Fungal Extracellular Vesicles Research Articles

Extracellular vesicles from Candida albicans modulate immune cells function and play a protective role in fungal keratitis

Fungal keratitis (FK) is a highly blinding infectious corneal disease caused by pathogenic fungi. Candida albicans (C. albicans) is one of the main pathogens of fungal keratitis. Extracellular vesicles (EVs), lipid bilayer compartments released by almost all living cells, including fungi, have garnered attention for their role in pathogenic microbial infection and host immune responses in recent years. Studies have reported that pretreating the host with fungal EVs can reduce the inflammatory response of the host when attacked by fungi and reduce the lethality of fungal infection. However, there are no studies that have evaluated whether C. albicans EVs can modulate the inflammatory response associated with C. albicans keratitis. Our study revealed that C. albicans EVs could activate the polymorphonuclear cells (PMNs) and promote their secretion of proinflammatory cytokines and nitric oxide (NO), enhance their phagocytic and fungicidal abilities against C. albicans. C. albicans EVs also induced a proinflammatory response in RAW264.7 cells, which is characterized by increased production of inflammatory cytokines and elevated expression of the chemokine CCL2. Similarly, stimulation of C. albicans EVs to RAW264.7 cells also enhanced the phagocytosis and killing ability of cells against C. albicans. Besides, in vivo experiments, after receiving subconjunctival injection of C. albicans EVs, C57BL/6 mice were infected with C. albicans. The results demonstrated that pre-exposure to C. albicans EVs could effectively diminish the severity of keratitis, reduce fungal load and improve prognosis. Overall, we conclude that C. albicans EVs can modulate the function of immune cells and play a protective role in C. albicans keratitis.

Extracellular vesicles from virulent P. brasiliensis induce TLR4 and dectin-1 expression in innate cells and promote enhanced Th1/Th17 response

ABSTRACT Extracellular vesicles (EVs) are membrane-enclosed nanoparticles that transport several biomolecules and are involved in important mechanisms and functions related to the pathophysiology of fungal diseases. EVs from Paracoccidioides brasiliensis, the main causative agent of Paracoccidioidomycosis (PCM), modulate the immune response of macrophages. In this study, we assessed the EVs proteome from a virulent P. brasiliensis isolated from granulomatous lesions and compared their immunomodulatory ability with EVs isolated from the fungus before the animal passage (control EVs) when challenging macrophages and dendritic cells (DCs). Proteome showed that virulent EVs have a higher abundance of virulence factors such as GP43, protein 14-3-3, GAPDH, as well as virulence factors never described in PCM, such as aspartyl aminopeptidase and a SidJ analogue compared with control EVs. Virulent extracellular vesicles induced higher expression of TLR4 and Dectin-1 than control EVs in macrophages and dendritic cells (DCs). In opposition, a lower TLR2 expression was induced by virulent EVs. Additionally, virulent EVs induced lower expression of CD80, CD86 and TNF-α, but promoted a higher expression of IL-6 and IL-10, suggesting that EVs isolated from virulent P. brasiliensis-yeast promote a milder DCs and macrophage maturation. Herein, we showed that EVs from virulent fungi stimulated a higher frequency of Th1/Tc1, Th17, and Treg cells, which gives new insights into fungal extracellular vesicles. Taken together, our results suggest that P. brasiliensis utilizes its EVs as virulence bags that manipulate the immune system in its favour, creating a milder immune response and helping with fungal evasion from the immune system.

Corrected and republished from: "Extracellular Vesicle Formation in Cryptococcus deuterogattii Impacts Fungal Virulence".

Small molecules are components of fungal extracellular vesicles (EVs), but their biological roles are only superficially known. NOP16 is a eukaryotic gene that is required for the activity of benzimidazoles against Cryptococcus deuterogattii. In this study, during the phenotypic characterization of C. deuterogattii mutants expected to lack NOP16 expression, we observed a reduced EV production. Whole-genome sequencing, RNA-Seq, and cellular proteomics revealed that, contrary to our initial findings, these mutants expressed Nop16 but exhibited altered expression of 14 genes potentially involved in sugar transport. Based on this observation, we designated these mutant strains as Past1 and Past2, representing potentially altered sugar transport. Analysis of the small molecule composition of EVs produced by wild-type cells and the Past1 and Past2 mutant strains revealed not only a reduced number of EVs but also an altered small molecule composition. In a Galleria mellonella model of infection, the Past1 and Past2 mutant strains were hypovirulent. The hypovirulent phenotype was reverted when EVs produced by wild-type cells, but not mutant EVs, were co-injected with the mutant cells in G. mellonella. These results connect EV biogenesis, cargo, and cryptococcal virulence.

