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The mechanisms by which macromolecules are transported through the cell wall of fungi are not known. A central question in the biology of Cryptococcus neoformans, the causative agent of cryptococcosis, is the mechanism by which capsular polysaccharide synthesized inside the cell is exported to the extracellular environment for capsule assembly and release. We demonstrate that C. neoformans produces extracellular vesicles during in vitro growth and animal infection. Vesicular compartments, which are transferred to the extracellular space by cell wall passage, contain glucuronoxylomannan (GXM), a component of the cryptococcal capsule, and key lipids, such as glucosylceramide and sterols. A correlation between GXM-containing vesicles and capsule expression was observed. The results imply a novel mechanism for the release of the major virulence factor of C. neoformans whereby polysaccharide …