Characterizing extracellular vesicles of human fungal pathogens

Abstract

Since their discovery in 2007, there has been growing awareness of the importance of fungal extracellular vesicles (EVs) for fungal physiology, host–pathogen interactions and virulence. Fungal EVs are nanostructures comprising bilayered membranes and molecules of various types that participate in several pathophysiological processes in fungal biology, including secretion, cellular communication, immunopathogenesis and drug resistance. However, many questions remain regarding the classification of EVs, their cellular origin, passage across the cell wall, experimental models for functional and compositional analyses, production in vitro and in vivo and biomarkers for EVs. Here, we discuss gaps in the literature of fungal EVs and identify key questions for the field. We present the history of fungal EV discovery, discuss five major unanswered questions in fungal EV biology and provide future perspectives for fungal EV research. We primarily focus our discussion on human fungal pathogens, but also extend it to include knowledge of other fungi, such as plant pathogens. With this Perspective we hope to stimulate new approaches and expand studies to understand the biology of fungal EVs.

Extracellular vesicles (EVs) are produced by all major classes of organism studied to date¹. Fungal EVs—as in other organisms—are bilayered membrane structures comprising several types of biomolecules, including proteins, polysaccharides, RNA, pigments and small molecules (Fig. 1).

Fig. 1 |. Composition of fungal EVs.

Fungal EVs can carry a wide variety of biomolecules, including proteins, polysaccharides, RNA, pigments and small molecules. The membrane structure of fungal EVs (inset) is still poorly understood, although microscopic evidence suggests a bilayered lipid membrane adorned with proteins and glycans. The presence of integral membrane proteins, as well as outer polysaccharides and mannoproteins decorating the vesicular surface, has been described in fungal EVs⁵. Although other membrane components remain poorly characterized, it has been suggested that fungal EVs can be coated with lectins and pathogen-associated molecular patterns^109–111.

The International Society for Extracellular Vesicles defines EVs as particles released from cells, surrounded by a lipid bilayer and unable to replicate independently². The most common subtypes of eukaryotic EVs are exosomes and microvesicles¹. Exosomes originate from the endosomal system and microvesicles arise through blebbing from the plasma membrane. Microvesicles are typically larger than exosomes. In most studies reporting on EVs, a broad population of vesicles is probably being investigated rather than a specific subtype, unless the subcellular origin of the EVs can be demonstrated. Based on the International Society for Extracellular Vesicles’ recommendations, classification of fungal EVs into exosomes or microvesicles is not recommended. Although there is evidence that fungal EVs can include these and other subtypes depending on the organism and analytical conditions^3–5, the precise subcellular origin of fungal EVs has not yet been determined. We recommend the general term fungal EVs, since it aligns with the definition of lipid-bilayered particles released from cells with no ability to replicate independently.

In eukaryotes, EV formation generally starts intracellularly or at the plasma membrane level, resulting in vesicle release to the extracellular milieu⁶. However, for cells with complex cell walls, such as prokaryotic bacteria and archaea, as well as eukaryotic fungi and plants, understanding the production and release of EVs to the outer environment is challenging. This is because their transit through the cell wall is yet to be defined. The cell wall is a dynamic but dense layer mostly comprising polysaccharides and glycoproteins (in plants and fungi)⁷ or hybrid glycans (in Gram-positive bacteria and mycobacteria)⁸. In all of these cases, the cell wall is a hydrophilic structure with viscoelastic properties that must be compliant to the trafficking of membrane-containing structures.

The release of EVs across the cell wall has been associated with physiological processes in fungi such as secretion and prion propagation, as well as immunological and immunopathological fungus–host interactions⁹. Notably, fungal EVs trigger interferon signalling¹⁰. However, essential questions regarding the physiological relevance of fungal EVs and their functions during host–pathogen interactions remain unanswered¹¹. In this Perspective we explore the properties of fungal EVs and discuss the knowledge gaps and future research directions in this field.