Candida albicans Biofilm-Derived Extracellular Vesicles Are Involved in the Tolerance to Caspofungin, Biofilm Detachment, and Fungal Proteolytic Activity.

It has been repeatedly reported that the cells of organisms in all kingdoms of life produce nanometer-sized lipid membrane-enveloped extracellular vesicles (EVs), transporting and protecting various substances of cellular origin. While the composition of EVs produced by human pathogenic fungi has been studied in recent decades, another important challenge is the analysis of their functionality. Thus far, fungal EVs have been shown to play significant roles in intercellular communication, biofilm production, and modulation of host immune cell responses. In this study, we verified the involvement of biofilm-derived EVs produced by two different strains of Candida albicans-C. albicans SC5314 and 3147 (ATCC 10231)-in various aspects of biofilm function by examining its thickness, stability, metabolic activity, and cell viability in the presence of EVs and the antifungal drug caspofungin. Furthermore, the proteolytic activity against the kininogen-derived antimicrobial peptide NAT26 was confirmed by HPLC analysis for C. albicans EVs that are known to carry, among others, particular members of the secreted aspartic proteinases (Saps) family. In conclusion, EVs derived from C. albicans biofilms were shown to be involved in biofilm tolerance to caspofungin, biofilm detachment, and fungal proteolytic activity.

Traversing the Cell Wall: The Chitinolytic Activity of Histoplasma capsulatum Extracellular Vesicles Facilitates Their Release.

Histoplasma capsulatum is the causative agent of histoplasmosis. Treating this fungal infection conventionally has significant limitations, prompting the search for alternative therapies. In this context, fungal extracellular vesicles (EVs) hold relevant potential as both therapeutic agents and targets for the treatment of fungal infections. To explore this further, we conducted a study using pharmacological inhibitors of chitinase (methylxanthines) to investigate their potential to reduce EV release and its subsequent impact on fungal virulence in an in vivo invertebrate model. Our findings revealed that a subinhibitory concentration of the methylxanthine, caffeine, effectively reduces EV release, leading to a modulation of H. capsulatum virulence. To the best of our knowledge, this is the first reported instance of a pharmacological inhibitor that reduces fungal EV release without any observed fungicidal effects.

Immunomodulatory Potential of Fungal Extracellular Vesicles: Insights for Therapeutic Applications.

Extracellular vesicles (EVs) are membranous vesicular organelles that perform a variety of biological functions including cell communication across different biological kingdoms. EVs of mammals and, to a lesser extent, bacteria have been deeply studied over the years, whereas investigations of fungal EVs are still in their infancy. Fungi, encompassing both yeast and filamentous forms, are increasingly recognized for their production of extracellular vesicles (EVs) containing a wealth of proteins, lipids, and nucleic acids. These EVs play pivotal roles in orchestrating fungal communities, bolstering pathogenicity, and mediating interactions with the environment. Fungal EVs have emerged as promising candidates for innovative applications, not only in the management of mycoses but also as carriers for therapeutic molecules. Yet, numerous questions persist regarding fungal EVs, including their mechanisms of generation, release, cargo regulation, and discharge. This comprehensive review delves into the present state of knowledge regarding fungal EVs and provides fresh insights into the most recent hypotheses on the mechanisms driving their immunomodulatory properties. Furthermore, we explore the considerable potential of fungal EVs in the realms of medicine and biotechnology. In the foreseeable future, engineered fungal cells may serve as vehicles for tailoring cargo- and antigen-specific EVs, positioning them as invaluable biotechnological tools for diverse medical applications, such as vaccines and drug delivery.

The multiple frontiers in the study of extracellular vesicles produced by fungi

The production of extracellular vesicles (EVs) by fungi has been recognized for about a decade. Here we discuss the roles played by fungal EVs in biofilm formation, antifungal resistance, and release of immunogens with vaccine potential. We also explore their significance in promoting international collaboration and understanding of fungal biology.

Fungal extracellular vesicle-mediated regulation: from virulence factor to clinical application.

Invasive fungal disease (IFD) poses a significant threat to immunocompromised patients and remains a global challenge due to limited treatment options, high mortality and morbidity rates, and the emergence of drug-resistant strains. Despite advancements in antifungal agents and diagnostic techniques, the lack of effective vaccines, standardized diagnostic tools, and efficient antifungal drugs contributes to the ongoing impact of invasive fungal infections (IFI). Recent studies have highlighted the presence of extracellular vesicles (EVs) released by fungi carrying various components such as enzymes, lipids, nucleic acids, and virulence proteins, which play roles in both physiological and pathological processes. These fungal EVs have been shown to interact with the host immune system during the development of fungal infections whereas their functional role and potential application in patients are not yet fully understood. This review summarizes the current understanding of the biologically relevant findings regarding EV in host-pathogen interaction, and aim to describe our knowledge of the roles of EV as diagnostic tools and vaccine vehicles, offering promising prospects for the treatment of IFI patients.

