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There is an urgent unmet need for novel antifungals. In this study, we searched for novel antifungal activities in the Pandemic Response Box, a collection of 400 structurally diverse compounds in various phases of drug discovery. We identified five molecules which could control the growth of Cryptococcus neoformans, Cryptococcus deuterogattii, and the emerging global threat Candida auris. After eliminating compounds which demonstrated paradoxical antifungal effects or toxicity to mammalian macrophages, we selected compound MMV1593537 as a nontoxic, fungicidal molecule for further characterization of antifungal activity. Scanning electron microscopy revealed that MMV1593537 affected cellular division in all three pathogens. In Cryptococcus, MMV1593537 caused a reduction in capsular dimensions. Treatment with MMV1593537 resulted in increased detection of cell wall chitooligomers in these three species. Since chitooligomers are products of the enzymatic hydrolysis of chitin, we investigated whether surface chitinase activity was altered in response to MMV1593537 exposure. We observed peaks of enzyme activity in C. neoformans and C. deuterogattii in response to MMV1593537. We did not detect any surface chitinase activity in C. auris. Our results suggest that MMV1593537 is a promising, nontoxic fungicide whose mechanism of action, at least in Cryptococcus spp, requires chitinase-mediated hydrolysis of chitin. IMPORTANCE The development of novel antifungals is a matter of urgency. In this study, we evaluated antifungal activities in a collection of 400 molecules, using highly lethal fungal pathogens as targets. One of these molecules, namely, MMV1593537, was not toxic to host cells and controlled the growth of isolates of Cryptococcus neoformans, C. deuterogattii, C. gattii, Candida auris, C. albicans, C. parapsilosis, and C. krusei. We tested the mechanisms of antifungal action of MMV1593537 in the Cryptococcus and C. auris models and concluded that the compound affects the cell wall, a structure which is essential for fungal life. At least in Cryptococcus, this effect involved chitinase, an enzyme which is required for remodeling the cell wall. Our results suggest that MMV1593537 is a candidate for future antifungal development.

Extracellular vesicles (EVs) produced by members of the Cryptococcus genus are associated with fundamental processes of fungal physiology and virulence. However, several questions about the properties of cryptococcal EVs remain unanswered, mostly because of technical limitations. We recently described a fast and efficient protocol of high-yield EV isolation from solid medium. In this study, we aimed at using the solid medium protocol to address some of the open questions about EVs, including the kinetics of EV production, the diversity of EVs produced by multiple isolates under different culture conditions, the separation of vesicles in a density gradient followed by the recovery of functional EVs, the direct detection of EVs in culture supernatants, and the production of vesicles in solid cultures of Titan cells. Our results indicate that the production of EVs is directly impacted by the culture medium and time of growth, resulting in variable detection of EVs per cell and a peak of EV detection at 24 h of growth. Nanoparticle tracking analysis (NTA) of EV samples revealed that multiple isolates produce vesicles with variable properties, including particles of diverging dimensions. EVs were produced in the solid medium in amounts that were separated on a centrifugation density gradient, resulting in the recovery of functional EVs containing the major cryptococcal capsular antigen. We also optimized the solid medium protocol for induction of the formation of Titan cells, and analyzed the production of EVs by NTA and transmission electron microscopy. This analysis confirmed that EVs were isolated from solid cultures of cryptococcal enlarged cells. With these approaches, we expect to implement simple methods that will facilitate the analysis of EVs produced by fungal cells. IMPORTANCE Fungal extracellular vesicles (EVs) are considered to be important players in the biology of fungal pathogens. However, the limitations in the methodological approaches to studying fungal EVs impair the expansion of knowledge in this field. In the present study, we used the Cryptococcus genus as a model for the study of EVs. We explored the simplification of protocols for EV analysis, which helped us to address some important, but still unanswered, questions about fungal EVs.