Abstract
Whereas extracellular vesicle (EV) research has become commonplace in different biomedical fields, this field of research is still in its infancy in mycology. Here we provide a robust set of data regarding the structural and compositional aspects of EVs isolated from the fungal pathogenic species Cryptococcus neoformans, C. deneoformans and C. deuterogattii. Using cutting-edge methodological approaches including cryogenic electron microscopy and cryogenic electron tomography, proteomics, and flow cytometry, we revisited cryptococcal EV features and suggest a new EV structural model, in which the vesicular lipid bilayer is covered by mannoprotein-based fibrillar decoration, bearing the capsule polysaccharide as its outer layer. About 10% of the EV population is devoid of fibrillar decoration, adding another aspect to EV diversity. By analyzing EV protein cargo from the three species, we characterized the typical Cryptococcus EV proteome. It contains several membrane-bound protein families, including some Tsh proteins bearing a SUR7/PalI motif. The presence of known protective antigens on the surface of Cryptococcus EVs, resembling the morphology of encapsulated virus structures, suggested their potential as a vaccine. Indeed, mice immunized with EVs obtained from an acapsular C. neoformans mutant strain rendered a strong antibody response in mice and significantly prolonged their survival upon C. neoformans infection.
Competing Interest Statement
The authors have declared no competing interest.
Footnotes
↵# Members of the data sharing transparency group on fungal extracellular vesicles
First, additional cryo-EM analyses of EVs isolated from two additional Cryptococcus species have been performed, confirming the diversity of fungal EVs and the presence of decoration in most of them. These experiments also revealed species specificities in EV size and decoration thickness. Second, our new experiments showed that proteinase K treatment strongly affect ConA labelling of EVs, supporting our model in which mannoproteins are at the surface of EVs. Third, we performed nano flow cytometry analyses and cryo-EM experiments to study N-and O-glycosylation mutant (alg3, ktr3, hoc3) EVs. These experiments revealed that ALG3 deletion is associated with reduced ConA labelling and decoration thickness. Fourth, we performed additional nano flow cytometry and cryo-EM analysis on an mp88Δ mutant strain revealing the impact of this mutation on EV ConA labelling and decoration thickness. Fifth, in this new version of the manuscript, we studied the proteomes of C. albicans and S. cerevisiae EVs showing that mannoproteins and GPI anchor proteins, including immunogenic ones, are common features of fungal EVs. Overall, all these data strongly support our previous conclusions and strengthen our new structural model of fungal EVs.