A RAPID METHOD FOR THE ISOLATION OF EXTRACELLULAR VESICLES IN Cryptococcus

Fungal cells are encaged within a thick cell wall. Extracellular export of different molecules in fungi, therefore, includes trans-cell wall transport. How fungal cells export macromolecules across the cell wall remained unknown until very recently, when fungal extracellular vesicles (EVs) were first described. EVs are vehicles for the extracellular export of hundreds of molecules, including proteins, lipids, pigments, nucleic acids, and polysaccharides. Several of these molecules are immunologically active and / or play fundamental pathogenic roles. In pathogenic species of Cryptococcus, EVs participate in long-distance communication between fungal cells and in the extracellular transport of glucuronoxylomannan, a major pathogenic determinant. Methods used for isolation of fungal EVs are typically laborious, time-consuming and usually result in very low yields. In this study, we screened a collection of 53 Cryptococcus isolates for the production of extracellular GXM, with the further selection of hyper and hypo producers of the polysaccharide for EV isolation. Fungal cells were incubated in 96-well plates containing of RPMI for 24 h at 37oC under a 5% CO2 atmosphere, a condition that was previously demonstrated to induce GXM secretion. Culture supernatants were collected and screened for the presence of GXM by ELISA. A high diversity in extracellular GXM production was observed, which allowed the selection of two C. neoformans isolates producing high (Cn6) and low (Cn5) amounts of extracellular GXM. These cells were incubated under similar but scaled up conditions in 150 cm2 culture flasks containing 100 ml of RPMI. Supernatants were collected by centrifugation, filtered through 0.45 um membranes, concentrated 3-fold in centrifugal filter devices (100 kDa cut-off) and finally ultracentrifuged at 100,000 g, followed by the application of antibody-based methods for removal of non-vesicular GXM. Vesicle production was analyzed by nanoparticle tracking analysis, which revealed peaks concentrated at the 100 nm range. Although the correlation between GXM and EV release was not clear, these results suggest that fungal EVs can be isolated and analyzed from low-volume cultures in 24 to 48 h. This fast method has the potential to allow the establishment of novel protocols for the analysis of EV functions in distinct populations of fungal pathogens.