Fungal Extracellular Vesicles: New Perspectives in Intercellular Communication, Pathogenicity, and Host-Pathogen Interactions

Fungal Extracellular Vesicles (FEVs) are now recognized as crucial vectors of intercellular communication within fungal ecosystems and during host interactions. They transport proteins, lipids, and nucleic acids—modulating virulence, facilitating tissue invasion, and promoting immune evasion. This review consolidates recent findings on the definition, classification, and communication processes of FEVs and their pathogenic and immunomodulatory roles. Methods for vesicle characterization, potential therapeutic applications, and the challenges arising from their variability are also discussed. All this knowledge paves the way for interdisciplinary approaches to better understand and combat fungal infections.

Abbreviations

DUC: Differential Ultracentrifugation; Erk1/2: Extracellular Signal-Regulated Kinase 1/2; EVs: Extracellular Vesicles; FEVs: Fungal Extracellular Vesicles; GDF15: Growth Differentiation Factor 15; GXM: Glucuronoxylomannan; HPCA1: Hydrogen Peroxide-induced Ca²⁺ increase 1 (or Hydrogen Peroxide-Responsive Calcium Channel 1); IL: Interleukin; IL-1β: Interleukin-1 beta; IL-6: Interleukin-6; IL-10: Interleukin-10; IL-12: Interleukin-12; LC-MS/MS: Liquid Chromatography-tandem Mass Spectrometry; MAPK: Mitogen-Activated Protein Kinase; mRNA: Messenger Ribonucleic Acid; MVBs: Multivesicular Bodies; NF-κB: Nuclear Factor kappa-light-chain-enhancer of activated B cells; NO: Nitric Oxide; OMVs: Outer Membrane Vesicles; PCD: Programmed Cell Death; PMN: Polymorphonuclear Neutrophil; PRRs: Pattern Recognition Receptors; RNA: Ribonucleic Acid; RNAi: RNA interference; SAR: Systemic Acquired Resistance; SEC: Size-Exclusion Chromatography; sEVs: small Extracellular Vesicles; SILAC: Stable Isotope Labeling by Amino acids in Cell culture; siRNA: Small Interfering Ribonucleic Acid; Th1: T helper 1; Th2: T helper 2; TLRs: Toll-Like Receptors; TNF-α: Tumor Necrosis Factor alpha; Treg: Regulatory T cells; tRNA: Transfer Ribonucleic Acid

Introduction

Interest in Fungal Extracellular Vesicles (FEVs) has been growing steadily, demonstrating their emergence as critical mediators of intercellular communication and host response modulation, a concept initially explored in non-pathogenic models and opportunistic fungi [1,2]. Their involvement in pathogenesis, by facilitating the transmission of virulence factors and altering immune balance, warrants special attention. Understanding their roles is crucial to advance fundamental knowledge and drive the development of innovative therapeutic tools against fungal infections [3]. Recent comprehensive reviews underscore the expanding scope of FEV research, particularly in the context of fungal-host interactions, their biogenesis, and burgeoning therapeutic potential [4,5].

Definition, classification, and nuances of Fungal Extracellular Vesicles (FEVs)

Fungal Extracellular Vesicles (FEVs) are heterogeneous, membrane-bound structures actively released by fungal cells into their extracellular environment, pivotal for intercellular communication, pathogenesis, stress response, and fungal adaptation [5,6]. While mammalian systems initially inspired their classification, it is continually evolving to reflect fungal-specific biology. Traditionally, FEVs are categorized based on biogenesis, size, and content. Exosomes (typically 30 nm - 150 nm) originate from the endosomal pathway, formed as intraluminal vesicles within multivesicular bodies that fuse with the plasma membrane for release, carrying a complex cargo reflecting cell physiology [7,8]. Microvesicles (or ectosomes, typically 100 - 1000 nm) are larger, formed by direct outward budding of the plasma membrane, with a cargo distinct from exosomes [9]. The release of larger vesicles (> 1000 nm), termed apoptotic bodies, during programmed cell death has also been documented in fungi like Saccharomyces cerevisiae and pathogens such as Candida albicans under stress or antifungal treatment, containing cellular debris and considered markers of cell death rather than active communication vesicles from healthy cells [10,11]. Furthermore, “stress-induced vesicles” describe FEV populations whose production and cargo are significantly modulated by environmental stressors like nutrient limitation or antifungal exposure, often enriching for molecules that facilitate survival and host interaction [12,13]. It is important to clarify that the term “Outer Membrane Vesicles” (OMVs) is specific to Gram-negative bacteria; fungi, possessing a plasma membrane and cell wall, release FEVs that must traverse this cell wall, a unique process involving cell wall remodeling enzymes [8,14]. This classification allows parallels with vesicles from other cell types [15], including observations of conserved biogenesis pathways compared to mammalian systems [16] and functional plasticity comparable to tumor exosomes (Figure 1) [17].