The characterization of RNA-binding proteins and RNA metabolism-related proteins in fungal extracellular vesicles.

RNA-binding proteins (RBPs) are essential for regulating RNA metabolism, stability, and translation within cells. Recent studies have shown that RBPs are not restricted to intracellular functions and can be found in extracellular vesicles (EVs) in different mammalian cells. EVs released by fungi contain a variety of proteins involved in RNA metabolism. These include RNA helicases, which play essential roles in RNA synthesis, folding, and degradation. Aminoacyl-tRNA synthetases, responsible for acetylating tRNA molecules, are also enriched in EVs, suggesting a possible link between these enzymes and tRNA fragments detected in EVs. Proteins with canonical RNA-binding domains interact with proteins and RNA, such as the RNA Recognition Motif (RRM), Zinc finger, and hnRNP K-homology (KH) domains. Polyadenylate-binding protein (PABP) plays a critical role in the regulation of gene expression by binding the poly(A) tail of messenger RNA (mRNA) and facilitating its translation, stability, and localization, making it a key factor in post-transcriptional control of gene expression. The presence of proteins related to the RNA life cycle in EVs from different fungal species suggests a conserved mechanism of EV cargo packing. Various models have been proposed for selecting RNA molecules for release into EVs. Still, the actual loading processes are unknown, and further molecular characterization of these proteins may provide insight into the mechanism of RNA sorting into EVs. This work reviews the current knowledge of RBPs and proteins related to RNA metabolism in EVs derived from distinct fungi species, and presents an analysis of proteomic datasets through GO term and orthology analysis, Our investigation identified orthologous proteins in fungal EVs on different fungal species.

The emerging role of extracellular vesicles in fungi: a double-edged sword.

Fungi are eukaryotic microorganisms found in nature, which can invade the human body and cause tissue damage, inflammatory reactions, organ dysfunctions, and diseases. These diseases can severely damage the patient's body systems and functions, leading to a range of clinical symptoms that can be life-threatening. As the incidence of invasive fungal infections has progressively increased in the recent years, a wealth of evidence has confirmed the "double-edged sword" role of fungal extracellular vesicles (EVs) in intercellular communication and pathogen-host interactions. Fungal EVs act as mediators of cellular communication, affecting fungal-host cell interactions, delivering virulence factors, and promoting infection. Fungal EVs can also have an induced protective effect, affecting fungal growth and stimulating adaptive immune responses. By integrating recent studies, we discuss the role of EVs in fungi, providing strong theoretical support for the early prevention and treatment of invasive fungal infections. Finally, we highlight the feasibility of using fungal EVs as drug carriers and in vaccine development.

Extracellular Vesicles from Candida haemulonii var. vulnera Modulate Macrophage Oxidative Burst

Members of the Candida haemulonii species complex are multidrug-resistant emergent yeast pathogens able to cause superficial and invasive infections in risk populations. Fungal extracellular vesicles (EVs) play a critical role in the pathogenicity and virulence of several species and may perform essential functions during infections, such as carrying virulence factors that behave in two-way communications with the host, affecting survival and fungal resistance. Our study aimed to describe EV production from Candida haemulonii var. vulnera and evaluate whether murine macrophage RAW 264.7 cells respond to their stimuli by generating an oxidative response after 24 h. For this purpose, reactive oxygen species detection assays demonstrated that high concentrations of yeast and EVs (1010 particles/mL) of Candida haemulonii did not change macrophage viability. However, the macrophages recognized these EVs and triggered an oxidative response through the classical NOX-2 pathway, increasing O2•- and H2O2 levels. However, this stress did not cause lipid peroxidation in the RAW 264.7 cells and neither lead to the activation of the COX-2-PGE2 pathway. Thus, our data suggest that low concentrations of C. haemulonii EVs are not recognized by the classical pathway of the oxidative burst generated by macrophages, which might be an advantage allowing the transport of virulence factors via EVs, not identified by the host immune system that could work as fine tube regulators during infections caused by C. haemulonii. In contrast, C. haemulonii var. vulnera and high EV concentrations activated microbicidal actions in macrophages. Therefore, we propose that EVs could participate in the virulence of the species and that these particles could be a source of antigens to be exploited as new therapeutic targets.

