Extracellular vesicles (EVs) are produced by all domains of life. In fungi, these structures were first described in Cryptococcus neoformans and, since then, they were characterized in several pathogenic and non-pathogenic fungal species. Cryptococcal EVs participate in the export of virulence factors that directly impact the Cryptococcus–host interaction. Our knowledge of the biogenesis and pathogenic roles of Cryptococcus EVs is still limited, but recent methodological and scientific advances have improved our understanding of how cryptococcal EVs participate in both physiological and pathogenic events. In this review, we will discuss the importance of cryptococcal EVs, including early historical studies suggesting their existence in Cryptococcus, their putative mechanisms of biogenesis, methods of isolation, and possible roles in the interaction with host cells.

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.

The human diseases caused by the fungal pathogens Cryptococcus neoformans and Cryptococcus gattii are associated with high indices of mortality and toxic and/or cost-prohibitive therapeutic protocols. The need for affordable antifungals to combat cryptococcal disease is unquestionable. Previous studies suggested benzimidazoles as promising anticryptococcal agents combining low cost and high antifungal efficacy, but their therapeutic potential has not been demonstrated so far. In this study, we investigated the antifungal potential of fenbendazole, the most effective anticryptococcal benzimidazole. Fenbendazole was inhibitory against 17 different isolates of C. neoformans and C. gattii at a low concentration. The mechanism of anticryptococcal activity of fenbendazole involved microtubule disorganization, as previously described for human parasites. In combination with fenbendazole, the concentrations of the standard antifungal amphotericin B required to control cryptococcal growth were lower than those required when this antifungal was used alone. Fenbendazole was not toxic to mammalian cells. During macrophage infection, the anticryptococcal effects of fenbendazole included inhibition of intracellular proliferation rates and reduced phagocytic escape through vomocytosis. Fenbendazole deeply affected the cryptococcal capsule. In a mouse model of cryptococcosis, the efficacy of fenbendazole to control animal mortality was similar to that observed for amphotericin B. These results indicate that fenbendazole is a promising candidate for the future development of an efficient and affordable therapeutic tool to combat cryptococcosis.

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.

There is an urgent need to develop novel antifungals. In this study, we screened 1600 compounds for antifungal activity against Cryptococcus neoformans and Candida auris. We evaluated 4 promising compounds against 24 additional isolates of C. neoformans, C. auris, Cryptococcus deuterogattii, and Cryptococcus gattii. The 4 compounds, dequalinium chloride (DQC), bleomycin sulfate (BMS), pentamidine isethionate salt (PIS), and clioquinol (CLQ), varied in their efficacy against these pathogens but were generally more effective against cryptococci. The compounds exerted their antifungal effect via multiple mechanisms including interference with the capsule of cryptococci and induction of hyphal-like morphology in C. auris. Our results indicate that DQC, BMS, PIS, and CLQ represent potential prototypes for the future development of antifungals.

Small molecules are components of fungal extracellular vesicles (EVs), but their biological roles are only superficially known. NOP16 is a eukaryotic gene that is required for the activity of benzimidazoles against Cryptococcus deuterogattii . In this study, during the phenotypic characterization of C. deuterogattii mutants lacking NOP16 expression, we observed that this gene was required for EV production.

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Small molecules are components of fungal extracellular vesicles (EVs), but their biological roles are only superficially known. NOP16 is a eukaryotic gene that is required for the activity of benzimidazoles against Cryptococcus deuterogattii. In this study, during the phenotypic characterization of C. deuterogattii mutants lacking NOP16 expression, we observed that this gene was required for EV production. Analysis of the small molecule composition of EVs produced by wild-type cells and two independent nop16Δ mutants revealed that the deletion of NOP16 resulted not only in a reduced number of EVs but also an altered small molecule composition. In a Galleria mellonella model of infection, the nop16Δ mutants were hypovirulent. The hypovirulent phenotype was reverted when EVs produced by wild-type cells, but not mutant EVs, were co-injected with the nop16Δ cells in G. mellonella. These results reveal a role for NOP16 in EV biogenesis and cargo, and also indicate that the composition of EVs is determinant for cryptococcal virulence.