Fungal Extracellular Vesicles

Leonardo Nimrichter, University of Rio de Janeiro, Rio de Janeiro, Brazil

Allan J Guimarães, Federal Fluminense University, Rio de Janeiro, Brazil
Marcio L Rodrigues, Federal University of Rio de Janeiro and Carlos Chagas Institute of the Oswaldo Cruz Foundation (ICC-Fiocruz),
Rio de Janeiro, Brazil
r 2018 Elsevier Inc. All rights reserved.

Abbreviations GXM Glucuronoxylomannan

AE Atopic eczema HBMEC Human brain microvascular endothelial cells
APT1 Aminophospholipid translocase 1 HSP60 Heat shock protein 60
CD Cluster of differentiation IL-10 Interleukin-10
Chitin b-1,4-poly-N-acetyl-D-glucosamine IL-12 Interleukin-12
CHS Chitin synthase mAb Monoclonal antibody
Cln1 G1/S cyclin 1 MHCII Major histocompatibility complex class II
DC-SIGN Dendritic cell-specific intercellular adhesion MP65 Mannoprotein 65
molecule-3-grabbing non-integrin PBMC Peripheral blood mononuclear cells
DLS Dynamic light scattering PCA3 Prostate cancer antigen 3
EVs Extracellular vesicles PSA Prostate–specific antigen
GAPDH Glyceraldehyde 3-phosphate dehydrogenase RNA Ribonucleic acid
GlcCer Glucosylceramide TGFb Transforming growth factor b
GRASP Golgi reassembly and stacking protein TNFa Tumor necrosis factor a
GSL Glycosphingolipids WT Wild type

Glossary Lipid raft Membrane micro-compartment enriched in

Cell wall Structural network that surrounds the lipidic cell sterols, sphingolipids, glycolipids and GPI-anchored
membrane in bacteria, fungi and plants, and that confers proteins that regulate functions such as endocytosis and
structural maintenance (rigidity) and osmotic stability to cellular signaling.
the cells. Macrophages Specific type of white blood cells
Chitin Polysaccharide obtained after the polymerization (leukocyte) specialized in the uptake and degradation of
of N-acetyl glucosamine. exogenous particles. There are two types of macrophages:
Dendritic cells Phagocytic cells whose main function is M1 (also known as classical or proinflammatory
the presentation of antigens from internalized pathogens to macrophages), which have a strong antimicrobial activity,
other immune cells (mainly CD4, CD8 and B cells). and M2 (also known as alternative or anti-inflammatory
Endocytosis Process that results in the internalization of macrophages), which have an immunoregulatory function.
exogenous components by cells. When the cells internalize Mannan Polysaccharide formed by polymerization of
solid particles, it is referred as phagocytosis. mannose monomers. Can be divided in N-linked mannan
Exosomes Small intraluminal vesicles (30–150 nm) (polymer attached to an asparagine through a nitrogen
released when multivesicular bodies (MVBs) fuse with the atom) or O-linked mannans (attached to a serine or
plasma membrane. threonine through an oxygen atom).
Extracellular vesicles Any bilayered compartment Microvesicles Extracellular vesicles that are formed by
released by living cells. direct plasma membrane budding.
Galleria mellonella A lepidopteran species widely used as Th1 and Th2 immune response Defense mechanism
an alternative model of host in infectious disease research. elicited by hosts that protect from external challenges,
Glucan Polysaccharide composed of glucose monomers mainly microbes and toxic compounds. Th1 and Th2 are
bound through glycosidic bonds. different types of immune response, being Th1 those known
Laccase Enzyme also known as diphenol oxidase, which is as inflammatory, and Th2 as anti-inflammatory.
involved in some fungi in melanin synthesis from specific
substrates, such as L-DOPA.

