The genus Malassezia comprises lipophilic, yeast-like fungi within the phylum Basidiomycota^[1]. These dimorphic microorganisms, which predominantly occur as budding yeast cells, although under certain conditions some species may also produce short hyphal elements^[2], were first described in 1846, with their widely recognized successful isolation and cultivation reported in 1927^[3,4]. Then, research on Malassezia species continued, although early investigations were significantly constrained by methodological limitations, particularly difficulties in cultivation and in establishing appropriate growth conditions. Subsequent studies focused on resolving taxonomic classification challenges, as well as on the detailed characterization of the biological and physiological properties of Malassezia species and their involvement in various pathological conditions^[5]. The nomenclatural problems, resulting in the classification of these fungi in the genus Pityrosporum, were largely resolved in 1995, when particular species were more precisely assigned to the genus Malassezia based on sequencing of the large-subunit rRNA and nuclear DNA complementarity studies^[5]. On the basis of these analyses, seven species of Malassezia were defined and formally recognized, including Malassezia furfur, Malassezia sympodialis, Malassezia obtusa, Malassezia globosa, Malassezia restricta, Malassezia slooffiae, and Malassezia pachydermatis^[6]; then, after 2002, next seven species of Malassezia were described, including Malassezia dermatis, Malassezia equina, Malassezia japonica, Malassezia nana, Malassezia yamatoensis, Malassezia caprae, and Malassezia cuniculi^[7]. The aspects concerning the history, nomenclature, and subsequent research on Malassezia, involving the host immune response to fungal colonization, are comprehensively summarized in the review by Ashbee and Evans^[8].

First evidence suggesting an etiological role for Malassezia species in skin disorders such as dandruff, seborrheic dermatitis (also referred to as seborrheic eczema), and seborrheic manifestations associated with rosacea dates back to 1927^[9]. Furthermore, over the past few decades Malassezia yeasts have attracted increased attention because of emerging evidence that they may also inhabit or translocate to extra-cutaneous anatomical sites or systemic compartments and influence inflammatory diseases beyond the skin^[10,11]. Therefore, recent research has reframed Malassezia yeasts as opportunistic pathogens whose interactions with host immunity and bacterial microbiota could be commensal or pathogenic, depending on host genetics, immune status, and environmental factors^[12].

The skin mycobiome of healthy adults is typically dominated by Malassezia species, among which M. restricta, M. globosa, M. sympodialis, and M. furfur are the most prevalent; however, colonization dynamics, as well as species presence and abundance, vary across the lifespan^[13,14]. The highest prevalence is observed right after birth and during puberty coinciding with increased activity of the sebaceous gland^[15]. Most Malassezia species are unable to synthesize saturated fatty acids due to the absence of fatty acid synthase (FAS) genes; consequently, their unique lipid requirements have driven adaptation to sebaceous, lipid-rich microenvironments (although M. pachydermatis is an exception with the ability to grow on conventional culture media)^[8,16]. Given their reliance on host-derived lipids, Malassezia species hydrolyze and metabolize saturated fatty acids into shorter-chain molecules, thereby influencing the composition of the cutaneous lipid environment^[12,17,18]. Currently diagnosed dermatological syndromes associated with Malassezia include pityriasis versicolor, seborrheic dermatitis, dandruff, and Malassezia-associated folliculitis^[19,20]. In addition, the involvement of Malassezia yeasts has been suggested in psoriasis and atopic dermatitis (AD), as antifungal therapy has repeatedly been reported to alleviate the severity of disease symptoms; however, the causative role of fungal presence in these conditions remains an active area of investigation^[21-24]. Current studies have not consistently demonstrated a direct correlation between the abundance of Malassezia cells on the skin and the clinical severity of AD^[7,21,25-28]; however, the increased level of anti-Malassezia immunoglobulin E (IgE) antibodies (sensitization) was observed to be significantly corelated with disease severity in AD patients^[29-32]. The role of Malassezia in the pathogenesis of AD is hypothesized to involve an additional contribution to excessive immune cell reactivity, as alterations in skin architecture, increased transepidermal water loss, and subsequent changes in the lipid profile influence Malassezia colonization. Since barrier disruption enables microorganisms to penetrate deeper layers of the epidermis, thereby enhancing immune responses following exposure to fungal allergens, Malassezia yeasts may additionally contribute to AD pathogenesis^[21,33].