Aspergillus fumigatus Extracellular Vesicles Display Increased Galleria mellonella Survival but Partial Pro-Inflammatory Response by Macrophages

Fungal extracellular vesicles (EVs) mediate intra- and interspecies communication and are critical in host-fungus interaction, modulating inflammation and immune responses. In this study, we evaluated the in vitro pro- and anti-inflammatory properties of Aspergillus fumigatus EVs over innate leukocytes. A. fumigatus EVs induced a partial proinflammatory response by macrophages, characterized by increased tumor necrosis factor-alpha production, and increased gene expression of induced nitric oxide synthase and adhesion molecules. EVs induce neither NETosis in human neutrophils nor cytokine secretion by peripheral mononuclear cells. However, prior inoculation of A. fumigatus EVs in Galleria mellonella larvae resulted in increased survival after the fungal challenge. Taken together, these findings show that A. fumigatus EVs play a role in protection against fungal infection, although they induce a partial pro-inflammatory response.

2nd Workshop in Microbial Molecular Biology

2nd Workshop in Microbial Molecular Biology

The RNA Content of Fungal Extracellular Vesicles: At the "Cutting-Edge" of Pathophysiology Regulation.

The role of extracellular vesicles (EVs) in interkingdom communication is widely accepted, and their role in intraspecies communication has been strengthened by recent research. Based on the regulation promoted by EV-associated molecules, the interactions between host and pathogens can reveal different pathways that ultimately affect infection outcomes. As a great part of the regulation is ascribable to RNA contained in EVs, many studies have focused on profiling RNAs in fungal and host EVs, tracking their accumulation during infection, and identifying potential target genes. Herein, we overview the main classes of RNA contained in fungal EVs and the biological processes regulated by these molecules, portraying a state-of-the-art picture of RNAs loaded in fungal EVs, while also raising several questions to drive future investigations. Our compiled data show unambiguously that EVs act as key elements in signaling pathways, and play a crucial role in pathosystems. A complete understanding of the processes that govern RNA content loading and trafficking, and its effect on recipient cells, will lead to improved technologies to ward off infectious agents that threaten human health.

Extracellular Vesicle Formation in Cryptococcus deuterogattii Impacts Fungal Virulence and Requires the NOP16 Gene.

Small molecules are components of fungal extracellular vesicles (EVs), but their biological roles are only superficially known. NOP16 is a eukaryotic gene that is required for the activity of benzimidazoles against Cryptococcus deuterogattii. In this study, during the phenotypic characterization of C. deuterogattii mutants lacking NOP16 expression, we observed that this gene was required for EV production. Analysis of the small molecule composition of EVs produced by wild-type cells and two independent nop16Δ mutants revealed that the deletion of NOP16 resulted not only in a reduced number of EVs but also an altered small molecule composition. In a Galleria mellonella model of infection, the nop16Δ mutants were hypovirulent. The hypovirulent phenotype was reverted when EVs produced by wild-type cells, but not mutant EVs, were coinjected with the nop16Δ cells in G. mellonella. These results reveal a role for NOP16 in EV biogenesis and cargo, and also indicate that the composition of EVs is determinant for cryptococcal virulence.

Bioinformatics strategies for studying the molecular mechanisms of fungal extracellular vesicles with a focus on infection and immune responses.

Fungal extracellular vesicles (EVs) are released during pathogenesis and are found to be an opportunistic infection in most cases. EVs are immunocompetent with their host and have paved the way for new biomedical approaches to drug delivery and the treatment of complex diseases including cancer. With computing and processing advancements, the rise of bioinformatics tools for the evaluation of various parameters involved in fungal EVs has blossomed. In this review, we have complied and explored the bioinformatics tools to analyze the host-pathogen interaction, toxicity, omics and pathogenesis with an array of specific tools that have depicted the ability of EVs as vector/carrier for therapeutic agents and as a potential theme for immunotherapy. We have also discussed the generation and pathways involved in the production, transport, pathogenic action and immunological interactions of EVs in the host system. The incorporation of network pharmacology approaches has been discussed regarding fungal pathogens and their significance in drug discovery. To represent the overview, we have presented and demonstrated an in silico study model to portray the human Cryptococcal interactions.

From fundamental biology to the search for innovation: The story of fungal extracellular vesicles

Fungal extracellular vesicles (EVs) have attracted increased attention in recent years. Originated from a serendipitous discovery, the initial observation of fungal EVs resulted in a set of data repetitively rejected by several scientific journals, which raised questions about their authenticity. However, after the most fundamental experimental issues related to their observation were addressed, fungal EVs were characterized in dozens of species and became an emerging field. In this essay, we will discuss these fundamental findings and the potential of fungal EVs for the development of vaccines and antifungals.

