Date Published: December 3, 2015
Publisher: Public Library of Science
Author(s): Marcio L. Rodrigues, Rodrigo M. C. Godinho, Daniel Zamith-Miranda, Leonardo Nimrichter, Deborah A. Hogan.
Extracellular vesicle (EV) release in fungi was described for the first time in 2007 in the yeast-like pathogen Cryptococcus neoformans . Since then, the phenomenon of EV production, which is present in all domains of life, has been observed in many different fungal species, including yeast cells and hyphae. Composition of EVs, the impact of their release on fungal pathogenesis, and their potential use as protective immunogens have been explored in a number of original studies and comprehensive reviews (see Fig 1 and  for a summary). However, many aspects related to the biological properties of fungal EVs remain obscure. In this manuscript, we will focus our discussion on three fundamental but still unanswered questions about fungal EVs.
It remains unknown whether fungal EVs are produced in vivo, which is likely linked to the lack of protocols and molecular markers for isolation of these membranous compartments from body fluids. Vesicle properties related to their stability in tissues are also obscure. C. neoformans EVs are rapidly disrupted by serum albumin at physiological concentrations . This observation argues against the stability of EVs in vivo, but not against their potential functions. EV disruption might result in the release of internal and potentially immunomodulatory compounds into the extracellular space, possibly impacting the physiology of host cells.
Exosomes and ectosomes are major EVs produced by eukaryotic cells. Exosomes consist of small (40–100 nm) vesicles originated by invagination of the endosomal compartments membrane, which is driven by a protein complex named endosomal sorting complex required for transport (ESCRT) . This complex regulates the release of small vesicles inside the lumen of the endosome, generating the so-called multivesicular bodies (MVBs). Upon fusion with the plasma membrane, MVBs release exosomes as EVs to the outer space . Unlike exosomes, ectosomes are larger (up to 1 μm), ubiquitous vesicles that are assembled at and released from the plasma membrane . In fungi, mechanisms of vesicle biogenesis and extracellular release are still obscure. Therefore, these extracellular membranous compartments are still collectively called EVs.
The conclusion that fungal EVs are originated either in the cytoplasm or at the plasma membrane levels implies that, to reach the outer space, passage through polysaccharide layers in the cell wall is required. In C. neoformans, three hypotheses are suggested to explain how fungal EVs traverse the cell wall , including (i) movement through channels, (ii) remodeling of the wall to facilitate EV transit, and (iii) mechanical pressure to force vesicle passage through small cell wall pores.
Since the first isolation of fungal EVs in 2007, the progress made in the field was unquestionable. It is now clear that fungal EVs are part of a general mechanism of macromolecule export that results in the extracellular release of immunologically active components with the potential to modulate host responses either in favor of infection control or of fungal dissemination. Major questions, however, remain unanswered. The currently available literature clearly points to the need of improvement of protocols supporting the generation of knowledge on the role of fungal EVs in vivo, as well as on their biogenesis pathways and mechanisms by which they reach the outer space. The availability of sophisticated microscopy tools and well-established protocols for genetic manipulation in a number of fungal pathogens supports the notion that, although still long, the way to understanding the biological roles of fungal EVs can be shortened soon.