Figure 1: The Multifaceted World of Fungal Extracellular Vesicles: From Pathogenesis to Therapeutic Frontier. This figure illustrates the diverse roles of fungal Extracellular Vesicles (EVs) in fungal biology and host interactions. It highlights their involvement in processes such as virulence factor transport, modulation of the host immune response, biofilm formation, intercellular communication, and their potential applications in diagnostics and therapeutics.

Role of fungal extracellular vesicles in intercellular communication

FEVs are pivotal in intercellular communication, mediating interactions within fungal populations and with host organisms by transporting diverse bioactive molecules. During infection, FEVs are central to the fungal-host dialogue, carrying proteins, lipids, nucleic acids (including non-coding RNAs), and virulence factors that facilitate invasion, colonization, and adaptation [4,18]. Proteomic analyses have identified hundreds of proteins in Candida albicans FEVs, many linked to pathogenicity [19]. FEVs typically deliver their loading via endocytosis or ligand-receptor interactions, as demonstrated in Cryptococcus neoformans infections, where FEVs engage host cells to modulate outcomes [20]. Upon delivery, FEV components can alter host intracellular signaling pathways like NF-κB and MAPK, disturb cellular homeostasis, and influence apoptosis and inflammation. The composition and quantity of FEVs are dynamic, significantly altered by stress conditions, often leading to enhanced virulence factor secretion [13,21]. This adaptability allows FEVs to induce cytokine secretion and create an immunomodulatory environment conducive to infection, a strategy with functional parallels to tumor-derived exosomes that remodel their local environment and suppress T-cell activity [17,22]. For instance, microRNAs, such as those analogous to miR-21, found in FEVs from fungi like Aspergillus fumigatus, can dampen host immune responses [22].

The influence of FEVs extends to cross-kingdom communication, notably through the transfer of small RNAs. For example, Botrytis cinerea vesicles deliver small interfering RNAs (siRNAs) to plant cells to suppress host RNA interference (RNAi) machinery by targeting plant defense genes, thereby enhancing fungal colonization [23,24]. RNA-loaded vesicles represent a conserved strategy across kingdoms for inter-organismal gene regulation, also observed in other fungal-plant and fungal-bacterial interactions where FEVs can deliver sRNAs to modulate recipient gene expression [25,26]. FEVs also mediate intra-fungal communication, contributing to population-level behaviors. Cryptococcus neoformans utilizes vesicles to transfer virulence factors between cells [27], and a vesicle-mediated quorum-sensing-like mechanism in this species coordinates biofilm formation and antifungal resistance [28]. Through tissue dissemination, FEVs establish distant cell-to-cell communication, distributing pathogenic signals and collectively reshaping host cell structures. A thorough understanding of these communication mechanisms is crucial for unveiling novel antifungal strategies targeting these vesicular pathways (Figure 1) [5].

Fungal extracellular vesicles in pathogenicity, host-pathogen interaction, and immune modulation

FEVs are critically involved in the arsenal of pathogenic fungi deployed for invasion and subverting host immune responses. A primary role is the delivery of virulence factors, including hydrolytic enzymes like proteases and lipases, which degrade tissue barriers, facilitating fungal dissemination [27,29]. Beyond direct damage, FEV components modulate host immune signaling pathways such as NF-κB and MAPK, reducing cellular resistance and impairing infection clearance [4]. Some fungi, like Cryptococcus gattii, use vesicles to transport regulatory RNAs that control collective fungal virulence [30]. FEVs significantly contribute to biofilm formation and maintenance, especially in pathogens like Candida albicans. Biofilms are structured microbial communities encased in an extracellular matrix that protects against antifungal agents and immune responses [18]. FEVs deliver matrix components and signaling molecules essential for biofilm integrity; for instance, C. albicans FEVs contain proteins and polysaccharides crucial for matrix development and can influence cell adherence and cohesion within the biofilm, and vesicles from this yeast have been shown to contribute to drug resistance through matrix modification [13,31].

