ALT The role of fungal EVs in antifungal resistance. (A and D) In S. cerevisiae and C. auris, caspofungin treatment leads to increased EV production. Complementation assays using wild-type EVs can restore the caspofungin susceptibility of cell wall defective mutants in S. cerevisiae and increase resistance to amphotericin B in C. auris. (B and C) In C. albicans and C. tropicalis, EVs contribute to drug resistance, biofilm formation, and the production of the biofilm extracellular matrix. Inhibitors of the vesicular trafficking pathway, such as turbinmicin, decrease biofilm biomass and EV production in C. albicans. In C. tropicalis, complementation with biofilm-derived EVs increases drug resistance and biofilm biomass. (E) In C. neoformans, fluconazole exposure decreases EV yield. Serial passaging in the absence of antifungal pressure restores EV production and drug susceptibility. Illustration created with BioRender.

ALT Model of EV biogenesis regulation and FLC susceptibility in C. neoformans. Specific gene mutations and FLC exposure can trigger diverse cellular responses, including changes in the expressions of drug efflux-associated genes, of the azole target gene, and in EVs biogenesis. Gat5 inhibits the expression of AFR1 and AFR3, which can affect the intracellular FLC levels, while subinhibitory concentration of FLC can inhibit EV production. Hap2/3/5 and Gat5 regulate EV production, and cellular lipid homeostasis. The differential regulation of these cellular processes may determine FLC resistance phenotypes in this fungal pathogen. Schematic model created in BioRender.

ALT Screening strategy for 1,4-benzodiazepines (1,4-BZDs) and control compounds. (A) Fluconazole (FLC) susceptibility profiles of Candida albicans isolates SC5314, SP-945, and P60002 on disk diffusion assays, including their mean RAD50, RAD20, and fraction of growth at 20% inhibition (FoG20) levels. (B) A lawn of C. albicans cells was plated on YPD agar and grown in the presence of a drug disk (FLC, 25 µg) for 48 hours. The size of the radius at 50% and 20% growth inhibition (RAD50 and RAD20, respectively) was measured as a proxy for drug susceptibility. The FoG20 was measured as a proxy for tolerance.

ALT Colonies and cell morphology of the three C. albicans strains