The proteins in fungal EVs mainly include cytoplasmic and membrane-associated proteins. They also include various types of enzymes, which can increase intercellular communication, by secreting proteins to the surrounding environment. From recent studies, it became established that the plasma membrane proteins Sur7 and EVp1, the actin-binding protein Apb1, and the cell wall mannosylated protein are present in Candida albicans EVs (Nimrichter et al., 2016; Dawson et al., 2020). Proteomic analysis from different fungal EVs revealed the presence of virulence factors and a large number of proteins involved in biofilm formation, pathogenicity, and host-cell interactions (Sun et al., 2020; Zarnowski et al., 2021; Kulig et al., 2022). Proteins involved in vesicle biogenesis, or in the regulation of its size, production, and cargo, were also identified in a mouse model of aflatoxin endophthalmitis (Gandhi et al., 2022a). In recent years, the exploration of fungal virulence-associated proteins has become a hot topic of research. Cryptococcus EVs include heat shock protein (HSP70, etc.), hospholipase B, urease, and laccase (Marina et al., 2020; Park et al., 2020; Parreira et al., 2021; Jung et al., 2022). Analysis of Histoplasma capsulatum vesicles showed the presence of catalase B and superoxide dismutase precursors in these vesicles (Albuquerque et al., 2008). Proteins with antifungal effects, such as histone G and aspergillin, were found in EVs released by Aspergillus fumigatus-triggered polymorphonuclear granulocytes, which inhibited mycelial extension and affected fungal growth (Shopova et al., 2020). Recent studies have established that proteins from Penicillium marneffei and Paracoccidioides brasiliensis-derived vesicles are classified into six categories, based on their functions. These functions include protein/amino acid metabolism, stress/immune response, signal transduction/vesicle formation, carbohydrate/lipid metabolism, cellular organization/biogenesis, and others (Vallejo et al., 2012a; Yang et al., 2020).

Many studies have shown that fungal-derived EVs contain nucleic acids, including RNA (ribonucleic acid) and DNA (deoxyribonucleic acid), which have many different functions. A variety of RNA molecules are present in fungal EVs, including ncRNA, mRNA, snoRNA, snRNA, tRNA, microRNA, miRNA, and asRNA (De Toledo et al., 2019; Munhoz da Rocha et al., 2020). Less extensive research has been conducted on their DNA content, which remains to be explored in depth. EVs mediate nucleic acid export, participate in fungal intercellular communication, and in other cellular processes. Cryptococcus neoformans, Paraccidioides brasiliensis, Candida albicans, and EVs from Saccharomyces cerevisiae could export RNA molecules with different properties and biological functions, including interstrain virulence transfer in Cryptococcus (Peres da Silva et al., 2015; Marina et al., 2020). Cellular small RNA levels differed before and after treatment with the antifungal drug caspofungin. This observation provided useful material for the regulation of gene expression and intrinsic multidrug resistance mechanisms in Candida auris. Differences in virulence among distinct fungal isolates have been related to the RNA composition and content of the respective EVs. Moreover, RNAs from Candida auris EVs were shown to play a role in drug-induced stress signaling and in cell wall maintenance (Peres da Silva et al., 2019; Munhoz da Rocha et al., 2021; Zamith-Miranda et al., 2021; Amatuzzi et al., 2022). In addition, bioinformatic analysis of small RNAs carried by Malassezia EVs showed that these RNAs were not dependent on the biogenesis pathway of the RNAi (Rayner et al., 2017). Several studies have shown that EVs have a different RNA composition from their originating fungal cells. For example, one of the most enriched transcripts in EV exhibits low levels of expression in cells (CNAG_06651 amidohydrolase). there are 73 different ncRNA sequences in Histoplasma capsulatum and EVs of strain G186AR (Peres da Silva et al., 2018; Alves et al., 2019). These results reveal the specificity of nucleic acid transport in fungal vesicles.

