Search

Abstract Members of the genus Cryptococcus are the causative agents of cryptococcal meningitis, a disease mainly associated with HIV-induced immunosuppression. Patients with cryptococcal meningitis are at a serious risk of death. Most patients suffering from cryptococcosis belong to neglected populations. With reduced support for research, new therapies are unlikely to emerge. In this essay, we used the Policy Cures/G-finder platform as a reference database for funding research on cryptococcal disease. Funding for cryptococcal research started being tracked by G-finder in 2013 and has continued to appear in the annual reports ever since. In total, 15 institutions were reported as major funders for research on cryptococcal disease over the years. The US National Institutes of Health (NIH) was the main funder, followed by the UK's Wellcome Trust. The annual analysis suggested slow yearly growth in funding from 2013 to 2021. The development of new tools to prevent and fight cryptococcal disease is urgent but requires improved funding.

ABSTRACT Emergomyces africanus is a highly fatal fungal pathogen affecting individuals with advanced HIV disease. Molecular patterns and ultrastructural aspects of E. africanus are unknown, and pathogenic models have not been investigated in detail. Since the cell wall of fungi is a determinant for interaction with the host and antifungal development, we characterized the ultrastructural aspects of E. africanus and the general properties of cell wall components under different conditions of growth in vitro and in vivo. We also tested the pathogenic potential of E. africanus in a Galleria mellonella model of infection. Transmission electron microscopy revealed the common intracellular, ultrastructural features of fungi in association with a thick cell wall. Scanning electron microscopy revealed a smooth cell surface, with no apparent decorative structures. Yeast cultures of E. africanus showed the distribution of chitin, chitooligomers, and mannoproteins commonly observed in fungi. However, in mixed microenvironments containing yeast and filamenting forms of E. africanus, the detection of chitooligomers was increased in comparison with isolated yeast cells, while the detection of these components in filamenting forms was markedly reduced. These observations were suggestive of the ability of E. africanus to change its cell wall composition in response to different microenvironments. Although E. africanus was unable to kill G. mellonella, this infection model allowed us to isolate infected hemocytes for further analysis of mannoproteins, chitin, and chitooligomers. Once again, the detection of E. africanus chitooligomers was markedly increased. These results reveal previously unknown ultrastructural features of E. africanus and suggest a high plasticity in the cell wall of this lethal pathogen.IMPORTANCEThe epidemiology of fungal infections is very dynamic, and novel health emergencies are hard to predict. New fungal pathogens have been continuously emerging for the last few decades, and Emergomyces africanus is one of these threats to human health. This complex scenario points to the need for generating knowledge about emerging pathogens so that new therapeutic strategies can be designed. In this study, we characterized the general cellular and pathogenic properties of the emerging fungal pathogen E. africanus. Our results reveal that E. africanus manifests some of the typical properties of fungal cells but also exhibits some unique characteristics that might be helpful for the future development of therapeutic strategies.

ABSTRACT Systemic candidiasis remains a significant public health concern worldwide, with high mortality rates despite available antifungal drugs. Drug-resistant strains add to the urgency for alternative therapies. In this context, vaccination has reemerged as a prominent immune-based strategy. Extracellular vesicles (EVs), nanosized lipid bilayer particles, carry a diverse array of native fungal antigens, including proteins, nucleic acids, lipids, and glycans. Previous studies from our laboratory demonstrated that Candida albicans EVs triggered the innate immune response, activating bone marrow-derived dendritic cells (BMDCs) and potentially acting as a bridge between innate and adaptive immunity. Vaccination with C. albicans EVs induced the production of specific antibodies, modulated cytokine production, and provided protection in immunosuppressed mice infected with lethal C. albicans inoculum. To elucidate the mechanisms underlying EV-induced immune activation, our study investigated pathogen-associated molecular patterns (PAMPs) and pattern recognition receptors (PRRs) involved in EVs-phagocyte engagement. EVs from wild-type and mutant C. albicans strains with truncated mannoproteins were compared for their ability to stimulate BMDCs. Our findings revealed that EV decoration with O- and N-linked mannans and the presence of β-1,3-glucans and chitin oligomers may modulate the activation of specific PRRs, in particular Toll-like receptor 4 (TLR4) and dectin-1. The protective effect of vaccination with wild-type EVs was found to be dependent on TLR4. These results suggest that fungal EVs can be harnessed in vaccine formulations to selectively activate PRRs in phagocytes, offering potential avenues for combating or preventing candidiasis.IMPORTANCESystemic candidiasis is a serious global health concern with high mortality rates and growing drug resistance. Vaccination offers a promising solution. A unique approach involves using tiny lipid-coated particles called extracellular vesicles (EVs), which carry various fungal components. Previous studies found that Candida albicans EVs activate the immune response and may bridge the gap between innate and adaptive immunity. To understand this better, we investigated how these EVs activate immune cells. We demonstrated that specific components on EV surfaces, such as mannans and glucans, interact with receptors on immune cells, including Toll-like receptor 4 (TLR4) and dectin-1. Moreover, vaccinating with these EVs led to strong immune responses and full protection in mice infected with Candida. This work shows how harnessing fungal EVs might lead to effective vaccines against candidiasis.