History of fungal EVs

Box 1 describes the accumulation of microscopic evidence for the occurrence of fungal EVs over time. Fungal EVs were discovered and formally defined in 2007¹²; however, there were hints of their existence long before that. To our knowledge, the first visual suggestion of fungal EVs dates back to 1958 in the wood-decay fungus Polystictus versicolor¹³, now known as Coriolus versicolor (the turkey tail mushroom). In this model, Girbardt described cellular components that were defined as different-shaped structures located outside the plasma membrane¹³. Using the current terminology, these structures resemble vesicles being released into the periplasmic space after fusion of multivesicular body (MVB)-like organelles with the plasma membrane. MVBs originate from endosome maturation and contain multiple internal vesicles formed by the inward folding of their membrane. Similar MVB-like structures fused with the plasma membrane were observed by freeze-fracture transmission electron microscopy (TEM) for the encapsulated pathogen Cryptococcus neoformans in 1973 during murine infection¹⁴. In this study, Takeo and colleagues describe a “bag-like paramural body showing multivesicles”, with some of the “paramural bodies found to be multivesicular systems”. These observations were associated with a large number of vesicles during infection compared with in vitro growth and a “high-secretion activity in C. neoformans grown in the parasitic state”. However, Takeo et al. restricted their observations to the cell wall and inner capsule and did not venture into the space outside the capsule, which presumably was thought to provide a barrier to extracellular transit.

BOX 1. Timeline of microscopic evidence for the occurrence of fungal EVs.

MVB-like structures associated with the plasma membrane were reported in 1958 for C. versicolor¹³. Microvesicle-like structures were reported in 1972 for A. nidulans¹⁵. MVB-like structures associated with the cell wall were detected in 1973 for C. neoformans using freeze-fracture electron microscopy¹⁴. Isolated chitosomes produced by Mucor rouxii were described in 1976 (top image) and shown to catalyse the synthesis of cell wall fibres (bottom image)¹⁹. EV-like structures associated with the outer cell wall were reported in 1990 for C. albicans²⁰ and at the cell wall–plasma membrane interface in 2000 for C. neoformans¹⁶. EVs were first isolated from fungal supernatants in 2007 (top image), when they were also detected in the inner layers of the cell wall (bottom image)¹². With the advent of cryo-EM, fungal EVs were analysed in detail in vitro and in vivo. EVs of Golovinomyces orontii were observed in the periplasmic space and interfacial matrix between a G. orontii haustorium and an Arabidopsis thaliana epidermal cell in 2011¹¹². EVs were detected (arrowhead) within the apical cell wall of an N. crassa hypha in 2020⁵⁶ (the white asterisk indicates a Spitzenkörper macrovesicle). In 2021, cryo-electron tomography revealed two vesicle populations with either no surface decoration (top image) or fibrillar decoration (bottom image) in C. neoformans⁵. In 2022, EVs of Colletotrichum lindemuthianum were observed in the periplasmic space (black arrowheads in top image) and interfacial matrix between infection hyphae and Phaseolus vulgaris cells (black asterisk in bottom image)²⁴. Note that several other studies reported EVs by electron microscopy after 2007. Owing to space limitations, we have only included images generated by cryo-EM. For C. neoformans (2021), the images to the right show a magnified view of the parts of the images to the left indicated by a dashed box. CW, cell wall; FP, fungal plasma membrane; FW, fungal wall; PP, plant plasma membrane. Photographs adapted with permission from (top left to bottom right): C. versicolor, ref. 13, Springer Nature Limited; A. nidulans, ref. 15, Society for General Microbiology; C. neoformans (1973), ref. 14, American Society for Microbiology; M. rouxii (top and bottom), ref. 19, National Academy of Sciences; C. albicans, ref. 20, American Society for Microbiology; C. neoformans (2000), ref. 16, American Society for Microbiology; C. neoformans (2007, top and bottom), ref. 12, American Society for Microbiology; C. neoformans (2021, top and bottom), ref. 5 under a Creative Commons license CC BY 4.0; C. lindemuthianum (top and bottom), ref. 24 under a Creative Commons license CC BY 4.0. Photograph of N. crassa courtesy of Rob Roberson.

Other mechanisms resulting in EV release were also documented before 2007. Vesicles possibly budding out of the plasma membrane were reported in 1972 for the filamentous fungus Aspergillus nidulans¹⁵ and in 2000 for C. neoformans¹⁶. In A. nidulans, small vesicles were observed near the plasma membrane, in addition to outer membrane blebbing¹⁵, resembling the shedding process currently associated with microvesicle formation¹. However, it cannot be ruled out that the structures reported in this study were membrane tubules rather than vesicles or blebs, as previously reported in fungal cells undergoing plasmolysis¹⁷—a process through which cells lose water in a hypertonic solution. Interestingly, the authors of the A. nidulans study¹⁵ suggested that the vesicles may be involved in wall formation. Membrane shedding resulting in EVs associated with cell wall synthesis was demonstrated decades later in the fungal pathogen Aspergillus fumigatus¹⁸, but earlier studies by Bracker and colleagues¹⁹ in the 1970s demonstrated vesicle-like structures named chitosomes in association with the synthesis of cell wall fibres. It remains unknown whether chitosomes are related to EVs, but their morphology and possible involvement with cell wall synthesis are consistent with this hypothesis.

