Purifying the Purified: Isolation and characterisation of extracellular vesicles from Candida

Abstract

Extracellular vesicles (EVs) produced by fungi are thought to influence pathogenesis and host responses to infection, rendering them potentially important in a medical context, particularly as targets for antivirulence drugs, and candidates for vaccines, for fungal diseases. Among other genera of fungi, EVs have been extracted from Candida, which are responsible for a large proportion of serious and fatal fungal disease cases. However, characterisation of EVs purportedly isolated from culture supernatants of Candida albicans may have resulted in erroneous conclusions being drawn regarding their physical attributes, functions and molecular cargo, due to a lack of purification of these structures from contaminants such as protein aggregates. This work aimed to investigate whether the ultracentrifugation-based extraction methods in previous studies were likely adequate enough to isolate C. albicans EVs from other entities, or whether additional purification procedures, namely size-exclusion chromatography in this case, are needed to achieve this feat and thereby permit analyses of only true EVs and EV-associated molecules. "Crude" preparations of EVs extracted from supernatants of C. albicans cultures via similar methods to those used previously, and fractions of these preparations subjected to size-exclusion chromatography, were analysed for the presence of EVs, protein and nucleic acid. Crude EV preparations and fractions from cultures of the newly emerged drug-resistant pathogen C. auris were also analysed as such. Objects in crude preparations that were possibly EVs were visualised by transmission electron microscopy. Nanoparticle tracking analysis revealed these preparations contained many particles, some of which may be EVs, between 50 and 150 nm in diameter, while whole protein, RNA and DNA quantitation assays showed that small amounts of these biomolecules were also present. Size-exclusion chromatography and subsequent analyses of individual fractions revealed a large amount of the protein in crude preparations was not associated with the possible EVs, while most of the nucleic acid was. Gel electrophoresis of fractions revealed the profiles of protein that was and was not co-purified with the particles detected by NTA differed only to a small extent, with several proteins differentially present in these factions later being identified by mass spectrometry. These findings suggest additional purification procedures following the collection of crude EV preparations are necessary for accurate characterisation of EVs and their contents, particularly in the case of protein, and such procedures should be undertaken in studies on fungal EVs in future.