Abstract 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 nop16 Δ 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 nop16 Δ mutants were hypovirulent. The hypovirulent phenotype was reverted when EVs produced by wild-type cells, but not mutant EVs, were co-injected with the nop16 Δ 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.

Extracellular vesicle formation inCryptococcus deuterogattiiimpacts fungal virulence and requires theNOP16gene