In C. neoformans¹⁶, the detection of glycosphingolipids on the cell wall was associated with possible vesicle transport across this structure¹⁶. Glycolipid-containing vesicles were observed at the cell surface and were shown to move across the periplasmic space and deposit cell membrane constituents on the cell wall¹⁶. Structures resembling EVs in association with the plasma membrane–cell wall interface, in addition to the outer layers of the cell wall, were also reported in 1990 for the opportunistic fungal pathogen Candida albicans²⁰. In this study, external EV-like structures were described as “emerged blebs enclosed by a discernible double membrane”.

Although there might be additional earlier studies suggesting the existence of fungal EVs that we do not discuss here, these reports underscore the notion that EVs were visualized but not characterized as a distinct transport system before 2007. These studies mostly relied on TEM and were accompanied by concerns that vesicle-like structures could be artefacts of microscopy and lipid aggregation, whereas the cell wall was considered a barrier impenetrable to such large structures, contradicting the existence of EVs²¹.

In 2007, a study on the trans-cell-wall transport of polysaccharides in C. neoformans demonstrated membrane-containing structures associated with the fungal cell wall, leading to the hypothesis that these membranous particles could be isolated from culture supernatants¹². Ultracentrifugation protocols in association with electron microscopy revealed that these particles could be isolated from culture fluids of C. neoformans and they contained polysaccharides that were used by the fungus for assembly of the cell surface^12,21. Initial investigations with human fungal pathogens were extended to a wide range of species, including plant fungal pathogens. For instance, in Botrytis cinerea, TEM of infectious hyphae and hyphae grown in vitro revealed EVs of various sizes and densities²². In Ustilago maydis, EV-enriched fractions contained messenger RNAs that were upregulated during infection²³.

Post-2007 research on fungal EVs was based on cryo-fixation techniques to preserve membranes in their near-native state. Cryoelectron microscopy (cryo-EM) can be used for imaging isolated fungal EVs and has revealed new features, including fibrillar decorations on the EV surface. These decorations mainly correspond to mannoproteins of still unknown functions⁵. Plunge freezing or high-pressure freezing followed by freeze substitution and resin embedding are suitable for cryo-EM imaging of EVs in thicker samples, such as cultured hyphae and infected plant tissues.

What is the origin of EVs?

The exact subcellular origin of fungal EVs has not yet been identified. There are multiple possible origins of fungal EVs, as illustrated in Fig. 2 and discussed below.

Fig. 2 |. Potential mechanisms of EV biogenesis in fungi.

Exosome biogenesis starts with plasma membrane invagination (i) and the subsequent endocytosis of external substances (represented as blue, irregular particles). This process leads to the formation of endosomes, followed by degradation of the ingested molecules. Endosomes can generate intraluminal vesicles (red spheres) by inward folding of their membranes, forming MVBs. MVBs can fuse with the plasma membrane and release intraluminal vesicles extracellularly. Alternatively, microvesicle-like EVs can form by membrane budding (ii). In fungi, these vesicles are deposited in the periplasmic space between the plasma membrane and cell wall, where they are directed to the outer space by as yet unknown mechanisms.

Endosomal origin of fungal EVs

The functions of eukaryotic MVBs in sorting and trafficking molecules, in addition to exosome formation, are widely known. In fungi, as in other eukaryotes, morphological studies suggest that MVBs fuse with the plasma membrane, releasing intraluminal vesicles into the periplasmic space (Fig. 2)^14,24. From here, the fungal EVs could traverse the cell wall to the extracellular space. Interestingly, a C. neoformans mutant lacking P4-ATPase Apt1p—one of the eukaryotic flippases responsible for regulating lipid asymmetry in biological membranes—exhibits aberrant MVBs and produces EVs with altered dimension and composition^25,26. This suggests that flippases are indirect regulators of EV formation in fungi, implying that this class of enzymes could be targeted using genetic and/or pharmacological tools for functional studies of fungal EVs.

