Lac2</i>), however, <i>Ure1</i> were down-regulated after RF treatment. Finally, in mouse models of intranasal and intravenous infection, RF treatment significantly reduced the fungal burden in multiple organs, reduced lung and brain damage, and decreased the levels of plasma interferon (IFN)-γ, tumor necrosis factor (TNF)-α, and interleukin (IL)-4 in the early stage of infection. These results showed that RF exerted significant antifungal effects for treatment of <i>C. neoformans</i> infection.