Regular protocols for the isolation of fungal extracellular vesicles (EVs) are time-consuming, hard to reproduce, and produce low yields. In an attempt to improve the protocols used for EV isolation, we explored a model of vesicle production after growth of Cryptococcus gattii and Cryptococcus neoformans on solid media. Nanoparticle tracking analysis in combination with transmission electron microscopy revealed that C. gattii and C. neoformans produced EVs in solid media. The properties of cryptococcal vesicles varied according to the culture medium used and the EV-producing species. EV detection was reproduced with an acapsular mutant of C. neoformans, as well as with isolates of Candida albicans, Histoplasma capsulatum, and Saccharomyces cerevisiae. Cryptococcal EVs produced in solid media were biologically active and contained regular vesicular components, including the major polysaccharide glucuronoxylomannan (GXM) and RNA. Since the protocol had higher yields and was much faster than the regular methods used for the isolation of fungal EVs, we asked if it would be applicable to address fundamental questions related to cryptococcal secretion. On the basis that polysaccharide export in Cryptococcus requires highly organized membrane traffic culminating with EV release, we analyzed the participation of a putative scramblase (Aim25; CNBG_3981) in EV-mediated GXM export and capsule formation in C. gattii. EVs from a C. gattii aim25Δ strain differed from those obtained from wild-type (WT) cells in physical-chemical properties and cargo. In a model of surface coating of an acapsular cryptococcal strain with vesicular GXM, EVs obtained from the aim25Δ mutant were more efficiently used as a source of capsular polysaccharides. Lack of the Aim25 scramblase resulted in disorganized membranes and increased capsular dimensions. These results associate the description of a novel protocol for the isolation of fungal EVs with the identification of a previously unknown regulator of polysaccharide release. IMPORTANCE Extracellular vesicles (EVs) are fundamental components of the physiology of cells from all kingdoms. In pathogenic fungi, they participate in important mechanisms of transfer of antifungal resistance and virulence, as well as in immune stimulation and prion transmission. However, studies on the functions of fungal EVs are still limited by the lack of efficient methods for isolation of these compartments. In this study, we developed an alternative protocol for isolation of fungal EVs and demonstrated an application of this new methodology in the study of the physiology of the fungal pathogen Cryptococcus gattii. Our results describe a fast and reliable method for the study of fungal EVs and reveal the participation of scramblase, a phospholipid-translocating enzyme, in secretory processes of C. gattii.

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.

22Regular protocols for the isolation of fungal extracellular vesicles (EVs) are time-consuming, 23 hard to reproduce, and produce low yields. In an attempt to improve the protocols used for 24 EV isolation, we explored a model of vesicle production after growth of Cryptococcus gattii 25 and C. neoformans on solid media. Nanoparticle tracking analysis in combination with 26 transmission electron microscopy revealed that C. gattii and C. neoformans produced EVs in 27 solid media. These results were reproduced with an acapsular mutant of C. neoformans, as 28 well as with isolates of Candida albicans, Histoplasma capsulatum, and Saccharomyces 29 cerevisiae. Cryptococcal EVs produced in solid media were biologically active and contained 30 regular vesicular components, including the major polysaccharide glucuronoxylomannan 31 (GXM) and RNA. Since the protocol had higher yields and was much faster than the regular 32 methods used for the isolation of fungal EVs, we asked if it would be applicable to address 33 fundamental questions related to cryptococcal secretion. On the basis that polysaccharide 34 export in Cryptococcus requires highly organized membrane traffic culminating with EV 35 release, we analyzed the participation of a putative scramblase (Aim25, CNBG_3981) in EV-36 mediated GXM export and capsule formation in C. gattii. EVs from a C. gattii aim25 strain 37 differed from those obtained from wild-type (WT) cells in physical-chemical properties and 38 cargo. In a model of surface coating of an acapsular cryptococcal strain with vesicular GXM, 39EVs obtained from the aim25 mutant were more efficiently used as a source of capsular 40 polysaccharides. Lack of the Aim25 scramblase resulted in disorganized membranes and 41 increased capsular dimensions. These results associate the description of a novel protocol 42 for the isolation of fungal EVs with the identification of a previously unknown regulator of 43 polysaccharide release. 44 45 IMPORTANCE. Extracellular vesicles (EVs) are fundamental components of the physiology of 46 cells from all kingdoms. In pathogenic fungi, they participate in important mechanisms of 47 results describe a fast and reliable method for the study of fungal EVs and reveal the 53 participation of scramblase, a phospholipid translocating enzyme, in secretory processes of 54 C. gattii. 55 56 57

Pathogenic species of Cryptococcus kill approximately 200,000 people each year. The most important virulence mechanism of C. neoformans and C. gattii, the causative agents of human and animal cryptococcosis, is the ability to form a polysaccharide capsule. Acapsular mutants of C. neoformans are avirulent in mice models of infection, and extracellularly released capsular polysaccharides are deleterious to the immune system. The principal capsular component in the Cryptococcus genus is a complex mannan substituted with xylosyl and glucuronyl units, namely glucuronoxylomannan (GXM). The second most abundant component of the cryptococcal capsule is a galactan with multiple glucuronyl, xylosyl, and mannosyl substitutions, namely glucuronoxylomannogalactan (GXMGal). The literature about the structure and functions of these two polysaccharides is rich, and a number of comprehensive reviews on this topic are available. Here, we focus our discussion on the less explored glycan components associated with the cryptococcal capsule, including mannoproteins and chitin-derived molecules. These glycans were selected for discussion on the basis that i) they have been consistently detected not only in the cell wall but also within the cryptococcal capsular network and ii) they have functions that impact immunological and/or pathogenic mechanisms in the Cryptococcus genus. The reported functions of these molecules strongly indicate that the biological roles of the cryptococcal capsule go far beyond the well-known properties of GXM and GXMGal.

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