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.

Zinc is an essential nutrient for all living organisms because it is a co-factor of several important proteins. Furthermore, zinc may play an essential role in the infectiousness of microorganisms. Previously, we determined that functional zinc metabolism is associated with Cryptococcus gattii virulence. Here, we characterized the ZIP zinc transporters in this human pathogen. Transcriptional profiling revealed that zinc levels regulated the expression of the ZIP1, ZIP2 and ZIP3 genes, although only the C. gattii zinc transporter Zip1 was required for yeast growth under zinc-limiting conditions. To associate zinc uptake defects with virulence, the most studied cryptococcal virulence factors (i.e., capsule, melanin and growth at 37 °C) were assessed in ZIP mutant strains; however, no differences were detected in these classical virulence-associated traits among the mutant and WT strains. Interestingly, higher levels of reactive oxygen species were detected in the zip1Δ and in the zip1Δ zip2Δ double mutants. In line with these phenotypic alterations, the zip1Δ zip2Δ double mutant displayed attenuated virulence in a murine model of cryptococcosis. Together, these results indicate that adequate zinc uptake is necessary for cryptococcal fitness and virulence.

Cryptococcus gattii has the ability to escape from the host’s immune system through poorly understood mechanisms and can lead to the death of healthy individuals. The role of mannoproteins in C. gattii pathogenicity is not completely understood. The present work characterized a protein, Kpr1, that is essential for the maintenance of C. gattii main virulence factor, the polysaccharide capsule. Our data contribute to the understanding of the role of Kpr1 in capsule structuring, mainly by modulating the distribution of glucans in C. gattii cell wall.

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

These findings suggest that macrophages may exhibit zinc depletion in response to C. neoformans infection.