To date, thirteen allergens derived from Malassezia species have been formally recognized and included in the official allergen nomenclature. These are Mala f 2, Mala f 3 (both are peroxisomal membrane proteins) and Mala f 4 (mitochondrial malate dehydrogenase) from M. furfur, and from M. sympodialis Mala s 1, Mala s 5, Mala s 6 (cyclophilin), Mala s 7-9, Mala s 10 (heat shock protein 70), Mala s 11 (manganese superoxide dismutase), Mala s 12 [glucose-methanol-choline (GMC) oxidoreductase] and Mala s 13 (thioredoxin)^[34,35]. However, analyses of patient sera have also revealed the presence of numerous additional antigens associated with these fungi, with varying degrees of immunological relevance^[8]. Furthermore, species of this genus secrete a variety of enzymes, including proteases and lipases, which may be strongly involved in the modulation of host-microbial interactions^[36,37]. An example of a protease produced by Malassezia is MgSAP1, secreted by M. globosa, which has been shown to disrupt biofilm formation by Staphylococcus aureus, a bacterial pathogen frequently implicated in soft tissue infections and also associated with AD^[38,39]. MgSAP1 has been detected on the skin surface of healthy volunteers and its degradative activity against S. aureus protein A, which is crucial for bacterial biofilm formation, indicated that fungal-secreted protease may influence S. aureus pathogenicity and contribute to the balance of the human skin environment^[13,40]. While the host may benefit from MgSAP1, its homolog MfSAP1 from M. furfur exhibited degradative activity against skin-associated extracellular matrix (ECM) proteins, efficiently degrading vitronectin, human epidermal keratin, thrombospondin, and fibronectin even at low enzyme concentrations^[36]. A knockout mutant strain lacking the protease MfSAP1 exhibited reduced cell adhesion and dispersal, and subsequently caused less edema in a human 3D reconstituted epidermis model. This was later confirmed in a murine model, which demonstrated that MfSAP1 promotes inflammation^[37]. In addition, in M. globosa and M. sympodialis several lipases, phospholipases C, and acid sphingomyelinases have been identified^[35].

Apart from Malassezia association with human skin, the role of commensal fungi in inflammatory bowel disease (IBD) has also been hypothesized. Samples extracted from sigmoid colon and cecum of patients with Crohn’s disease were significantly depleted from Ascomycota, with enrichment of Basidiomycota, especially M. restricta and M. globosa^[41]. The polymorphism in CARD9 (caspase recruitment domain-containing protein 9) gene encoding a signaling protein involved in antifungal immunity, recognized in IBD patients, was shown to be significantly corelated with the presence of Malassezia^[41]. Studies indicate that macrophage and dendritic cell responses to Malassezia yeasts are mediated in part through Dectin-2 and CARD9 signaling, leading to pro-interleukin-1β (pro-IL-1β) production and activation of the NACHT, LRR and PYD domains-containing protein 3 (NLRP3) inflammasome; this highlights the importance of functional CARD9-dependent pathways in coordinating innate immune recognition of these fungi^[42,43]. Furthermore, it has been hypothesized that conditions associated with intestinal malabsorption may create a lipid-rich environment favorable for Malassezia expansion, potentially linking elevated fungal colonization levels with IBD pathogenesis, although direct evidence remains limited^[44].

Even considering current reports and proposed hypotheses, the direct mechanisms by which Malassezia fungi contribute to the onset of systemic diseases have not been fully characterized, particularly with respect to yeast-host cell interactions and the mechanisms of virulence factors transfer and activity. However, growing interest in cross-kingdom communication mediated by extracellular vesicles secreted by diverse cell types has led to the hypothesis that these structures produced by Malassezia species may represent a mechanism underlying fungal involvement in disease development.

Extracellular vesicles produced by fungal cells were first thoroughly characterized in 2007 for the human fungal pathogen Cryptococcus neoformans^[45-47], and have been subsequently described also in other fungi, including Candida albicans and non-albicans Candida species^[48,49]. Fungal vesicles have been identified as nano-sized structures (with a size range of 50-600 nm) capable of transporting enzymes, virulence factors, cytoplasmic proteins, toxins, pigments, quorum sensing molecules, and small RNAs across the fungal cell wall, thereby playing a critical role not only in the regulation of fungal biology, adaptation to environmental stress, biofilm formation and communication among fungi, but also in the interactions with the host^[50-52]. Even with recent advances, numerous aspects of fungal extracellular vesicle characteristics and functionality remain to be fully elucidated^[53]. To date, most studies have focused on the isolation and characterization of populations of extracellular vesicles, examining their size, composition, and molecular cargo. Additionally, comprehensive research has been initiated to explore their functional properties, including roles in intercellular and interkingdom communication, modulation of host immune responses, and contributions to both physiological homeostasis and pathological processes such as infection or inflammation. However, the precise regulatory pathways and factors determining cargo selection, vesicle release, and target specificity remain largely unresolved, highlighting a significant gap in our current knowledge^[47,53,54].