Summary: Extracellular vesicles (EVs) have been identified in diverse fungi, including human pathogens. In this protocol, we present two techniques for isolating and analyzing fungal EVs. The first is for high-throughput screening, and the second is for yielding concentrated samples suitable for centrifugation-based density gradients. We describe steps for analytical assays such as nano-flow cytometry and nanoparticle tracking analysis to measure EV dimensions and concentration. EV suspensions can serve diverse assays, including electron microscopy, compositional determination, and cell-to-cell communication assays.For complete details on the use and execution of this protocol, please refer to Rizzo et al.,1 Rizzo et al.,2 Reis et al.,3 and Reis et al.4 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

ABSTRACT We conducted a comprehensive comparative analysis of extracellular vesicles (EVs) from two Acanthamoeba castellanii strains, Neff (environmental) and T4 (clinical). Morphological analysis via transmission electron microscopy revealed slightly larger Neff EVs (average = 194.5 nm) compared to more polydisperse T4 EVs (average = 168.4 nm). Nanoparticle tracking analysis (NTA) and dynamic light scattering validated these differences. Proteomic analysis of the EVs identified 1,352 proteins, with 1,107 common, 161 exclusive in Neff, and 84 exclusively in T4 EVs. Gene ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) mapping revealed distinct molecular functions and biological processes and notably, the T4 EVs enrichment in serine proteases, aligned with its pathogenicity. Lipidomic analysis revealed a prevalence of unsaturated lipid species in Neff EVs, particularly triacylglycerols, phosphatidylethanolamines (PEs), and phosphatidylserine, while T4 EVs were enriched in diacylglycerols and diacylglyceryl trimethylhomoserine, phosphatidylcholine and less unsaturated PEs, suggesting differences in lipid metabolism and membrane permeability. Metabolomic analysis indicated Neff EVs enrichment in glycerolipid metabolism, glycolysis, and nucleotide synthesis, while T4 EVs, methionine metabolism. Furthermore, RNA-seq of EVs revealed differential transcript between the strains, with Neff EVs enriched in transcripts related to gluconeogenesis and translation, suggesting gene regulation and metabolic shift, while in the T4 EVs transcripts were associated with signal transduction and protein kinase activity, indicating rapid responses to environmental changes. In this novel study, data integration highlighted the differences in enzyme profiles, metabolic processes, and potential origins of EVs in the two strains shedding light on the diversity and complexity of A. castellanii EVs and having implications for understanding host-pathogen interactions and developing targeted interventions for Acanthamoeba-related diseases.IMPORTANCEA comprehensive and fully comparative analysis of extracellular vesicles (EVs) from two Acanthamoeba castellanii strains of distinct virulence, a Neff (environmental) and T4 (clinical), revealed striking differences in their morphology and protein, lipid, metabolites, and transcripts levels. Data integration highlighted the differences in enzyme profiles, metabolic processes, and potential distinct origin of EVs from both strains, shedding light on the diversity and complexity of A. castellanii EVs, with direct implications for understanding host-pathogen interactions, disease mechanisms, and developing new therapies for the clinical intervention of Acanthamoeba-related diseases.