Biogenesis of Fungal EVs

EVs are produced virtually by all living organisms, including bacteria, fungi, plantae, protozoa and mammals (Marcilla et al., 2014;
Brown et al., 2015; Rodrigues et al., 2015; Yanez-Mo et al., 2015). The term “EVs” is used to designate any bilayered compartment
released by alive cells. In fungal organisms it includes exosome-like particles, microvesicles (also named ectosomal or shedding

Reference Module in Life Sciences doi:10.1016/B978-0-12-809633-8.12093-X 1

2 Fungal Extracellular Vesicles

Fig. 1 Fungal EVs biogenesis. EVs are formed through at least three distinct mechanisms: (A) multivesicular bodies fusion with plasma
membrane releasing exosomes, (B) plasma membrane budding and (C) cytoplasmic subtraction. In all cases the EVs could reach the extracellular
environment intact.

vesicles) and compartments generated by cytoplasmic subtractions (Gyorgy et al., 2011; Rodrigues et al., 2013) (Fig. 1). Exosomes
are small intraluminal vesicles (30–150 nm) released when multivesicular bodies (MVBs) fuse with the plasma membrane
(Fig. 1(A)) (Yanez-Mo et al., 2015; Rashed et al., 2017) (Fig. 1(A)). Microvesicles are usually larger (100–1000 nm) and assembled
by direct plasma membrane budding (Fig. 1(B)). Cytoplasmic subtractions due to plasma membrane reshaping were recently
described in Saccharomyces cerevisiae (Rodrigues et al., 2013). They are formed through an invagination process followed by a
reorientation of the invaginating membrane to the plasma membrane, with subsequent membrane fusion and release of vesicles
into the periplasmic space (Fig. 1(C)). Fungal EVs usually consist of one or two populations ranging between 50–250 and 300–800
nm (Albuquerque et al., 2008; Rodrigues et al., 2008; Oliveira et al., 2010; Wolf et al., 2014, 2015; Brown et al., 2015; Vargas et al.,
2015). Differences in electron density between fungal EVs have been demonstrated, supporting the idea that EVs could have
multiple biogenesis pathways resulting in distinct cargo (Rodrigues et al., 2008). Remarkably, even strains of the same species can
produce EVs with distinct sizes, confirming the heterogeneity of these compartments (Vargas et al., 2015).
Major components of the cell wall include chitin, b-1,3 and b-1,6 glucans and mannoproteins (regularly found as an outer cell
wall layer of C. albicans and S. cerevisiae).

Fungal EVs Cargo

The mechanisms mentioned above are compatible with distinct vesicle sizes and high diversity of structures carried by fungal EVs,
including proteins lacking secretory signals, metabolic enzymes, RNA, pigment and polysaccharides (reviewed in Nimrichter et al.,
2016). A number of EV components are conserved among different fungal species and strains, but some are species-specific
(Vallejo et al., 2012a, b). The major lipids characterized in fungal EVs are glucosylceramide (GlcCer), sterols and phospholipids
(Rodrigues et al., 2007; Albuquerque et al., 2008; Vallejo et al., 2012a, b; Vargas et al., 2015). The variety of proteins carried
by fungal EVs is enormous (Table 1) and includes proteins involved with multiple metabolic pathways (Rodrigues et al., 2008;
Vallejo et al., 2012a, b; Rodrigues et al., 2014; Wolf et al., 2014; Gil-Bona et al., 2015; Vargas et al., 2015; Wolf et al., 2015; Matos
Baltazar et al., 2016; Nimrichter et al., 2016).
Unexpectedly, major hits found in EVs from distinct fungal species included enzymes involved with glycolysis, fermentation,
gluconeogenesis, pentose phosphate, tricarboxylic acid, and glyoxylate cycles (reviewed in Nimrichter et al., 2016). At least some
of these enzymes are involved in fungal adhesion to host cells, such as enolase, GAPDH and phosphoglycerate mutase (Nimrichter
et al., 2016). However, their participation during EV recognition by host cells has never been investigated. It is plausible that
 Fungal Extracellular Vesicles 3

Table 1 Related functions for proteins characterized in fungal

extracellular vesicles

Related functions for proteins characterized in fungal extracellular vesicles:

Pathogenesis
Lipid metabolism
Carbohydrate metabolism
Cell well architecture
Amino acid and Protein metabolism
Ribosomal proteins
Cellular organization and biogenesis
Cell signaling
Response to stress
Endocytic route

release of fungal EVs loaded with metabolic enzymes can be a mechanism for a rapid change in cell metabolism. Polymers such as
polysaccharides and melanin are also carried by EVs (Rodrigues et al., 2007; Eisenman et al., 2009). Recently, the presence of RNA
was characterized in fungal EVs from several species (da Silva et al., 2015). RNA-containing vesicles could be responsible for
biological processes triggered in host cells during fungal infection or participate during communication among fungi or even with
other organisms.