The host defense system typically responds to pathogen invasion through phagocytic activity and pro-inflammatory cytokine secretion (e.g., TNF-α, IL-6), initiating an inflammatory cascade [32]. Pattern recognition receptors (PRRs), including Toll-like receptors (TLRs), recognize fungal cell wall components like β-glucans and chitin [33], leading to the activation of adaptive immunity involving T and B lymphocytes. However, pathogenic fungi have evolved sophisticated immune evasion methods, and FEVs are instrumental in these processes. FEVs facilitate immune detection evasion while promoting entry into host tissues by transporting antigens, enzymes, and immunomodulatory molecules that can decrease immune recognition and proinflammatory cytokine production. Cryptococcus neoformans secretes vesicles containing capsular polysaccharides (e.g., glucuronoxylomannan, GXM) that act as decoys, saturating antibodies and complement, thereby reducing phagocytosis and blunting cytokine responses [34,35].

Moreover, FEVs containing microRNAs or non-coding RNAs can directly modify host gene expression to promote immunosuppression. Candida albicans vesicles can induce immune tolerance by suppressing IL-1β production and polarizing macrophages towards an anti-inflammatory M2-like profile [19,36]. Similarly, microRNAs in Aspergillus fumigatus vesicles decrease key pro-inflammatory cytokines like IL-1β and TNF-α [22]. Pathogens like Paracoccidioides brasiliensis utilize vesicles to deliver molecules that prevent phagosome acidification, aiding their intracellular survival [37]. These mechanisms highlight how FEVs are vital modulators of host-pathogen interactions, often creating local immunosuppression [38]. Such vesicle-mediated strategies are not unique to fungi; bacteria release vesicles delivering toxins or enzymes [39,40], and lipoproteins in Mycobacterium tuberculosis vesicles can block phagocytosis [41]. Some bacterial vesicles can induce immune tolerance, aiding persistence [42]. In plant ecosystems, plant-derived extracellular vesicles also mediate immune signaling and interactions with microbes [43]. For example, exosomes from Arabidopsis plants can inhibit Botrytis cinerea sporulation and activate defense genes [44]. The host also uses extracellular vesicles for immune regulation; helminth-infected intestinal cells release exosomes, stimulating TGF-β production, promoting immune tolerance [45]. The diverse roles and mechanisms of FEVs vary significantly across different fungal species, as summarized in Table 1, which highlights key findings for several prominent pathogenic and model fungi.

Methodologies for fungal extracellular vesicle isolation, purification, and analysis

The comprehensive characterization of FEVs relies on robust isolation and purification, followed by in-depth compositional analysis, especially proteomics [2,41]. Purification is essential, initially involving centrifugation to separate FEVs from cells and debris. Differential Ultracentrifugation (DUC) is common, separating particles by size, shape, and density [7,41], but can co-purify contaminants and potentially damage vesicles [43,67]. While filtration removes debris or concentrates vesicles, it may also lead to partial FEV loss [68]. Size-Exclusion Chromatography (SEC) separates molecules by hydrodynamic volume, generally yielding higher purity FEV preparations than DUC by removing soluble protein contaminants and preserving vesicle integrity, though yield can be variable [68,69]. Affinity chromatography, using specific ligands for FEV surface markers, offers high purity [70]; when markers are unknown, broader specificity ligands like lectins (binding common fungal glycans potentially on FEVs) or exploratory approaches to identify novel ligands are employed [71]. Often, combined techniques are needed to achieve high yield and purity [72], and standardization of protocols is crucial for reproducibility [5]. Mass spectrometry (MS/MS) remains the gold standard for comprehensive FEV proteome profiling [34], providing insights into biogenesis, cargo, and effector functions. FEV proteomics can reveal biomarkers for fungal infections and elucidate mechanisms of host immune modulation and tissue penetration [19,49]. For instance, vesicular proteins in C. albicans FEVs can reduce cytokine activity [19]. Integrating proteomic data with other omics provides a holistic view of FEV-mediated interactions [72]. Comparative proteomics, such as studies on virus-infected cell exosomes showing altered protein profiles [73], suggests similar shifts in FEVs from stressed fungi or infected hosts, influencing immune responses [30].