Using a proteomics approach, Oliveira et al.²⁷ concluded that Saccharomyces cerevisiae mutants lacking proteins responsible for MVB formation still produce EVs but have altered protein composition. In fact, only a few studies point to direct connections between the functionality of MVBs and EV formation in fungi. The subunits of the endosomal sorting complexes required for transport (ESCRT) are necessary for EV formation in eukaryotes²⁸. In a C. albicans model of biofilm formation, 16 out of 21 mutants defective in orthologues of ESCRT proteins (Vps27, Hse1, Vps23, Vps28, Mvb12, Srn2, Vps22, Vps25, Vps36, Vps2, Vps20, Snf7, Vps24, Bro1, Doa4 and Vps4) showed slightly reduced EV production²⁹. Together, these observations suggest that fungal cells produce EVs that could be related to mammalian exosomes.

Plasma membrane origin of fungal EVs

Plasma membrane blebbing in association with the cell wall has been repeatedly reported in fungi, including S. cerevisiae³⁰, Schizosaccharomyces pombe³¹, C. neoformans¹⁶ and C. albicans²⁰. Indeed, some of the essential eukaryotic components for constructing the plasma membrane play a crucial role in the formation of fungal EVs. In C. albicans, genes associated with phospholipid biosynthesis influence both the morphology and cargo of EVs³². Deletion of AIM25, a gene encoding a presumed scramblase, impacts the generation of cryptococcal EVs³³. An Δaim25 mutant strain produced EVs that exhibited larger dimensions and differences in small RNA cargo compared with those from the wild-type strain³³.

A major challenge in studying the formation of EVs at the plasma membrane level in fungi arises owing to the presence of a thick and complex cell wall. In 1998, Osumi³¹ noted structures resembling microvesicles in protoplasts (cells with an artificially degraded cell wall) of S. pombe. More recently, Rizzo et al.¹⁸ used A. fumigatus protoplasts to investigate the release of EVs from the plasma membrane. More recently, the integration of fluorescence microscopy, super-resolution fluorescence microscopy, high-resolution scanning electron microscopy and TEM has made it possible to image plasma membrane projections and the release of particles resembling EVs in greater detail¹⁸.

The idea that fungal EVs can originate at the level of the plasma membrane is supported by early studies that were not specifically investigating EV biology. In C. neoformans, plasma membrane projections forming vesicle-like structures in contact with the cell wall were identified two decades ago¹⁶. Similarly, in S. cerevisiae, plasma membrane invaginations were observed to lead to the formation of vesicular structures containing cytoplasmic material that were subsequently transported into the periplasmic space³⁴. This observation aligns with the identification of cytoplasmic proteins in fungal EVs^5,24,35–37. Of note, these plasma membrane invaginations resemble eisosomes, which comprise plasma membrane protein complexes that mark the site of endocytosis in some eukaryotes³⁸. As reported for C. albicans, the eisosome component Sur7 is present in EVs^24,39,40. However, further studies are necessary to investigate whether the EVs derived from cytoplasmic substrates in S. cerevisiae are associated with eisosome formation. Recent studies indicate that eiosomes do not directly participate in endocytosis, but rather play diverse roles in fungal lipid homeostasis, plasma membrane organization, cell wall synthesis regulation and stress responses^41,42. However, the molecular mechanisms necessary for EV formation at the plasma membrane are largely unknown.

How do EVs cross the cell wall bidirectionally?

In cell-wall-containing organisms, such as plants, fungi and bacteria, crossing the cell wall is necessary for molecules to reach the extracellular space. In addition, several fungal species produce extracellular matrices (three-dimensional frameworks comprising carbohydrates, proteins, lipids and nucleic acids)⁴³, which may pose a further barrier to EV movement. For decades, it has been known that cell wall-containing organisms efficiently export various molecules, as exemplified by the ability of yeasts to secrete biologically active invertases⁴⁴. In fungi, the cell wall is recognized for its viscoelastic nature and its ability to withstand hydrostatic turgor pressure^45,46. This property results from a combination of rigid chitin chains with flexible β-1,3-glucans⁴⁷, which are cross-linked among themselves and with other cell wall components. This semi-permeable barrier enables fungal cells to regulate their volume and facilitate or restrict the diffusion of macromolecules. In C. neoformans, the cell wall contains small pores at the nanometre scale, even in the presence of dense melanization⁴⁸. EV passage through the cell wall therefore requires the interaction of these membrane structures with fungal cell wall components.