Fungal extracellular vesicles (EVs) were first described in human pathogens. In a few years, the field of fungal EVs evolved to include several studies with plant pathogens, in which extracellularly released vesicles play fundamental biological roles. In recent years, solid progress has been made in the determination of the composition of EVs produced by phytopathogens. In addition, EV biomarkers are now known in fungal plant pathogens, and the production of EVs during plant infection has been demonstrated. In this manuscript, we review the recent progress in the field of fungal EVs, with a focus on plant pathogens. [Graphic: see text] The author(s) have dedicated the work to the public domain under the Creative Commons CC0 “No Rights Reserved” license by waiving all of his or her rights to the work worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law, 2023.

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 release of extracellular vesicles (EVs) has been implicated as an alternative transport mechanism for the passage of macromolecules through the fungal cell wall, a phenomenon widely reported in yeasts but poorly explored in mycelial cells. In the present work, we have purified and characterized the EVs released by mycelia of the emerging, opportunistic, widespread and multidrug-resistant filamentous fungus Scedosporium apiospermum. Transmission electron microscopy images and light scattering measurements revealed the fungal EVs, which were observed individually or grouped with heterogeneous morphology, size and electron density. The mean diameter of the EVs, evaluated by the light scattering technique, was 179.7 nm. Overall, the structural stability of S. apiospermum EVs was preserved during incubation under various storage conditions. The lipid, carbohydrate and protein contents were quantified, and the EVs’ protein profile was evidenced by SDS-PAGE, revealing proteins with molecular masses ranging from 20 to 118 kDa. Through immunoblotting, ELISA and immunocytochemistry assays, antigenic molecules were evidenced in EVs using a polyclonal serum (called anti-secreted molecules) from a rabbit inoculated with conditioned cell-free supernatant obtained from S. apiospermum mycelial cells. By Western blotting, several antigenic proteins were identified. The ELISA assay confirmed that the anti-secreted molecules exhibited a positive reaction up to a serum dilution of 1:3200. Despite transporting immunogenic molecules, S. apiospermum EVs slightly induced an in vitro cytotoxicity effect after 48 h of contact with either macrophages or lung epithelial cells. Interestingly, the pretreatment of both mammalian cells with purified EVs significantly increased the association index with S. apiospermum conidia. Furthermore, EVs were highly toxic to Galleria mellonella, leading to larval death in a typically dose- and time-dependent manner. Collectively, the results represent the first report of detecting EVs in the S. apiospermum filamentous form, highlighting a possible implication in fungal pathogenesis.

ABSTRACT Resistance to fluconazole (FLC), the most widely used antifungal drug, is typically achieved by altering the azole drug target and/or drug efflux pumps. Recent reports have suggested a link between vesicular trafficking and antifungal resistance. Here, we identified novel Cryptococcus neoformans regulators of extracellular vesicle (EV) biogenesis that impact FLC resistance. In particular, the transcription factor Hap2 does not affect the expression of the drug target or efflux pumps, yet it impacts the cellular sterol profile. Subinhibitory FLC concentrations also downregulate EV production. Moreover, in vitro spontaneous FLC-resistant colonies showed altered EV production, and the acquisition of FLC resistance was associated with decreased EV production in clinical isolates. Finally, the reversion of FLC resistance was associated with increased EV production. These data suggest a model in which fungal cells can regulate EV production in place of regulating the drug target gene expression as a first line of defense against antifungal assault in this fungal pathogen. IMPORTANCE Extracellular vesicles (EVs) are membrane-enveloped particles that are released by cells into the extracellular space. Fungal EVs can mediate community interactions and biofilm formation, but their functions remain poorly understood. Here, we report the identification of the first regulators of EV production in the major fungal pathogen Cryptococcus neoformans. Surprisingly, we uncover a novel role of EVs in modulating antifungal drug resistance. Disruption of EV production was associated with altered lipid composition and changes in fluconazole susceptibility. Spontaneous azole-resistant mutants were deficient in EV production, while loss of resistance restored initial EV production levels. These findings were recapitulated in C. neoformans clinical isolates, indicating that azole resistance and EV production are coregulated in diverse strains. Our study reveals a new mechanism of drug resistance in which cells adapt to azole stress by modulating EV production.