Importantly, despite increasing interest in fungal extracellular vesicles, knowledge regarding vesicles produced by Malassezia species remains remarkably limited. The aim of this review is therefore to consolidate and discuss the currently available evidence, highlighting the potential biological roles of these vesicles, including their interactions with host cells and with other members of the skin microbiota such as S. aureus.

Extracellular vesicles secreted by Malassezia species were first described in 2011 in M. sympodialis and designated by the authors as MalaEx (Malassezia extracellular vesicles)^[55]. The methodology and results concerning their characterization to date are summarized in Table 1^[55-60].

Research on extracellular vesicles produced by Malassezia species is relatively recent, and the available information regarding their composition and biological functions remains limited. Visualization using transmission electron microscopy (TEM)^[55,56,59], field-emission transmission electron microscopy (FE-TEM)^[60] and cryo-electron tomography^[57] has revealed a heterogeneous population of oval-shaped, lipid-bilayer-enclosed structures in the preparations of MalaEx, which is in line with findings reported for other fungal species^[45-54]. TEM analysis of vesicles secreted by M. furfur further demonstrated variations in electron density, implying compositional differences among MalaEx^[59]. Further size analysis of MalaEx secreted by M. restricta using field-emission scanning electron microscopy (FE-SEM) indicated vesicle diameters ranging from 50-150 nm, whereas FE-TEM revealed sizes of 100-200 nm^[60]_. Nanoparticle tracking analysis (NTA) of M. furfur MalaEx showed a size range of 40-400 nm, with a mean diameter of 112 nm^[59], which is consistent with NTA measurements of M. sympodialis MalaEx, revealing vesicles with diameters ranging from 70-580 nm with a mean diameter of 154 nm^[58]. These sizes are also within the range reported for extracellular vesicles produced by other fungal species^[45-54].

NTA analysis performed by Johansson et al. further demonstrated that culture conditions influence vesicle size^[57]. Fungal cells cultured in RPMI 1640 medium produced vesicles with a mean diameter of 171 nm, whereas MalaEx produced by M. sympodialis cultured for 72 h in mDixon broth had an average size of 245 nm^[57]. After sucrose gradient purification, Gehrmann et al. reported vesicle sizes ranging from 50 to 200 nm for M. sympodialis MalaEx^[55]. Using immunoelectron microscopy (iEM), the authors observed antigen localization on the surface of MalaEx, which was subsequently confirmed by Western blot analysis using polyclonal rabbit immunoglobulin G (IgG) against M. sympodialis^[55]. Among the identified antigens, an approximately 70 kDa allergen was detected and shown to be recognized by IgE-containing serum from a patient with atopic eczema sensitized to M. sympodialis^[55].

Further proteomic analyses revealed that M. sympodialis MalaEx isolated from cells grown in mDixon broth at 32 °C for 3 days contain multiple allergens and metabolic enzymes^[55,57]. Among the identified allergens, MalaEx were enriched in Mala s 1 and Mala s 7, both of which may contribute to the pathogenesis of inflammatory skin disorders such as AD. Mala s 1, a phosphoinositide-binding protein primarily localized to the fungal cell surface, has been suggested to participate in post-secretory modification of secondary metabolites. Mala s 7 belongs to an amplified allergen gene family. It is believed that gene duplication promotes the diversification of functions, therefore influence the virulence in M. sympodialis^[55,57]. Additional allergens including Mala s 5, s 6, and s 8-13, were also identified in MalaEx but not enriched relative to cellular protein contrary to Mala s 1 and Mala s 7^[55,57]. These findings could imply selective sorting of particular allergens to vesicles^[57].

Mass spectrometry-based proteomic analysis further revealed that M. sympodialis vesicles are enriched in enzymes associated with diverse metabolic and catalytic processes^[57]. These include hydrolases (lysophospholipases), particularly involved in hydrolysis of sebum lipids leading to the release of fatty acids. These are thought to exert pro-inflammatory effect, contributing to skin irritation and inflammation, characteristic of Malassezia-associated dermatoses^[12,61]. Importantly, Malassezia vesicles have been shown to contain aspartic proteinases^[12,13], along with carboxypeptidases, phosphatases, and glucosidases, enzymes involved in cell wall remodeling, as well as proteins associated with nucleic acid repair and metabolism^[57]. The broad spectrum of processes in which these vesicle-associated proteins participate supports the concept that MalaEx function as molecular packages for intercellular communication^[57]_.