Fungal Extracellular Vesicles Are Involved in Intraspecies Intracellular Communication.

ABSTRACTFungal infections are associated with high mortality rates in humans. The risk of fungal diseases creates the urgent need to broaden the knowledge base regarding their pathophysiology. In this sense, the role of extracellular vesicles (EVs) has been described to convey biological information and participate in the fungus-host interaction process. We hypothesized that fungal EVs work as an additional element in the communication routes regulating fungal responses in intraspecies interaction systems. In this respect, the aim of this study was to address the gene regulation profiles prompted by fungal EVs in intraspecies recipient cells. Our data demonstrated the intraspecies uptake of EVs in pathogenic fungi, such as Candida albicans, Aspergillus fumigatus, and Paracoccidioides brasiliensis, and the effects triggered by EVs in fungal cells. In C. albicans, we evaluated the involvement of EVs in the yeast-to-hypha transition, while in P. brasiliensis and A. fumigatus the function of EVs as stress transducers was investigated. P. brasiliensis and A. fumigatus were exposed to an inhibitor of glycosylation or UV light, respectively. The results demonstrated the role of EVs in regulating the expression of target genes and triggering phenotypic changes. The EVs treatment induced cellular proliferation and boosted the yeast to hyphal transition in C. albicans, while they enhanced stress responsiveness in A. fumigatus and P. brasiliensis, establishing a role for EVs in fungal intraspecies communication. Thus, EVs regulate fungal behavior, acting as potent message effectors, and understanding their effects and mechanism(s) of action could be exploited in antifungal therapies.

Extracellular Vesicles from Fusariumgraminearum Contain Protein Effectors Expressed during Infection of Corn.

Fusarium graminearum (Fgr) is a devastating filamentous fungal pathogen that causes diseases in cereals, while producing mycotoxins that are toxic for humans and animals, and render grains unusable. Low efficiency in managing Fgr poses a constant need for identifying novel control mechanisms. Evidence that fungal extracellular vesicles (EVs) from pathogenic yeast have a role in human disease led us to question whether this is also true for fungal plant pathogens. We separated EVs from Fgr and performed a proteomic analysis to determine if EVs carry proteins with potential roles in pathogenesis. We revealed that protein effectors, which are crucial for fungal virulence, were detected in EV preparations and some of them did not contain predicted secretion signals. Furthermore, a transcriptomic analysis of corn (Zea mays) plants infected by Fgr revealed that the genes of some of the effectors were highly expressed in vivo, suggesting that the Fgr EVs are a mechanism for the unconventional secretion of effectors and virulence factors. Our results expand the knowledge on fungal EVs in plant pathogenesis and cross-kingdom communication, and may contribute to the discovery of new antifungals.

Host cell membrane microdomains and fungal infection.

Lipid microdomains or lipid rafts are dynamic and tightly ordered regions of the plasma membrane. In mammalian cells, they are enriched in cholesterol, glycosphingolipids, Glycosylphosphatidylinositol-anchored and signalling-related proteins. Several studies have suggested that mammalian pattern recognition receptors are concentrated or recruited to lipid domains during host-pathogen association to enhance the effectiveness of host effector processes. However, pathogens have also evolved strategies to exploit these domains to invade cells and survive. In fungal organisms, a complex cell wall network usually mediates the first contact with the host cells. This cell wall may contain virulence factors that interfere with the host membrane microdomains dynamics, potentially impacting the infection outcome. Indeed, the microdomain disruption can dampen fungus-host cell adhesion, phagocytosis and cellular immune responses. Here, we provide an overview of regulatory strategies employed by pathogenic fungi to engage with and potentially subvert the lipid microdomains of host cells. TAKE AWAY: Lipid microdomains are ordered regions of the plasma membrane enriched in cholesterol, glycosphingolipids (GSL), GPI-anchored and signalling-related proteins. Pathogen recognition by host immune cells can involve lipid microdomain participation. During this process, these domains can coalesce in larger complexes recruiting receptors and signalling proteins, significantly increasing their signalling abilities. The antifungal innate immune response is mediated by the engagement of pathogen-associated molecular patterns to pattern recognition receptors (PRRs) at the plasma membrane of innate immune cells. Lipid microdomains can concentrate or recruit PRRs during host cell-fungi association through a multi-interactive mechanism. This association can enhance the effectiveness of host effector processes. However, virulence factors at the fungal cell surface and extracellular vesicles can re-assembly these domains, compromising the downstream signalling and favouring the disease development. Lipid microdomains are therefore very attractive targets for novel drugs to combat fungal infections.