Chromoblastomycosis (CBM) is a neglected human implantation mycosis caused by several dematiaceous fungal species. Currently available therapy is usually associated with physical methods, especially surgery, and with high refractoriness. Therefore, drug discovery for CBM is essential. Drug repositioning is a strategy used to facilitate the discovery of new treatments for several diseases. The aim of this study was to discover substances with antifungal activity against CBM agents from a collection of drugs previously approved for use in human diseases. A screening was performed with the NIH Clinical Collection against Fonsecaea pedrosoi. Ten substances, with clinical applicability in CBM, inhibited fungal growth by at least 60%. The minimum inhibitory concentration (MIC) of these substances was determined against other CBM agents, and the benzimidazoles albendazole, mebendazole and thiabendazole presented the lowest MIC values. The selectivity index, based on MIC and cytotoxicity of these substances, revealed albendazole to be more selective. To investigate a possible synergism of this benzimidazole with itraconazole and terbinafine, the chequerboard method was used. All interactions were classified as indifferent. Our current results suggest that benzimidazoles have repositioning potential against CBM agents. Albendazole seems to be the most promising, since it presented the highest selectivity against all dematiaceous fungi tested.

ABSTRACT There is an urgent unmet need for novel antifungals. In this study, we searched for novel antifungal activities in the Pandemic Response Box, a collection of 400 structurally diverse compounds in various phases of drug discovery. We identified five molecules which could control the growth of Cryptococcus neoformans, Cryptococcus deuterogattii, and the emerging global threat Candida auris. After eliminating compounds which demonstrated paradoxical antifungal effects or toxicity to mammalian macrophages, we selected compound MMV1593537 as a nontoxic, fungicidal molecule for further characterization of antifungal activity. Scanning electron microscopy revealed that MMV1593537 affected cellular division in all three pathogens. In Cryptococcus, MMV1593537 caused a reduction in capsular dimensions. Treatment with MMV1593537 resulted in increased detection of cell wall chitooligomers in these three species. Since chitooligomers are products of the enzymatic hydrolysis of chitin, we investigated whether surface chitinase activity was altered in response to MMV1593537 exposure. We observed peaks of enzyme activity in C. neoformans and C. deuterogattii in response to MMV1593537. We did not detect any surface chitinase activity in C. auris. Our results suggest that MMV1593537 is a promising, nontoxic fungicide whose mechanism of action, at least in Cryptococcus spp, requires chitinase-mediated hydrolysis of chitin. IMPORTANCE The development of novel antifungals is a matter of urgency. In this study, we evaluated antifungal activities in a collection of 400 molecules, using highly lethal fungal pathogens as targets. One of these molecules, namely, MMV1593537, was not toxic to host cells and controlled the growth of isolates of Cryptococcus neoformans, C. deuterogattii, C. gattii, Candida auris, C. albicans, C. parapsilosis, and C. krusei. We tested the mechanisms of antifungal action of MMV1593537 in the Cryptococcus and C. auris models and concluded that the compound affects the cell wall, a structure which is essential for fungal life. At least in Cryptococcus, this effect involved chitinase, an enzyme which is required for remodeling the cell wall. Our results suggest that MMV1593537 is a candidate for future antifungal development.

ABSTRACT In this study, we investigated the influence of fungal extracellular vesicles (EVs) during biofilm formation and morphogenesis in Candida albicans. Using crystal violet staining and scanning electron microscopy (SEM), we demonstrated that C. albicans EVs inhibited biofilm formation in vitro. By time-lapse microscopy and SEM, we showed that C. albicans EV treatment stopped filamentation and promoted pseudohyphae formation with multiple budding sites. The ability of C. albicans EVs to regulate dimorphism was further compared to EVs isolated from different C. albicans strains, Saccharomyces cerevisiae, and Histoplasma capsulatum. C. albicans EVs from distinct strains inhibited yeast-to-hyphae differentiation with morphological changes occurring in less than 4 h. EVs from S. cerevisiae and H. capsulatum modestly reduced morphogenesis, and the effect was evident after 24 h of incubation. The inhibitory activity of C. albicans EVs on phase transition was promoted by a combination of lipid compounds, which were identified by gas chromatography-tandem mass spectrometry analysis as sesquiterpenes, diterpenes, and fatty acids. Remarkably, C. albicans EVs were also able to reverse filamentation. Finally, C. albicans cells treated with C. albicans EVs for 24 h lost their capacity to penetrate agar and were avirulent when inoculated into Galleria mellonella. Our results indicate that fungal EVs can regulate yeast-to-hypha differentiation, thereby inhibiting biofilm formation and attenuating virulence. IMPORTANCE The ability to undergo morphological changes during adaptation to distinct environments is exploited by Candida albicans and has a direct impact on biofilm formation and virulence. Morphogenesis is controlled by a diversity of stimuli, including osmotic stress, pH, starvation, presence of serum, and microbial components, among others. Apart from external inducers, C. albicans also produces autoregulatory substances. Farnesol and tyrosol are examples of quorum-sensing molecules (QSM) released by C. albicans to regulate yeast-to-hypha conversion. Here, we demonstrate that fungal EVs are messengers impacting biofilm formation, morphogenesis, and virulence in C. albicans. The major players exported in C. albicans EVs included sesquiterpenes, diterpenes, and fatty acids. The understanding of how C. albicans cells communicate to regulate physiology and pathogenesis can lead to novel therapeutic tools to combat candidiasis.

