Characterization of Extracellular Vesicles Produced by Aspergillus fumigatus Protoplasts

Abstract

Extracellular vesicles (EVs) are membranous compartments produced by yeast and mycelial forms of several fungal species. One of the difficulties in perceiving the role of EVs during the fungal life, and particularly in cell wall biogenesis, is caused by the presence of a thick cell wall. One alternative to have better access to these vesicles is to use protoplasts. This approach has been investigated here with Aspergillus fumigatus, one of the most common opportunistic fungal pathogens worldwide. Analysis of regenerating protoplasts by scanning electron microscopy and fluorescence microscopy indicated the occurrence of outer membrane projections in association with surface components and the release of particles with properties resembling those of fungal EVs. EVs in culture supernatants were characterized by transmission electron microscopy and nanoparticle tracking analysis. Proteomic and glycome analysis of EVs revealed the presence of a complex array of enzymes related to lipid/sugar metabolism, pathogenic processes, and cell wall biosynthesis. Our data indicate that (i) EV production is a common feature of different morphological stages of this major fungal pathogen and (ii) protoplastic EVs are promising tools for undertaking studies of vesicle functions in fungal cells. IMPORTANCE Fungal cells use extracellular vesicles (EVs) to export biologically active molecules to the extracellular space. In this study, we used protoplasts of Aspergillus fumigatus, a major fungal pathogen, as a model to evaluate the role of EV production in cell wall biogenesis. Our results demonstrated that wall-less A. fumigatus exports plasma membrane-derived EVs containing a complex combination of proteins and glycans. Our report is the first to characterize fungal EVs in the absence of a cell wall. Our results suggest that protoplasts represent a promising model for functional studies of fungal vesicles.

Updates in Paracoccidioides Biology and Genetic Advances in Fungus Manipulation

“…In fungi, EVs are also involved in the transport of macromolecules across the cell wall [113]. Various reports have described the release of EVs by several species of pathogenic fungi in both yeasts and filamentous fungi [114][115][116][117][118][119][120]. Conventional and unconventional secretory pathways have been proposed for the release of EVs by fungi.…”

RevisitingCryptococcusextracellular vesicles properties and their use as vaccine platforms

“…In these organisms, a large body of literature describes how EVs participate in intercellular signaling within and in organism-to-organism communication, including carcinogenesis and host-pathogen interactions (Xu et al 2018, Shopova et al 2020). In fungi, the first report of fungal EVs was published in 2007 (Rodrigues et al 2007), and, since then, their existence has been reported in many species of pathogenic and nonpathogenic fungi (Rizzo et al 2020).…”

Omics Approaches for Understanding Biogenesis, Composition and Functions of Fungal Extracellular Vesicles

“…Immunofluorescence of C. neoformans surface lipids revealed plasma membrane projections, suggesting that the plasma membrane could bud and release EVs ( Rodrigues et al, 2000 ). The participation of the plasma membrane in fungal EVs formation was also shown using “wall-less” Aspergillus fumigatus cells ( Rizzo et al, 2020a ). Ultra-resolution microscopy analyses of these fungal protoplasts demonstrated the occurrence of EVs budding from the plasma membrane.…”

“…In addition, the deletion of the sterol biosynthesis gene Erg6 induced changes in the lipid and protein content of C. neoformans EVs, suggesting a role for sterols in EVs formation ( Oliveira et al, 2020 ). Studies have shown that fungal EVs can originate from intracellular organelles, such as endosomes ( Oliveira et al, 2010 ; Zarnowski et al, 2018 ; Zhao et al, 2019 ; Park et al, 2020 ), or at the plasma membrane ( Rodrigues et al, 2000 , 2013 ; Rizzo et al, 2020a ). Morphological studies of C. neoformans showed structures resembling multivesicular bodies (MBVs) that can fuse to the plasma membrane, resulting in the release of intraluminal MBV vesicles into the fungal periplasm ( Takeo et al, 1973 ).…”

“…Shedding of these plasma membrane-derived EVs was increased during cell wall synthesis, suggesting their participation in this process. Accordingly, protoplast EVs contain cell-wall polysaccharides and polysaccharide synthases, which are plasma membrane-associated enzymes ( Rizzo et al, 2020a ). These morphological and compositional studies support the presence of plasma membrane-derived EVs in fungi similar to the mammalian microvesicles (or ectosomes; Raposo and Stoorvogel, 2013 ; Stahl and Raposo, 2019 ).…”