Traversing the Cell Wall

The plasma membrane is the interface between mammalian cells and the extracellular environment. In contrast, fungal organisms
are encaged in a thick cell wall, which demands EVs to cross this barrier to be released into the extracellular environment
(Rodrigues et al., 2013, 2015). In encapsulated fungal cells, such as those belonging to the Cryptococcus complex, EVs must also
traverse the capsule (Rodrigues et al., 2007). The mechanisms by which fungal EVs traverse fungal outer layers and reach the
extracellular environment is still unclear.
Cell wall composition can change considerably according to the fungal species and/or morphological stage (reviewed by Erwig
and Gow, 2016). However, some common compounds are widely known. These compounds form a network that is basically
composed by a matrix of polysaccharides where different classes of proteins and lipids are embedded (Nimrichter et al., 2005). In
most species the presence of melanin has been reported (Gomez and Nosanchuk, 2003). The major structural polysaccharides
include chitin and glucans and N or O-linked mannans (review in Latge, 2010). The most common prototypes for study of fungal
cell walls are S. cerevisiae and C. albicans, where a layer of mannoproteins is depicted at the edge of the cell wall (Erwig and Gow,
2016). However, other species can have polysaccharides or hydrophobins, such as Histoplasma capsulatum and Aspergillus conidia,
respectively, as outer layers (Erwig and Gow, 2016). In order to support turgor pressure and also permit budding and morpho-
logical changes the cell wall must be robust, elastic and very dynamic.
After their release at the periplasmic space, fungal EVs presumably interact with cell wall components. The exact number of EVs
produced by each fungal cell is not known. Although mechanical pressure could facilitate EV passage through the wall (Brown
et al., 2015), other mechanisms were proposed. For instance, Anderson and colleagues demonstrated the presence of channels at
the cell wall of C. albicans (Anderson et al., 1990). Cell wall protuberances were associated with the edge of these channels where
small vesicles were depicted. In addition, flask-shaped vesicles were observed underneath the base of these protuberances
(Anderson et al., 1990). In C. neoformans, Wolf and colleagues (Wolf et al., 2014) demonstrated that EVs interact directly with the
cell wall. Notably, they also observed multiple or single leaving events between the plasma membrane and the cell wall. Thus the
release of EVs in C. neoformans does not appear to be linked to channels.
EV-mediated cell wall hydrolysis was also proposed as a mechanism facilitating their passage through the cell wall. In fact, EVs
isolated from cultures of C. neoformans, Paracoccidioideis brasiliensis, H. capsulatum and C. albicans carry a number of glycosidases
including, chitinases, glucosidases and mannosidases (Nimrichter et al., 2016). Lipases and proteases are also transported by EVs.
Although the activity of cell wall-related enzymes was never reported in C. neoformans EVs, active laccase and urease were detected
biochemically, supporting the notion that other enzymes can be also functional (Rodrigues et al., 2008). However, direct EV-
mediated hydrolysis has never been demonstrated.
In fungi, production of chitin and glucans occurs at the plasma membrane level (Latge, 2007). Once the newly synthesized
polysaccharides are extruded to the cell wall they can be modified by cell wall enzymes. Polysaccharides are then cross-linked to
each other or to GPI-remnant proteins (De Groot et al., 2005; Latge, 2007). Proteomic analysis suggested that fungal EVs also
participate in cell wall synthesis (Albuquerque et al., 2008; Vallejo et al., 2012a, b; Rodrigues et al., 2014; Gil-Bona et al., 2015;
Vargas et al., 2015). Chitin synthases were reported in EVs released by H. capsulatum, C. neoformans and P. brasiliensis (Nimrichter
et al., 2016). Some chitin synthases are regulated by protease activity (Sburlati and Cabib, 1986). For instance, two chitin synthases
(CHS1 and CHS2) from S. cerevisiae are more active after trypsin or chymotrypsin treatment (Duran and Cabib, 1978; Sburlati and
Cabib, 1986) while the third one (CHS3) is inactivated (Orlean, 1987; Valdivieso et al., 1991). Thus, fungal EVs could potentially
4 Fungal Extracellular Vesicles