Candida auris has emerged as a serious worldwide threat by causing opportunistic infections that are frequently resistant to one or more conventional antifungal medications resulting in high mortality rates. Against this backdrop, health warnings around the world have focused efforts on understanding C. auris fungal biology and effective prevention and treatment approaches to combat this fungus. To date, there is little information about the differentially expressed genes when this fungus is treated with conventional antifungals, and caspofungin is a standard echinocandin deployed in the therapy against C. auris. In this work, we treated two distinct strains of C. auris for 24 h with caspofungin, and the cellular responses were evaluated at the morphological, translational and transcriptional levels. We first observed that the echinocandin caused morphological alterations, aggregation of yeast cells, and modifications in the cell wall composition of C. auris. Transcriptomic analysis revealed an upregulation of genes related to the synthesis of the cell wall, ribosome, and cell cycle after exposure to caspofungin. Supporting these findings, the integrated proteomic analysis showed that caspofungin-treated cells were enriched in ribosome-related proteins and cell wall, especially mannoproteins. Altogether, these results provide further insights into the biology of C. auris and expands our understanding regarding the antifungal activity of caspofungin and reveal cellular targets, as the mannose metabolism, that can be further explored for the development of novel antifungals.

Extracellular vesicles (EVs) are double-membrane particles associated with intercellular communication. Since the discovery of EV production in the fungus Cryptococcus neoformans, the importance of EV release in its physiology and pathogenicity has been investigated. To date, few studies have investigated the proteomic content of EVs from multiple fungal species. Our main objective was to use an orthology approach to compare proteins identified by EV shotgun proteomics in 8 pathogenic and 1 nonpathogenic species. Using protein information from the UniProt and FungiDB databases, we integrated data for 11,433 hits in fungal EVs with an orthology perspective, resulting in 3,834 different orthologous groups. OG6_100083 (Hsp70 Pfam domain) was the unique orthologous group that was identified for all fungal species. Proteins with this protein domain are associated with the stress response, survival and morphological changes in different fungal species. Although no pathogenic orthologous group was found, we identified 5 orthologous groups exclusive to S. cerevisiae. Using the criteria of at least 7 pathogenic fungi to define a cluster, we detected the 4 unique pathogenic orthologous groups. Taken together, our data suggest that Hsp70-related proteins might play a key role in fungal EVs, regardless of the pathogenic status. Using an orthology approach, we identified at least 4 protein domains that could be novel therapeutic targets against pathogenic fungi. Our results were compiled in the herein described ExVe database, which is publicly available at http://exve.icc.fiocruz.br.

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.

ABSTRACT Fungal 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. IMPORTANCE Here, we report a study about extracellular vesicles (EVs) as communication mediators in fungi. Our results demonstrated the role of EVs from Candida albicans, Aspergillus fumigatus, and Paracoccidioides brasiliensis regulating the expression of target genes and phenotypic features. We asked whether fungal EVs play a role as message effectors. We show that fungal EVs are involved in fungal interaction systems as potent message effectors, and understanding their effects and mechanisms of action could be exploited in antifungal therapies.