Lipids are important components of fungal EVs. The biogenesis of Aspergillus fumigatus EVs includes the formation of vesicles at the cell membrane level (Rizzo et al., 2020b). There is evidence that the lipid composition of fungal EVs also differs from that of its originating cell. For example, comparing the lipid composition of EVs from Candida albicans and Candida auris to the respective whole-cell composition indicates an enrichment of conserved hexosylceramide (HexCer, a lipid substance) in EVs of 67- and 109-fold, respectively (Zamith-Miranda et al., 2021). Therefore, the lipid composition could be a key factor to elucidating the EV biogenesis mechanism. Lipid components of fungal vesicles have the potential to modulate the interaction of fungi with their hosts. It has been shown that fatty acid and triglyceride-rich vesicles extracted from Paraccidioides brasiliensis induced a strong granulomatous response in mice (Alves et al., 1987). In Cryptococcus neoformans EVs, phosphatidylcholine (PC), phosphatidylethanolamine (PE), and sphingomyelin (SM) were the most differentially expressed lipids among the lipidomics associated with Cryptococcus infection. The deletion of the sterol biosynthetic gene Erg6 induced changes in the lipid and protein content of Cryptococcus neoformans EVs, suggesting a role for sterols in their formation (Oliveira et al., 2020; Zhang et al., 2021). In addition, the lipid profiles of EVs cultured in different media were clearly different. Moreover, differences in lipid composition caused by growth conditions may affect virulence and immune potential (Cleare et al., 2020).

Fungal EVs contain a variety of biologically active substances, including cytochromes, carbohydrates, and other compounds. Cryptococcus neoformans produces vesicles containing podoconjugates (the main virulence factor of Cryptococcus neoformans) which are composed of two polysaccharides, glucuronosyl xyloglucan (GXM) and galactomannan (GalXM; LaRocque-de-Freitas et al., 2018; Marina et al., 2020). The carbohydrate metabolites found in Histoplasma capsulatum EVs are also potentially interesting. These compounds have a different glucose content and function in Histoplasma capsulatum EVs cultured in different media sources, possibly due to increased synthesis of glucose in fungal cells or differences in the EV biogenesis mechanism (Cleare et al., 2020). EVs also act as vehicles for the transmission of pathogens, including prions, protein infection factors, and others. Prions from Saccharomyces cerevisiae sup35 could be vertically and horizontally transferred between yeast cells, causing transmissible spongiform encephalopathy (TSE; Kabani and Melki, 2016; Liu et al., 2017).

Fungal EVs affect the morphogenesis and biofilm formation capacity of Candida albicans, stimulating adaptive immune responses. It has been shown that Candida albicans EVs could in vitro modulate yeast to mycelial differentiation, thereby inhibiting biofilm formation and attenuating virulence. Candida albicans EVs treated for 24 h lost their ability to penetrate agar, reduced fungal load in invertebrate models of Candida albicans infection, and were nontoxic when inoculated into Galleria mellonella (Honorato et al., 2022). In addition, both the presence and absence of Candida albicans genes could have qualitative and quantitative effects on EV size, composition, and immunostimulatory phenotype. Genes involved in phospholipid synthesis could affect not only multiple secretory phenotypes, but also the release of EVs carrying microbial cargo. Factors affecting lipid synthesis are important aspects for future studies of secreted virulence factors (Wolf et al., 2015). The characteristics of Candida albicans filamentous cell-secreted EVs (HEV) were compared with yeast EVs (YEV). Interesting differences were observed in the proteomic analyses of the biogenesis and function of mycelial EVs. These were shown to differ from yeast EVs and could be used to discover novel diagnostic markers and targets for the treatment of Candida albicans infections (Martínez-López et al., 2022). To understand the pathogenesis properties of Candida in endophthalmitis, and to validate potential diagnostic and prognostic markers, EVs associated with transport, cell adhesion, membrane fusion, and cytoskeletal organization of differentially expressed proteins (such as aquaporin-5, CD5 antigen-like protein, Gasdermin and vesicle-associated proteins) were studied in a mouse model of Candida albicans and Aspergillus flavus endophthalmitis (Gandhi et al., 2022a,b). Under an atopic dermatitis (AD) setting, Candida albicans promoted altered glycosylation patterns in keratinocyte-derived EVs, to interact with inhibitory Siglecs on antigen-presenting cells. Strategies that target this pathway to enhance antifungal responses and limit pathogen transmission may provide new therapeutic options for AD cutaneous candidiasis (Kobiela et al., 2022). In Candida albicans EVs, the plasma membrane proteins Sur7 and EVp1 could be fungal equivalents of four transmembrane protein tags for EV studies (Dawson et al., 2020). The new antifungal drug turbinmicin disrupts the transport of EVs during biofilm growth and thus exhibits antifungal effects (Zhao et al., 2021). Candida auris EVs induced the activation of phagocytes and increased phagocytosis, thereby regulating host cell defense mechanisms (Zamith-Miranda et al., 2021; Chan et al., 2022). Saccharomyces cerevisiae EVs activated receptors on immune cells and could be used as a novel vaccine material for immune cell maturation (Higuchi et al., 2023).