contribute to chitin synthesis directly, by their chitin synthases, or indirectly, through their proteolytic activity. Furthermore, EVs
from C. neoformans carry chitin-deacetylase, an enzyme that converts chitin into chitosan. In this pathogen, chitosan is required for
cell wall integrity (Baker et al., 2007). The absence of chitosan affects capsular size and promotes a “leaky” melanin phenotype
(Baker et al., 2007).

Host Cell Modulation and Impact on Disease Development

Since fungal EVs carry a combination of virulence factors, it has been speculated that they could impact host cell responses
(Oliveira et al., 2010; Huang et al., 2012; Vargas et al., 2015; Wolf et al., 2015; da Silva et al., 2016). EVs from C. neoformans and
C. albicans co-localized with the lipid raft marker GM1 at the surface of macrophages and dendritic cells (Oliveira et al., 2010;
Vargas et al., 2015). Fusion between fungal EVs and the host plasma membrane was not detected. EVs were internalized after few
minutes of interaction, with a complete internalization after 1 h (Vargas et al., 2015). Exposure for longer periods demonstrated
that EVs from C. neoformans and C. albicans are not toxic to primary mammalian cells (Oliveira et al., 2010; Vargas et al., 2015).
Together these findings indicate that cell surface receptors are required for EV association with and internalization by phagocytes.
Accordingly, Peres da Silva and colleagues reported that EVs from P. brasiliensis display mannose and N-acetylglucosamine residues
that could be recognized by DC-SIGN (Peres da Silva et al., 2015). Internalization of fungal EVs by phagocytes culminated with
production of nitric oxide and cytokines such as IL-12, IL-10, TNFa and TGFb, confirming their immunomodulatory activity
(Oliveira et al., 2010; Vargas et al., 2015). In addition, the fungicidal activity of macrophages was increased after exposure to EVs
from C. neoformans or P. brasiliensis (Oliveira et al., 2010; da Silva et al., 2016). Furthermore, P. brasiliensis EVs promoted
M1-polarization in macrophages and reversed the M2-polarization stimulated by IL-4/IL-10 (da Silva et al., 2016). The
M1 phenotype is important for host defense during phagocytosis and killing by macrophages (Murray and Wynn, 2011). During
chronic paracoccidioidomycosis the majority of macrophages are M2, favoring a Th2 response and the susceptibility to the
infection (de Carli et al., 2016). Thus, EVs could contribute to paracoccidioidomycosis control.
The ability of fungal EVs to stimulate human cells was also observed. Gehrmann and colleagues showed that Malassezia
sympodialis, a commensal yeast linked to atopic eczema (AE), releases EVs loaded with allergens (Gehrmann et al., 2011).
M. sympodialis EVs were able to stimulate the production of TNFa and IL-4 by CD14- and CD34-depleted PBMC from healthy and
AE patients. Remarkably, the response was significantly higher in M. sympodialis sensitized patients (Gehrmann et al., 2011).
Collectively these results suggested that fungal EVs could activate cells of the innate immune system. Indeed, the capacity to
stimulate the immune system was confirmed using Galleria mellonella larvae. G. mellonella immune mechanisms share a high degree
of structural and functional homology to those used by the innate immune system of vertebrates (Browne et al., 2013). Treatment
of G. mellonella larvae with EVs from C. albicans protected the insect against a subsequent challenge with the pathogen (Vargas et al.,
2015). The number of viable yeasts recovered from EV-treated larvae was significantly reduced when compared to PBS-treated
larvae. These results were accompanied by a higher percentage of larvae survival in the presence of fungal EVs.
Besides impacting the innate immune response, host cell activation mediated by fungal EVs could also change the outcome of
the adaptive immune response as treatment of dendritic cells with EVs from C. albicans enhanced the surface expression of CD86
and MHC-II (Vargas et al., 2015). At least in part, this property could be linked to the high levels of MP65, a major protein in
C. albicans EVs (Gil-Bona et al., 2015; Vargas et al., 2015). MP65 is a major mannoprotein at C. albicans cell surface (Gil-Bona et al.,
2015). In vitro, MP65 stimulates dendritic cells inducing cytokine production, such as TNF-a and IL-6, and increases the expression
of costimulatory molecules. These results indicate that fungal EVs can be potential antigen carriers to be explored in vaccine
formulations.
The ability of fungal EVs to interact with host cells was also investigated in human brain microvascular endothelial cells
(HBMEC) (Huang et al., 2012). Remarkably, treatment of HBMEC with EVs from C. neoformans induced fusion of the host cells
and stimulated redistribution of membrane proteins in lipid rafts. The proteins CD44, a known receptor for C. neoformans in
HBMEC (Jong et al., 2008), and caveolin-1 were significantly enhanced in lipid rafts fractions after exposure to EVs (Huang et al.,
2012). In contrast, a significant reduction in raft-associated b-actin was observed. Thus, EVs from C. neoformans are able to activate
HBMEC and promote changes that facilitate fungal adhesion and their ability to cross the brain blood barrier in vitro. Experiments
in vivo confirmed that C. neoformans EVs could potentially impact disease development. Intravenous injection of EVs in a murine
model of cryptococcosis significantly increased the fungal burden in the brain (Huang et al., 2012).