Despite our understanding of the fungal cell wall, the mechanisms through which large molecules traverse it remain poorly understood. When fungal EVs were initially described, it was suggested that vesicular export might facilitate transport across the cell wall¹². The detection of various extracellular macromolecules in fungal EVs, including pigments, polysaccharides, proteins, RNAs and small molecules, supported this hypothesis (reviewed in refs. 49,50). A possible association between vesicles and the cell wall agrees with studies conducted in the 1960s and 1970s, which detected lipids in cell wall fractions of fungi^51,52. More recent studies demonstrated that fungal EVs indeed contribute with a substantial portion of cell wall lipids comprising the cell wall^53,54. Additionally, observations using different microscopy methods revealed vesicle-like structures associated with the cell wall, spanning both inner and outer layers^12,24,36,55. Although TEM data indicate the passage of EVs through yeast cell walls, there is limited evidence for EVs transiting the cell walls of filamentous fungi. This is surprising if one assumes that EVs are abundantly secreted by filamentous fungi. A rare example of EV passage through the cell wall was provided in Neurospora crassa in 2020⁵⁶. The study showed that membrane-bound spherical-to-ovoid inclusions are present within the apical cell⁵⁷.

The presence of cell-wall-associated vesicles in fungi, along with the description of cell wall pores⁵⁷, lends support to the existence of mechanisms to facilitate vesicle passage through the fungal cell wall. Several hypotheses have been proposed to explain this phenomenon. For example, proteomics studies have identified dozens of cell wall hydrolytic enzymes in fungal EVs^5,37,58,59. Thus, as suggested for bacteria, EVs might catalyse their own passage through the wall by breaking down some of its structural components in cases where polysaccharide-degrading enzymes are associated with the EV surface⁶⁰. Corroborating this hypothesis, EVs released by the fungal pathogen Histoplasma capsulatum carry substantial amounts of active chitinase-1 (Cts1)⁶¹. Inhibiting the enzymatic activity of Cts1 with methylxanthines significantly decreased the release of EVs by H. capsulatum yeasts, directly impacting fungal virulence⁶¹. Cts1 possesses a glycosylphosphatidylinositol attachment site, suggesting its presence in the external layer of the plasma membrane and possibly in EV membranes⁶². This observation, along with the fact that chitinase activity was detectable in intact EVs, suggests that Cts1 is localized on the surface of these EVs⁶¹. Although this study aids our understanding of the release of EVs by H. capsulatum, the chitinolytic activity detected in EVs from C. albicans, Candida parapsilosis and S. cerevisiae was very low or even absent, suggesting differences between species. In addition, it remains unclear whether the presence of hydrolases in EVs correlates with cell wall degradation.

Given that fungal EVs can traverse the cell wall, an important question arises: do EVs contribute to cell wall remodelling? As previously discussed, the passage of EVs through the cell wall of H. capsulatum may be influenced by chitinases⁶¹, but other enzymes capable of hydrolysing structural compounds, such as glucanases and proteases, have also been characterized as EV components³⁵. The direct effect of these enzymes on the cell wall network has not been thoroughly investigated. Nevertheless, proteomic analysis of fungal EVs revealed the presence of enzymes associated with glycan and polysaccharide synthesis, suggesting that EVs may contribute to cell wall synthesis and remodelling^18,59,61. Indeed, protoplasts of A. fumigatus grown under conditions stimulating cell wall regeneration contained a fibrillar material closely associated with EV-like compartments¹⁸. Notably, EVs isolated from cell wall regeneration conditions exclusively contained N-acetyl-galactosaminyl residues and exhibited a higher glucose content than EVs isolated from non-regenerating conditions¹⁸. These monosaccharides are the building units of the cell wall polysaccharides galactosaminogalactan and glucan, respectively, suggesting an association between EVs and cell wall synthesis. Work on S. cerevisiae also showed that the higher susceptibility of a mutant strain lacking chitin-synthase 3 to caspofungin was rescued with the addition of EVs from the wild-type strain and by strains overexpressing this enzyme⁵⁹. Understanding the role of fungal EVs in the cell wall may provide insights into the plasticity of this structure and shed light on the mechanisms for the release of proteins and other molecules.

EVs could also be spontaneously released from the periplasmic space during cell division, a process requiring the hydrolysis of cell wall layers to facilitate cell separation. Another possibility is that EVs present in the extracellular space might be propelled extracellularly during cell wall synthesis. This assumes that they would be deposited onto cell wall layers, subsequently becoming more external with cell wall thickening. Finally, the idea that the cell wall is more flexible than initially thought⁶³ aligns with the hypothesis that vesicle traffic could be driven by motor-like proteins, such as chitin synthases with myosin-like domains⁶⁴ and myosin-related proteins detected in association with EVs by proteomic approaches⁶⁵.