Fungal extracellular vesicles: A frontier for therapeutic and diagnostic innovation

The ability of FEVs to transport functional biomolecules and interact with host cells positions them as promising candidates for novel therapeutic and diagnostic applications. FEVs from pathogenic fungi like C. neoformans naturally contain key antigens such as GXM [37], and immunization with these FEVs has induced protective immunity in murine models [38,74]. Similarly, FEVs from Histoplasma capsulatum (enriched with Hsp60) and C. albicans (containing Als3, Eno1) are potential vaccine candidates [19,37]. However, the complexity of native FEVs poses challenges, as some components could induce excessive inflammation or hypersensitivity [75]; thus, careful characterization, purification, and potential engineering are necessary. The capacity of FEVs to traverse biological barriers also inspires their investigation as targeted drug delivery vehicles for antifungals or RNAi-based therapies, drawing from advances in human exosome research [5,76], though large-scale production and targeting in fungal systems require further development [77]. Alternatively, inhibiting FEV biogenesis/release or neutralizing their cargo offers an indirect therapeutic strategy [29]. The unique molecular signatures of FEVs (miRNAs, lipids, proteins) make them valuable for early, non-invasive diagnostics of fungal infections [5,49,78]. FEVs derived from probiotic Lactobacillus species, also show therapeutic potential by enhancing gut barrier function and reducing inflammation [79]. Furthermore, genetically engineering parental fungal cells via CRISPR/Cas9 technologies could create “designer” FEVs with tailored functionalities, such as enhanced targeting or specific therapeutic payloads [74,80], paving the way for personalized nanodevices for precision fungal medicine.

Future perspectives and challenges

Despite notable progress, FEV research faces challenges. FEV composition varies among species and environmental factors, hindering standardization [2,29]. Isolation yields are often low, and purification methods can introduce artifacts [72]. Deciphering the complex, bidirectional FEV-host cell interactions require interdisciplinary approaches [38]. Future research will benefit from advanced imaging and organoid models for in situ analysis of FEV-host interactions [78,81], and the integration of multi-omics datasets will provide a systematic biology view to identify new biomarkers and targets [74]. Genome editing and nanotechnologies are expected to optimize FEV targeting for personalized therapies [74]. These innovations should accelerate the clinical translation of fundamental discoveries, vital for addressing emerging and drug-resistant fungal pathogens (Figure 1).

Conclusion

In conclusion, FEVs represent essential mediators of fungal communication, pathogenicity, and host modulation. By transporting modulatory biomolecules, they impact tissue invasion and immune evasion, providing adaptive advantages [1,34]. While technical challenges and heterogeneity persist, Emerging advances in imaging, spectrometry, and integrative multi-omic analyses provide exciting opportunities for therapeutic targeting and vaccine development. Integrating these innovative approaches, including synthetic biology and nanotechnology, promises to transform our understanding of infection mechanisms and enrich the therapeutic arsenal against fungal diseases [3,74].

Despite significant advances in understanding Fungal Extracellular Vesicles (FEVs), several limitations remain. Current studies are disproportionately focused on a few model organisms, with limited cross-species comparisons that hinder broader biological generalization. Standardized methodologies for vesicle isolation, cargo profiling, and functional validation are still lacking, which complicates reproducibility across laboratories. Additionally, in vivo studies exploring FEV-host interactions, immune evasion, and therapeutic delivery remain scarce. Future research should prioritize establishing consensus protocols, expanding species diversity, integrating omics technologies, and validating findings in clinically relevant models. These directions will be essential to unlocking the diagnostic and therapeutic potential of FEVs in fungal pathobiology.

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