Genetically Modified Strains as Tools to Investigate Fungal EVs

As mentioned previously, fungal EVs were isolated for the first time from C. neoformans cultures (Rodrigues et al., 2007). To this date,
most of the information based on genetic alterations and EV release in fungal pathogens comes from this model. The attempts to
impair fungal EV release in genetically modified strains generally failed, although important differences in EV properties were
observed. The first gene investigated was SEC6, which encodes a protein that participates in the fusion of exocytic vesicles with the
plasma membrane in yeasts (TerBush et al., 1996). Panepinto and colleagues investigated the role of Sec6 during EV-mediated release
of virulence factors by C. neoformans (Panepinto et al., 2009), including the enzymes phospholipase, urease and laccase, and the
major capsular polysaccharide glucuronoxylomannan (GXM) (Rodrigues et al., 2008). Notably, laccase and urease activities were
 Fungal Extracellular Vesicles 5

significantly reduced in iSEC6 cells. In addition, the amount of secreted GXM was also decreased (Panepinto et al., 2009).
Sec6 suppression in C. neoformans led to vesicle accumulation in the cytoplasm and abrogated EV release. In iSEC6 cells laccase was
restricted to the cytoplasm within vesicular compartments. Although phospholipase activity and capsular size were not affected in the
SEC6 knockout, the iSEC6 strain was less virulent than WT cells. Fungal EVs appeared to be responsible for the correct transport of
virulence factors to the cell surface, contributing to the disease development.
The influence of proteins that regulate membrane asymmetry in EV properties was investigated in C. neoformans (Rizzo et al.,
2014). The gene APT1 encodes a putative aminophospholipid translocase, a class of proteins named as flippases. Their role
in membrane asymmetry helps to control trafficking events that precede secretion. Flippases were linked to EV production in
C. elegans (Tuck, 2011; Wehman et al., 2011). In C. neoformans Apt1 is required for protein secretion and pathogenicity (Hu and
Kronstad, 2010). Deletion of APT1 affected Golgi morphology and GXM secretion, but not capsule size and GXM composition
(Rizzo et al., 2014). The amount of GXM found in EVs from yeasts lacking Apt1, however, was considerably reduced when
compared to vesicles obtained from WT cells (Rizzo et al., 2014).
The connections between proteins involved with unconventional secretion mechanisms and export of fungal molecules were
explored in a few studies. In eukaryotes, Golgi reassembly and stacking protein (Grasp) participates in unconventional mechanisms
of secretion (Kinseth et al., 2007). Deletion of GRASP in C. neoformans affected Golgi morphology (Kmetzsch et al., 2011) and
reduced the dimensions of extracellular polysaccharides, as determined by dynamic light scattering (DLS). These results were
reverted by addition of Ca2 þ to the system, allowing GXM aggregation through calcium bridges (Nimrichter et al., 2007; Kmetzsch
et al., 2011). In agreement with the reduction in extracellularly released GXM the mutants also displayed a hypocapsular phe-
notype. Recent data from our laboratory suggested that GRASP deletion affects EV populations of lower dimensions, suggesting