In certain fungal species, the cell wall is not the final barrier for EV release. As mentioned earlier, members of the Cryptococcus genus are coated by a thick capsule and EVs promote capsular formation¹². However, both wild-type and acapsular strains of Cryptococcus produce EVs, although the capsule affects vesicular properties^5,58,66. A comparison between the dimensions of EVs produced by a wild-type strain and those produced by an acapsular mutant demonstrated that the majority of vesicles from mutant cells had a diameter of around 50 nm, with most in the range of 20–90 nm and only 4.9% larger than 90 nm⁶⁶. Vesicles from wild-type cells were much more variable in size, ranging from 20 nm to over 200 nm, with 50.6% displaying a diameter larger than 90 nm⁶⁶. Since cryptococcal EVs contain highly hydrophilic capsular polysaccharides¹², we hypothesize that the cargo component could cause alterations in dimensions, since hydrophilic compounds would contribute with the intra-vesicular retention of water.

Several studies have reported the presence of free EVs in cryptococcal supernatants. These observations imply the existence of uncharacterized mechanisms for EVs to traverse the capsule. The structure of the cryptococcal capsule is more porous than that of the fungal cell wall⁶⁷, suggesting that the capsular network is more permissive to vesicle passage, a property that can be influenced by multiple factors. For instance, capsules from C. neoformans cells cultivated for 15 days were significantly less permeable to dextrans than cells cultivated for two days⁶⁸, suggesting that ageing affects capsular permeability. It remains unknown whether similar effects are observed for cryptococcal EVs. It has been reported that the binding of antibodies to the capsule changes its permeability, implying that the host’s immune response might also affect EV passage through the capsule⁶⁹.

There are further potential mechanisms for EV release across the cell wall, some of which we propose here. EVs could be expelled by regulated processes resulting in programmed cell bursting. It is also possible that in some situations EVs do not actually cross the cell wall. After secretion to the periplasmic space, EVs could burst to release their contents, which could then diffuse across the fungal cell wall. Furthermore, turgor pressure could be important for EV passage, but so far these mechanisms have not been explored experimentally.

Previously, it was unclear whether cells of the same fungal species could communicate with each other by taking up EVs, particularly because the cell wall was considered chemically incompatible with the import of membranous structures. Demonstration that EVs serve as vehicles for prion transmission in yeast^70,71 supported the concept that fungal cells could take up vesicles. Further support came from the finding that lipid formulations of the antifungal amphotericin B, such as AmBisome, reach the plasma membrane of C. albicans in their intact form, suggesting that the fungal cell wall is permissive to vesicular structures, potentially through rapid remodelling⁴⁶. C. neoformans was equally permissive to liposome penetration unless the cell wall was melanized⁴⁶. Recent solid-state NMR studies have shown that amphotericin B forms extra membranous asymmetric head-to-tail dimer stacks, which act as sponges that extract ergosterol from the cell membrane⁷². This model does not require the AmBisome liposomal carrier to be transported to the fungal cell membrane, but it must be delivered through the layers of the cell wall. Previous studies⁴⁶ showed that the 60-nm-diameter AmBisome liposome can even transport non-elastic 15 nm colloidal gold particles in the lumen of the liposome through the inner and outer cell wall layers despite the predicted porosity of the cell wall being restrictive to particles above 5.8 nm in diameter⁴⁶.

EVs have also been shown to deliver their components intact to the fungal cytoplasm in human and plant pathogens. In the grey mould B. cinerea, small RNAs are protected and transported into the plant cells by EVs to induce cross-kingdom RNA interference of host genes⁷³. In the human pathogen Cryptococcus gattii, an avirulent strain regained its virulence by the addition of EVs produced by a virulent isolate of the same species⁷⁴. In a plant infection model, Arabidopsis EVs containing small RNA or messenger RNA were taken up by B. cinerea, resulting in disease control and attenuated pathogenicity^75,76. Notably, in the same model, fungal small RNAs were exported in EVs to enter plant cells⁷³. These results suggest the existence of mechanisms for the passage of fungal EVs through the plant cell wall. An exception are fungi that penetrate the wall and produce haustoria or intracellular hyphae in living plant cells.