Cryptococcus neoformans mutants lacking the gene coding for steryl glucosidase (Δsgl1), enriched in sterol glucosidase-catalyzed steryl glucoside (SGs), and containing polysaccharide glucuronide oxyglycans (GXM) extracellular vesicles could delay the acute lethality of Galleria mellonella infection to Cryptococcus neoformans. This observation demonstrated the potential use of Δsgl1 EVs as a vaccination strategy for Cryptococcus neoformans (Colombo et al., 2019; Rizzo et al., 2021). A vesicular peptide (isoleucine-proline-isoleucine, Ile-Pro-Ile) from EVs produced by Cryptococcus gattii improved the survival of Galleria mellonella infected by Cryptococcus (Reis et al., 2021). Immunization with EVs had a positive impact on mice infected with Paracoccidioides brasiliensis. Specifically, it induced the mobilization of activated T lymphocytes and natural killer T cells to the infected lungs, improved the production of pro-inflammatory cytokines and histopathological features, and reduced the fungal load (Baltazar et al., 2021). Sporothrix brasiliensis EVs co-cultured with macrophages could enhance the fungicidal activity (assessed through the phagocytic index) of the macrophages (Campos et al., 2021). Aspergillus EVs enhanced phagocytosis and macrophage cytotoxicity, triggering the release of a unique set of antifungal EVs (afEVs) by polymorphonuclear granulocytes (PMNs). Infection with differentiated PLB-985 neutrophil-derived extracellular vesicles limited the growth of Aspergillus fumigatus hyphae, which also demonstrated antifungal effects (Rafiq et al., 2022). As an immunological tool, pre-exposing mice to EVs from Aspergillus fumigatus induced the production of a fungal antigen-specific IgG-rich serum, reduced alveolar infiltration by neutrophils, prevented extensive lung tissue damage, and improved phagocytosis and fungal clearance (Souza et al., 2022). EVs released by Aspergillus flavus enhanced the phagocytosis and cytotoxicity of macrophages. In addition, proteins from these vesicles could produce marker molecules that provided information about the physiological and disease status induced by the fungal infection. These marker molecules could serve as novel prognostic targets for fungal fundus disease (Brauer et al., 2020; Gandhi et al., 2022a).

Candida albicans EVs can mediate the host inflammatory response. Short exposure of macrophages to EVs resulted in the internalization of these vesicles and in the production of nitric oxide (NO), IL-12, TGF-β, and IL-10. Similarly, dendritic cells treated with EVs produced IL-12, IL-10, and TNF-α (Honorato et al., 2022). Significantly elevated levels of IL-6, IL-1β, TNF-α, and IFN-γ were observed in EVs released from mice with Candida albicans endophthalmitis (Gandhi et al., 2022b). In addition, Candida albicans EVs activated the L-arginine/NO pathway to reduce the intracellular levels of reactive oxygen species (ROS) and apoptosis. This outcome was achieved by increasing NO levels and upregulating the expression of the NO dioxygenase gene YHB1, thereby promoting the growth of Candida albicans. During host cell infection, Candida albicans EVs synergistically enhanced the pathogenesis and destructive effects of the fungus on host cells, as well as in the RAW264.7, HOK, TR146, and HGEC cell lines (Wei et al., 2023). EVs originating from Candida glabrata, Candida parapsilosis, and Candida tropicalis induced pro-inflammatory responses in human immune cells and stimulated the secretion of TNF-α and IL-8 (Karkowska-Kuleta et al., 2020; Kulig et al., 2022). Moreover, Candida auris EVs enhanced yeast cell proliferation in macrophages, and Saccharomyces cerevisiae EVs were shown to transport pathogenic prions (Zamith-Miranda et al., 2021; Higuchi et al., 2023).