Beyond proteins, the nucleic acid content of Malassezia extracellular vesicles has also been investigated for M. sympodialis grown in mDixon broth supplemented with 50 mM MES [2-(N-morpholino)ethanesulfonic acid] for 48 h at 32 °C^[56]. Rayner et al. identified populations of small RNAs (16-22 nucleotides) within M. sympodialis MalaEx^[56]. Bioinformatic analysis of the mean free energy indicated that the enriched small RNAs do not belong to the miRNA population, as they do not readily form hairpin structures, and their biogenesis is therefore suggested to occur via an independent pathway^[56]. Previously, extracellular vesicles derived from other fungal species, such as Cryptococcus neoformans, Paracoccidioides brasiliensis, Candida albicans, and Saccharomyces cerevisiae were shown to contain predominantly RNAs shorter than 250 nucleotides, including 114 non-coding RNAs, among them snoRNAs, snRNAs, rRNAs, tRNAs, and miRNA-like sequences common to all species, as well as selected full-length mRNAs related to vesicle-mediated transport and metabolic pathways, supporting the idea that RNA-based communication may represent a shared feature among human fungal pathogens^[62]. Importantly, further studies are required to comprehensively characterize the composition of MalaEx, particularly with regard to their DNA and lipid content, as such data remain limited.

Investigating the functional roles of Malassezia extracellular vesicles is essential for understanding their impact on host immunity and microbial interactions. Their interactions with the skin under physiological conditions, as well as their potential involvement in individuals affected by AD, have been investigated. To date, only a limited number of such reports are available; they are discussed in detail below and summarized in Figure 1.

The enrichment of major allergens within MalaEx provides a possible mechanism for antigen delivery and sensitization. Because secreted vesicles are of nanometer size, they can diffuse through the stratum corneum more easily than intact yeast cells, allowing allergens to reach antigen-presenting cells in the epidermis and dermis. The activation of keratinocytes and monocytes suggests engagement of pattern recognition receptors such as Toll-like receptor 2 (TLR2), Toll-like receptor 4 (TLR4), and Dectin-1, all of which are known to detect fungal components^[43,70,71]. Cell wall components could serve as ligands for these receptors, initiating downstream signaling and cytokine production. In other fungal species, vesicle-derived small RNAs have been shown to interact with host cells and subsequently modulate host immunity^[72,73]. However, the functional impact of small RNAs contained in MalaEx has not yet been demonstrated. Future studies are therefore warranted to further characterize the nucleic acid cargo of MalaEx produced by different Malassezia species. Despite significant advances, the precise cellular targets of MalaEx and their uptake pathways remain incompletely understood, representing a compelling area for future investigation. As in other fungi, the biogenesis of extracellular vesicles remains insufficiently characterized, and the precise molecular mechanisms and environmental factors that influence this process have yet to be fully elucidated, which is particularly relevant in the context of pathogenic microorganisms.

Due to the significant enrichment of allergens and surface-presented antigens, MalaEx may serve as potential biomarkers in studies of fungal dermatitis, where quantitative profiling of specific vesicular components could improve disease diagnosis and monitoring of disease progression. Furthermore, as explored in the literature for vesicles derived from other organisms^[74-76], Malassezia-derived vesicles could be investigated as vaccine candidates or as delivery systems for bioactive molecules, particularly in the context of topical therapies. In addition, to comprehensively investigate Malassezia interactions with skin cells, diverse immune cell types, and the resident skin microbiota, and to accurately characterize the resulting immune responses, the development of physiologically relevant host-pathogen infection models is required, including systems that incorporate extracellular vesicles produced by all interacting members within the studied environment. In addition, it is important to emphasize that findings derived from murine models, especially concerning immune responses and skin architecture, may not fully recapitulate human physiology and should be interpreted with appropriate caution.

Although data on extracellular vesicles secreted by Malassezia species remain limited, current studies indicate that MalaEx have a significant impact on both the resident microbial community and the host. These vesicular carriers have been shown to contain proteins, enzymes, allergens, lipids, and small RNAs, which collectively influence skin physiology and immune responses. Functionally, MalaEx modulate keratinocyte activation and stimulate cytokine release from immune cells. Moreover, they appear to affect the bacterial-fungal balance on human skin, suggesting a potential role in preventing microbial overgrowth.