Plant cells incorporate fungal EVs through clathrin-mediated endocytosis⁷³, but whether similar mechanisms occur in fungal cells remains to be explored. The fact that EVs released by C. albicans yeasts can impede their own filamentation⁷⁷ suggests that EVs can traverse the cell wall to reach the intracellular space, as suggested for Paracoccidioides brasiliensis⁷⁸. Considering that the fungal cell wall can allow the passage of bacterial macromolecules⁷⁹ and even bacteria⁸⁰, these findings may not be surprising. However, the mechanisms by which fungal cells take up EVs remain unknown.

Are EVs produced in vivo?

Demonstrating conclusively that fungal EVs are produced during in vivo infection and have roles in fungal pathobiology is challenging. Standard methods for EV isolation from human or animal fluids will inevitably co-isolate host vesicles, thereby complicating the use of conventional techniques to ascertain the presence of fungal EVs. For example, nanoparticle tracking analysis or TEM cannot distinguish between host and fungal EVs. The detection of fungus-specific molecules (for example, ergosterol) is also problematic, as there remains a possibility that these fungal components were incorporated by host cells and subsequently released within EVs. Thus, there is a challenging need for precise methodologies to definitively demonstrate that fungal EVs are produced during infection.

Most studies characterizing fungal EVs at the structural and functional levels are based on the isolation of EVs from culture supernatants obtained in vitro. Fungal EVs are produced and efficiently detected in fungal cultures developed under diverse conditions, including variable composition of media, temperature, time of growth and fluidity of the cultivation medium (solid and liquid)⁹. EV cargo is strongly regulated by nutritional cues, such as protein availability, reducing agents and vitamins^81,82, indicating that EV production and release are regular physiological events during fungal growth. However, the lack of biomarkers specific to EVs from fungal pathogens of humans makes it challenging to demonstrate their production during infection. In contrast, for bacterial and protozoan EVs, specific biomarkers have supported the design of diagnostics for parasitic and bacterial diseases, including tuberculosis^83–85.

An EV biomarker, pounchless 1 (PLS1), was successfully identified in the plant-pathogenic fungus B. cinerea⁷³. PLS1 is a transmembrane tetraspanin protein that labels secreted fungal EVs. Both mammals and plants also utilize tetraspanin proteins (such as mammalian CD63 and CD9 and plant tetraspanins 8 and 9) as important biomarkers for their EVs, specifically exosomes or exosome-like EVs. Using a PLS1–YFP B. cinerea strain to infect transgenic Arabidopsis plants expressing tetraspanin 8-CFP, EVs from both the plant and the fungal pathogen were observed in the extracellular apoplastic fluid. Since tetraspanins are transmembrane proteins located on the EV surface, it is possible to purify fungal EVs using an affinity capture method, as has been successfully demonstrated in plants⁸⁶.

Indirect evidence supporting the idea that EVs from human fungal pathogens are produced in vivo was presented in early studies. For instance, components from EVs produced by C. neoformans, H. capsulatum and P. brasiliensis were recognized by sera obtained from infected human patients^36,37,58. Vesicle-like structures were microscopically detected in infected tissues in a C. neoformans murine infection model¹², as well as in Pneumocystis carinii-infected rat tissues⁸⁷, but the origin of these vesicles (host or fungi) could not be distinguished. In the plant infection model (citrus fruits), EVs released by Penicillium digitatum were detected in vivo as carriers of fungal toxins, including tryptoquialanines and fungisporin⁸⁸.

The recent use of mass spectrometry and serological tests using fungal EVs for the detection of fungal disease has provided direct evidence for fungal EV production during human infection^89,90. In urine samples from patients with invasive aspergillosis, EVs bearing galactofuranose have been detected^89,91. Notably, galactofuranose, the five-membered ring form of galactose, is abundantly produced by Aspergillus species, but has never been found in mammals⁹². These results strongly indicate that fungal EVs are produced during infection, although the possibility that fungal antigens were endocytosed by host cells and exported in human EVs cannot be ruled out. Similar findings were recently reported for other fungal pathogens, as EVs isolated from the serum and urine samples of patients with infections caused by C. albicans, C. neoformans and P. brasiliensis contained fungal molecules⁹⁰. These data highlight the need for universal EV biomarkers that could be applicable to other models.