The EVs secreted by Cryptococcus neoformans could disseminate virulence factors, including heat shock protein (Parreira et al., 2021), phospholipase B (Jung et al., 2022), urease (Marina et al., 2020), laccase (Park et al., 2020), and polysaccharides (Jung et al., 2022). These EVs could also induce an immune response on macrophages, to escape intracellular restriction (Zhang et al., 2021). Cryptococcus gattii promoted intracellular multiplication in macrophages, by secreting EVs and disseminating virulence factors (such as melanin, laccase, and podoconjugates) into the host (Goulart et al., 2010; De Sousa et al., 2022). Malassezia released EVs that induced IL-4 and TNF-α reactions in peripheral blood mononuclear cells (PBMCs; Gehrmann et al., 2011). These could also promote IL-6 production, by participating in NF-κB-dependent signaling pathways (Zhang et al., 2019; Vallhov et al., 2020). Histoplasma capsulatum EVs contain several virulence-related proteins, such as peroxidase (C0NND7) and alkaline phosphatase (C0NBI7; Cleare et al., 2020). Attenuated Paracoccidioides brasiliensis EVs induced in vitro expression of fungal virulence traits and enhanced infection in mice. In addition, EVs from virulent Paracoccidioides brasiliensis stimulated RAW 264.7 cells and bone marrow-derived macrophages to express high levels of inflammatory mediators, specifically TNF-α, IL-6, MCP-1 and NO (da Silva et al., 2016; Octaviano et al., 2022). Incubation of Penicillium marneffei EVs with macrophages resulted in elevated expression levels of ROS, NO, and several inflammatory factors, including IL-1β, IL-6, IL-10, and TNF-α. Proteomic studies have shown that they contain a number of virulence factors, including HSP, MP1p, and peroxidase (Yang et al., 2020). EVs isolated from Sporothrix brasiliensis stimulated the production of IL-12 and IL-6 by macrophages and increased the phagocytic index and fungal load in DCs (dendritic cells). They also increased the levels of immunogenic components and proteins in EVs, which, according to proteomics data, could be related to their virulence (Ikeda et al., 2018; Campos et al., 2021). Aspergillus fumigatus EVs mediated the β-glucan-stimulated IL-1α secretion by neutrophils (Ratitong et al., 2021). Aspergillus flavus EVs induced the production of inflammatory mediators, such as NO, TNF-α, IL-6, and IL-1β, by macrophages (Brauer et al., 2020).

Fungal-derived vesicles provide numerous outstanding advantages for the delivery of biological drugs. Specifically, they show unparalleled superiority as drug carriers, enhancing the solubility of insoluble molecules and their bioavailability, reducing the dose, and providing efficient drug targeting. In addition, bioengineered EVs offer advantages as drug delivery carriers: they have excellent hematological stability and biocompatibility, low toxicity and immunogenicity, as well as a long-distance and targeted transport capability, being also able to cross biological barriers (Figure 3).

Encapsulation of griseofulvin in deformable membrane vesicles (DMVs) for dermal administration has high drug permeability and skin retention, which can overcome its low bioavailability for oral administration. However, this approach also has many side effects and requires a long duration of the treatment (Indian patent application 208/DEL/2009; Aggarwal and Goindi, 2012). Multimolecular liposome/anionosomes containing clotrimazole (the vesicle gel system) were prepared by lipid hydrolysis to provide sustained and controlled release of appropriate drugs for local vaginal therapy (Ning et al., 2005). In addition, an effective ocular nano-vesicle carrier, which provides prolonged and controlled ocular delivery of clotrimazole (CLT) to the carrier system, has also been developed (Basha et al., 2013). Moreover, a novel nanosystem vesicle loaded with itraconazole enhanced corneal permeation and antimycotic activity, to combat Candida albicans infections (Elmeshad and Mohsen, 2016).

Although EV studies are becoming widespread in different biomedical fields, in mycology, this area of research is still in its infancy. Fungal resistance faced in the antibiotic era has created an urgent need for developing more vaccine types against fungal diseases, which are estimated to affect more than 1 billion people worldwide, with significant mortality (Bongomin et al., 2017; Rizzo et al., 2020a). Fungal EV vaccines have the following advantages: first, their high production yield, due to the rapid replication rates of fungal cells and their ability to secrete EVs in large quantities. Second, their multifunctionality indicates the possibility of modifying multiple antigens. Third, the low complexity of their composition. Fourth, their high stability.

The presence of known protective antigens on the surface of EVs suggests their potential for vaccine development. For example, Cryptococcus neoformans EVs contain proteins from the Mp88, Cda, and Gox families, which have immunomodulatory effects. Moreover, EVs obtained from Cryptococcus neoformans mutant strains produced a strong antibody response in mice, greatly prolonging the survival time of animals infected with Cryptococcus neoformans (Rizzo et al., 2021). In the last few years, substantial progress has been made in the host-fungal relationship. Recurrent vulvovaginal candidiasis (RVVC) is characterized by recurrent annual episodes. The virosomal vaccine (PEV7) and the immunotherapeutic vaccine (NDV-3A) can be used to prevent RVVC (de Bernardis et al., 2012; Gil-Bona et al., 2015). Through a comparative proteomic study of Candida albicans and its soluble secretory proteins, EVs from Candida albicans were identified as a new vaccine source by Ana et al. Interestingly, the Bgl2 protein found in vesicles and supernatants without EVs had the potential to prevent systemic candidiasis in a mouse model (Edwards et al., 2018).