ABSTRACT Emerging and reemerging pathogens are a worldwide concern, and it is predicted that these microbes will cause severe outbreaks. Candida auris affects people with weakened immune systems, particularly those who are hospitalized or are in health care facilities. Extracellular vesicles (EVs) are lipid bilayer structures released by organisms from all domains of life. EVs can deliver functional molecules to target cells, including proteins and nucleic acids, especially RNA molecules. EVs from several pathogenic fungi species play diverse biological roles related to cell-cell communication and pathogen-host interaction. In this study, we describe a data set which we produced by sequencing the RNA content of EVs from C. auris under normal growth conditions and in the presence of the antifungal caspofungin, a first-line drug to treat this fungus. To generate a more complete data set for future comparative studies, we also sequenced the RNA cellular content of EVs under the same conditions. This data set addresses a previously unexplored area of fungal biology regarding cellular small RNA and EV RNA. Our data will provide a molecular basis for the study of the aspects associated with antifungal treatment, gene expression response, and EV composition in C. auris. These data will also allow the exploration of small RNA content in the fungal kingdom and might serve as an informative basis for studies on the mechanisms by which molecules are directed to fungal EVs. IMPORTANCE Candida auris, a relevant emerging human-pathogenic yeast, is the first fungus to be called a global public health threat by the WHO. This is because of its rapid spread on all inhabited continents, together with its extremely high frequency of drug and multidrug resistance. In our study, we generated a large data set for 3 distinct strains of C. auris and obtained cellular small RNA fraction as well as extracellular vesicle RNA (EV-RNA) during normal growth conditions and after treatment with caspofungin, the first-line drug used to treat C. auris infection.

ABSTRACT Extracellular vesicles (EVs) produced by members of the Cryptococcus genus are associated with fundamental processes of fungal physiology and virulence. However, several questions about the properties of cryptococcal EVs remain unanswered, mostly because of technical limitations. We recently described a fast and efficient protocol of high-yield EV isolation from solid medium. In this study, we aimed at using the solid medium protocol to address some of the open questions about EVs, including the kinetics of EV production, the diversity of EVs produced by multiple isolates under different culture conditions, the separation of vesicles in a density gradient followed by the recovery of functional EVs, the direct detection of EVs in culture supernatants, and the production of vesicles in solid cultures of Titan cells. Our results indicate that the production of EVs is directly impacted by the culture medium and time of growth, resulting in variable detection of EVs per cell and a peak of EV detection at 24 h of growth. Nanoparticle tracking analysis (NTA) of EV samples revealed that multiple isolates produce vesicles with variable properties, including particles of diverging dimensions. EVs were produced in the solid medium in amounts that were separated on a centrifugation density gradient, resulting in the recovery of functional EVs containing the major cryptococcal capsular antigen. We also optimized the solid medium protocol for induction of the formation of Titan cells, and analyzed the production of EVs by NTA and transmission electron microscopy. This analysis confirmed that EVs were isolated from solid cultures of cryptococcal enlarged cells. With these approaches, we expect to implement simple methods that will facilitate the analysis of EVs produced by fungal cells. IMPORTANCE Fungal extracellular vesicles (EVs) are considered to be important players in the biology of fungal pathogens. However, the limitations in the methodological approaches to studying fungal EVs impair the expansion of knowledge in this field. In the present study, we used the Cryptococcus genus as a model for the study of EVs. We explored the simplification of protocols for EV analysis, which helped us to address some important, but still unanswered, questions about fungal EVs.

Antifungal resistance has become more frequent, either due to the emergence of naturally resistant species or the development of mechanisms that lead to resistance in previously susceptible species. Among these fungal species of global threat, Candida auris stands out for commonly being highly resistant to antifungal drugs, and some isolates are pan-resistant. The rate of mortality linked to C. auris infections varies from 28% to 78%. In this study, we characterized C. auris extracellular vesicles (EVs) in the presence of caspofungin, an echinocandin, which is the recommended first line antifungal for the treatment of infections due to this emerging pathogen. Furthermore, we also analyzed the protein and RNA content of EVs generated by C. auris cultivated with or without treatment with caspofungin. We observed that caspofungin led to the increased production of EVs, and treatment also altered the type and quantity of RNA molecules and proteins enclosed in the EVs. There were distinct classes of RNAs in the EVs with ncRNAs being the most identified molecules, and tRNA-fragments (tRFs) were abundant in each of the strains studied. We also identified anti-sense RNAs, varying from 21 to 55 nt in length. The differentially abundant mRNAs detected in EVs isolated from yeast subjected to caspofungin treatment were related to translation, nucleosome core and cell wall. The differentially regulated proteins identified in the EVs produced during caspofungin treatment were consistent with the results observed with the RNAs, with the enriched terms being related to translation and cell wall. Our study adds new information on how an echinocandin can affect the EV pathway, which is associated with the yeast cell being able to evade treatment and persist in the host. The ability of C. auris to efficiently alter the composition of EVs may represent a mechanism for the fungus to mitigate the effects of antifungal agents.