Biomarkers, especially those accessible on the EV surface, will also be invaluable for the affinity purification of fungal EVs from infected tissues, where host EVs probably predominate. In the case of plant-pathogenic fungi, many virulence-associated genes are only expressed during host interaction, at specific infection stages or in specialized fungal cell types. The corresponding proteins would not be found in EVs produced by cultured mycelia, so analysis of EVs purified from infected plants will be essential to understand their role in the interaction. Notable progress has been made in the field of plant infections, where the SNARE (Snc1 and Sso2), 14-3-3 (Bmh1) and tetraspanin (BcPLS1) proteins were characterized as fungal EV biomarkers^24,73. These findings encourage the development of similar studies in human fungal pathogens.

What are the functions of subpopulations of fungal EVs?

Most studies investigate a broad population of EVs with undetermined subcellular origin⁹³. This directly impacts functional studies, including those focused on determining biogenesis, drug resistance, biofilm formation and bioactive molecule incorporation by recipient cells, since it is unknown which EV subpopulation is responsible for each biological effect. This also applies to the determination of EV composition since molecules associated with different vesicle subpopulations will be detected^5,35,94. For instance, in immunological studies proposing EVs as vaccine candidates, the observed protective effects could be attenuated by the presence of EV subpopulations with opposite immune functions, so identifying the EV population responsible for a particular phenotype is complex. Furthermore, in studies analysing the composition of EVs, it may be unclear which components belong to each vesicle subpopulation. This is further complicated by the fact that fungal EVs are highly diverse within fungal isolates belonging to the same species³. This highlights the need for better methods to separate and analyse subpopulations of fungal EVs. One approach is to import and adapt methods that have been successfully used for other eukaryotes or bacteria, including gradient centrifugation, genetic and/or chemical screenings for altered EV production, chromatographic separation, affinity chromatography and others. Progress has been made by employing these approaches for separating EV fractions of different densities^3–5, but major uncertainties persist, since the reasons for the variable properties of fungal EVs are still unknown. Experimental evidence based on gradient centrifugation approaches suggests that fungal EV subpopulations may have variable antibody reactivity and morphology, which could be explored for the development of new analytic approaches^3,94.

What are the genetic regulators of fungal EVs?

A limited number of fungal mutant strains deficient in EV production have been identified^4,27,29,95. A screen identifying EV regulators⁴ revealed several transcription factors (Hap2, Gat5, Bzp2 and Liv4), which are positive regulators of EV production in C. neoformans. These transcription factors, as well as the other components of the heterotrimeric HAP complex (a key regulator of mitochondrial function; Hap3 and Hap5 proteins)⁹⁶ regulated EV production in C. neoformans⁴. Although the Δhap2 mutant strain produced less than 5% of the wild-type EV levels, it did not display any major growth defect, suggesting that a wild-type level of EV production is dispensable in this fungus under the conditions tested. The exact mechanisms by which these transcription factors regulate EV production are still unknown. We anticipate that the combination of omics strategies with biochemistry, in vivo and genetic analyses will help to answer some of the current questions concerning the production, composition, diversity and biological roles of fungal EVs.

Future perspectives

As the recognition of the biological importance of EVs produced by fungi grows, more questions emerge regarding their cellular origin, formation and in vivo function(s). By identifying the key knowledge gaps in the field, we hope to stimulate the development of new experimental approaches to generate insights into fungal EVs.

The challenges in identifying the genetic regulators of fungal EVs are significant, especially in filamentous or dimorphic fungi. The production of EVs by filamentous fungi was first described in 2014⁹⁷ and further confirmed in other investigations^{18,22,88,98–106}. However, most of the research on fungal EVs has been conducted in yeast cells. This reflects our limited understanding of how EVs are produced by multicellular fungi. Despite these challenges, progress has been made in this area. Mutant collections are now available for several filamentous fungi and recent advances have been made in the genetic manipulation of EV-producing dimorphic pathogens belonging to the Sporothrix¹⁰⁷ and Paracoccidioides¹⁰⁸ genera, which are known for their genetic complexity. We anticipate that the coming years will witness important advances in understanding how EVs are formed in different morphological stages of fungi.

Fungal EVs can be leveraged for diverse purposes, such as vaccine development and drug design, but deeper knowledge is required for applications to be successful. For example, to design future drugs and vaccines using fungal EVs as targets or prototypes, it is necessary to clarify the pathways involved in their formation and to enhance methods for their separation and functional analysis. Understanding how EVs are produced during infection will aid in designing new interventions. High-resolution microscopy methods combined with genetic approaches may reveal how EVs cross the fungal cell wall. Additionally, identifying biomarkers will enable the creation of widely applicable tools for fundamental biological models, fungal disease diagnosis and microbial pathogenesis.