Fungal Extracellular Vesicles are Recoverable Across Variable Ultracentrifugation Speeds but Display Species-specific Profiles of Sedimentation.

Extracellular vesicles (EVs) are central components of fungal biology, yet their isolation commonly relies on ultracentrifugation protocols originally developed for mammalian systems. In Cryptococcus, most EVs carry the capsular polysaccharide glucuronoxylomannan (GXM), raising the possibility that vesicle density, and therefore sedimentation, could be influenced by polysaccharide content. Here, we systematically evaluated EV recovery across sequential ultracentrifugation speeds (20,000 × g to 100,000 × g) in Cryptococcus deuterogattii, Candida auris, and Candida parapsilosis. In C. deuterogattii, GXM was detected across all ultracentrifugation fractions, and each fraction efficiently transferred polysaccharide to acapsular cells, demonstrating that its presence is independent of fraction density and does not correlate with sedimentation behavior. In the three fungal species, transmission electron microscopy and nanoparticle tracking analysis confirmed that bona fide EVs are recoverable across all centrifugation speeds. Qualitative and quantitative proteomic analyses revealed largely overlapping protein compositions among fractions within each species. However, proteomic profiles differed between species. In C. deuterogattii and C. auris, fractions displayed similar proteomic and predicted protein-protein interaction signatures across centrifugation speeds. In contrast, C. parapsilosis exhibited a clear partitioning pattern, with low-speed fractions (20,000 × g and 40,000 × g) clustering together and differing from higher-speed fractions (60,000-100,000 × g). These findings demonstrate that fungal EVs are recoverable across a broad range of ultracentrifugation speeds, but their sedimentation behavior is species-specific. Our study highlights the need for tailored EV isolation strategies and cautions against assuming methodological equivalence across fungal pathogens.