Abstract Whereas extracellular vesicle (EV) research has become commonplace in different biomedical fields, this field of research is still in its infancy in mycology. Here we provide a robust set of data regarding the structural and compositional aspects of EVs isolated from the fungal pathogenic species Cryptococcus neoformans, C. deneoformans and C. deuterogattii. Using cutting‐edge methodological approaches including cryogenic electron microscopy and cryogenic electron tomography, proteomics, and flow cytometry, we revisited cryptococcal EV features and suggest a new EV structural model, in which the vesicular lipid bilayer is covered by mannoprotein‐based fibrillar decoration, bearing the capsule polysaccharide as its outer layer. About 10% of the EV population is devoid of fibrillar decoration, adding another aspect to EV diversity. By analysing EV protein cargo from the three species, we characterized the typical Cryptococcus EV proteome. It contains several membrane‐bound protein families, including some Tsh proteins bearing a SUR7/PalI motif. The presence of known protective antigens on the surface of Cryptococcus EVs, resembling the morphology of encapsulated virus structures, suggested their potential as a vaccine. Indeed, mice immunized with EVs obtained from an acapsular C. neoformans mutant strain rendered a strong antibody response in mice and significantly prolonged their survival upon C. neoformans infection.

Candida auris

Extracellular vesicles (EVs) are lipid bilayer structures released by organisms from all kingdoms of life. The diverse biogenesis pathways of EVs result in a wide variety of physical properties and functions across different organisms. Fungal EVs were first described in 2007 and different omics approaches have been fundamental to understand their composition, biogenesis, and function. In this review, we discuss the role of omics in elucidating fungal EVs biology. Transcriptomics, proteomics, metabolomics, and lipidomics have each enabled the molecular characterization of fungal EVs, providing evidence that these structures serve a wide array of functions, ranging from key carriers of cell wall biosynthetic machinery to virulence factors. Omics in combination with genetic approaches have been instrumental in determining both biogenesis and cargo loading into EVs. We also discuss how omics technologies are being employed to elucidate the role of EVs in antifungal resistance, disease biomarkers, and their potential use as vaccines. Finally, we review recent advances in analytical technology and multi-omic integration tools, which will help to address key knowledge gaps in EVs biology and translate basic research information into urgently needed clinical applications such as diagnostics, and immuno- and chemotherapies to fungal infections.

AbstractThe genomes of a large number of Cryptococcus neoformansC. neoformansCryptococcuC. neoformansCryptococcus

The small molecule (molecular mass

During the postharvest period, citrus fruits can be affected by phytopathogens such as Penicillium digitatum