that Grasp participate in vesicle biogenesis (Luna S. Joffe, unpublished data). EVs are affected by other cryptococcal genes. A
considerable increase in EV release was observed in a C. neoformans strain lacking Cln1, a protein involved with cell cycle
progression (Garcia-Rodas et al., 2014). In addition, deletion of CAP10, a putative xylosyltransferase, resulted in EVs with
significantly reduced dimensions (Tefsen et al., 2014). Notably, increased laccase and acid phosphatase activities were observed at
the fungal cell surface.
The relationship between secretory genes and EV formation was more deeply explored in S. cerevisiae. Vesicle dimensions and
protein composition were analyzed in mutants lacking either conventional or unconventional secretory regulators. All mutants
produced EVs, but amounts, composition and dimensions varied depending on the gene deleted. No direct correlation was
established between secretory mechanisms (conventional or unconventional) and EV formation, since all genes were apparently
required for normal vesicle release. These results are summarized in Table 2.

EVs as Targets of New Antifungal Drugs and Protective Antibodies

The potential participation of fungal EVs in physiological events and disease development suggests that they can become
interesting targets for antifungal drugs and antibodies. As mentioned above GlcCer is a glycosphingolipid (GSL) component of
fungal EVs (Rodrigues et al., 2007; Vallejo et al., 2012a,b; Vargas et al., 2015). Besides its structural function, GlcCer is also a
virulence regulator in C. neoformans and C. albicans (Rittershaus et al., 2006; Noble et al., 2010). Recently, Mor and colleagues
identified a new class of antifungal drugs that indirectly impact the synthesis of GlcCer. One of the effects was the accumulation
of intracellular vesicles in yeasts of C. neoformans (Mor et al., 2015). Other drugs that impact lipid biosynthesis or proteins
involved with EVs biogenesis could impair release of EVs and change the fungal physiology. For instance, Knechtle and
colleagues identified a plant derived diyne-furan fatty acid named as EV-086 as a potent antifungal drug (Knechtle et al., 2014).
This compound targets delta-9-fatty acid desaturation increasing the ratio of saturated/unsaturated fatty acids. Accumulation of
saturated fatty acid would impact directly the membrane composition and fluidity (Ballweg and Ernst, 2017). Consequently, the
function of several proteins could be modified as well as the biogenesis, cargo and release of EVs. These possibilities require
experimental confirmation.
Administration of monoclonal antibodies (mAbs) that recognize structures carried by fungal EVs could be another strategy
to impact EV activity during disease development. Three probable mechanisms are expected for mAb to interfere with fungal
EVs in vivo, including (i) removal of released EVs, reducing their potential deleterious effect in host cells, (ii) preventing initial
passage through the cell wall or (iii) impacting gene regulation in fungal metabolism. All these three mechanisms were

Table 2 Effects of gene deletion on the properties of S. cerevisiae EVs

Gene product Associated secretory pathway Average EV dimensions Protein composition EV amount

Sec4 Conventional Increased Slightly affected Reduced

Bos1 Conventional Not determined Slightly affected Not determined
Sec1 Conventional Not determined Slightly affected Not affected
Snf7 Unconventional Not affected Heavily affected Not affected
Vps23 Unconventional Not determined Slightly affected Not determined
Grasp Unconventional Not determined Not determined Reduced
6 Fungal Extracellular Vesicles

previously described in models of fungal infections involving protective mAbs (Eisenman et al., 2005; McClelland and
Casadevall, 2012). Indeed, it could explain why mAb to GlcCer promotes a protective effect in a cryptococcosis murine model
(Rodrigues et al., 2007). Recently, Baltazar and colleagues investigated whether mAbs to HSP60 impact release and cargo of
EVs in H. capsulatum yeast cells (Matos Baltazar et al., 2016). Noteworthy, HSP60 is a major EV compound in the H. capsulatum
model (Albuquerque et al., 2008). Incubation of yeasts with mAb-HSP60 significantly changed the size of EVs, as well as their
protein content and enzymatic activities. Remarkably, changes in protein composition varied depending on the epitope
recognized by mAbs to HSP60 (Matos Baltazar et al., 2016). This observation confirms that antibody binding can modify the
EV release to the extracellular environment.

Challenges in the Field of Fungal EVs

Although the number of publications and findings regarding fungal EVs increased significantly over the last decade (Joffe et al.,
2016), there are unsolved questions that require further investigation. One major concern is whether EVs are released by fungal
cells in vivo, as well as their amounts, and whether they contribute or not to infection development. Isolation of fungal EVs from
infected tissues will likely represent a breakpoint in the field. On the basis of the current literature, it is plausible that EVs isolated
in vivo could show considerable changes in composition when compared with EVs from culture supernatants. Furthermore, one
must consider that EVs released during infection could express a mixture of fungal and host molecules, which makes these analyses
even more complex. Although a number of methods can be used for EV preparation, successful protocols require a high recovery of
EVs using small volumes of samples. The standard methods are differential and density gradient centrifugation, but the viscosity of
some biological fluids and the similar densities between some EVs limit the use of centrifugation methods. EV markers are now
known in mammalian samples (reviewed in Xu et al., 2016). For instance, survivin, PSA and PCA3 are exosome markers for
prostate cancer in urine samples (Nilsson et al., 2009). Monoclonal antibodies have been used as tools to isolate EVs, including
distinct subtypes, using immunological methods and commercial kits (Taylor et al., 2011; Lasser et al., 2012; Vlassov et al., 2012;
Ghosh et al., 2014; Hudson et al., 2014; Enderle et al., 2015; Lobb et al., 2015; Brett et al., 2017). The absence of a fungal EV marker
is challenging to the field. GlcCer seems to be an interesting fungal EV-marker candidate, although some species including C.
glabrata and S. cerevisiae are unable to produce this GSL (Tavares et al., 2008). Protein markers would also facilitate separation of
EVs originated from distinct biogenesis pathways. Finally, the participation of fungal EVs during cell-to-cell communication has
never been demonstrated, although indirect evidence suggested that it could occur in vitro during fungal differentiation (Leone
et al., 2017).

Acknowledgments

L.N., A.J.G., and M.L.R. are supported by Grants from Conselho Nacional de Desenvolvimento Tecnológico (CNPq, Brazil) and
Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ, Brazil). The authors gratefully
acknowledge Vanessa Koiky for her help with preparation of Fig. 1.

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Further Reading

Brown, L., Wolf, J.M., Prados-Rosales, R., Casadevall, A., 2015. Through the wall: Extracellular vesicles in Gram-positive bacteria, mycobacteria and fungi. Nat. Rev. Microbiol.
13 (10), 620–630.
Joffe, L.S., Nimrichter, L., Rodrigues, M.L., Del Poeta, M., 2016. Potential roles of fungal extracellular vesicles during infection. mSphere 1 (4).
Nimrichter, L., de Souza, M.M., Del Poeta, M., et al., 2016. Extracellular vesicle-associated transitory cell wall components and their impact on the interaction of fungi with
host cells. Front. Microbiol. 7, 1034.
Rodrigues, M.L., Oliveira, D.L., Vargas, G., et al., 2016. Analysis of yeast extracellular vesicles. Methods Mol. Biol. 1459, 175–190.

Relevant Website

https://www.isev.org
International Society For Extracellular Vesicles (ISEV).