The cell wall is a ubiquitous structure in the fungal kingdom, with some features varying depending on the species. Additional external structures can be present, such as the capsule of Cryptococcus neoformans (Cn), its major virulence factor, mainly composed of glucuronoxylomannan (GXM), with anti-phagocytic and anti-inflammatory properties. The literature shows that other cryptococcal species and even more evolutionarily distant species, such as the Trichosporon asahii, T. mucoides, and Paracoccidioides brasiliensis can produce GXM-like polysaccharides displaying serological reactivity to GXM-specific monoclonal antibodies (mAbs), and these complex polysaccharides have similar composition and anti-phagocytic properties to cryptococcal GXM. Previously, we demonstrated that the fungus Histoplasma capsulatum (Hc) incorporates, surface/secreted GXM of Cn and the surface accumulation of the polysaccharide enhances Hc virulence in vitro and in vivo. In this work, we characterized the ability of Hc to produce cellular-attached (C-gly-Hc) and secreted (E-gly) glycans with reactivity to GXM mAbs. These C-gly-Hc are readily incorporated on the surface of acapsular Cn cap59; however, in contrast to Cn GXM, C-gly-Hc had no xylose and glucuronic acid in its composition. Mapping of recognized Cn GXM synthesis/export proteins confirmed the presence of orthologs in the Hc database. Evaluation of C-gly and E-gly of Hc from strains of distinct monophyletic clades showed serological reactivity to GXM mAbs, despite slight differences in their molecular dimensions. These C-gly-Hc and E-gly-Hc also reacted with sera of cryptococcosis patients. In turn, sera from histoplasmosis patients recognized Cn glycans, suggesting immunogenicity and the presence of cross-reacting antibodies. Additionally, C-gly-Hc and E-gly-Hc coated Cn cap59 were more resistant to phagocytosis and macrophage killing. C-gly-Hc and E-gly-Hc coated Cn cap59 were also able to kill larvae of Galleria mellonella. These GXM-like Hc glycans, as well as those produced by other pathogenic fungi, may also be important during host-pathogen interactions, and factors associated with their regulation are potentially important targets for the management of histoplasmosis.

ABSTRACT Extracellular vesicles (EVs) are membranous compartments produced by yeast and mycelial forms of several fungal species. One of the difficulties in perceiving the role of EVs during the fungal life, and particularly in cell wall biogenesis, is caused by the presence of a thick cell wall. One alternative to have better access to these vesicles is to use protoplasts. This approach has been investigated here with Aspergillus fumigatus, one of the most common opportunistic fungal pathogens worldwide. Analysis of regenerating protoplasts by scanning electron microscopy and fluorescence microscopy indicated the occurrence of outer membrane projections in association with surface components and the release of particles with properties resembling those of fungal EVs. EVs in culture supernatants were characterized by transmission electron microscopy and nanoparticle tracking analysis. Proteomic and glycome analysis of EVs revealed the presence of a complex array of enzymes related to lipid/sugar metabolism, pathogenic processes, and cell wall biosynthesis. Our data indicate that (i) EV production is a common feature of different morphological stages of this major fungal pathogen and (ii) protoplastic EVs are promising tools for undertaking studies of vesicle functions in fungal cells. IMPORTANCE Fungal cells use extracellular vesicles (EVs) to export biologically active molecules to the extracellular space. In this study, we used protoplasts of Aspergillus fumigatus, a major fungal pathogen, as a model to evaluate the role of EV production in cell wall biogenesis. Our results demonstrated that wall-less A. fumigatus exports plasma membrane-derived EVs containing a complex combination of proteins and glycans. Our report is the first to characterize fungal EVs in the absence of a cell wall. Our results suggest that protoplasts represent a promising model for functional studies of fungal vesicles.

Extracellular vesicles (EVs) have garnered much interest in the cell biology and biomedical research fields. Many studies have reported the existence of EVs in all types of living cells, including in fifteen different fungal genera. EVs play diverse biological roles, from the regulation of physiological events and response to specific environmental conditions to the mediation of highly complex interkingdom communications. This review will provide a historical perspective on EVs produced by fungi and an overview of the recent discoveries in the field. We will also review the current knowledge about EV biogenesis and cargo, their role in cell-to-cell interactions, and methods of EV analysis. Finally, we will discuss the perspectives of EVs as vehicles for the delivery of biologically active molecules.

The fungal cell wall and membrane are the most common targets of antifungal agents, but the potential of membrane lipid organization in regulating drug-target interactions has yet to be investigated. Energy-dependent lipid transporters have been recently associated with virulence and drug resistance in many pathogenic fungi. To illustrate this view, we discuss (i) the structural and biological aspects of ATP-driven lipid transporters, comprising P-type ATPases and ATP-binding cassette transporters, (ii) the role of these transporters in fungal physiology and virulence, and (iii) the potential of lipid transporters as targets for the development of novel antifungals. These recent observations indicate that the lipid-trafficking machinery in fungi is a promising target for studies on physiology, pathogenesis and drug development. Keywords: Flippases, ABC transporters, Fungal infections, Antifungals, Phosphatidylserine, Phosphatidylethanolamine, Sterols

Cryptococcus neoformans mutants lacking each of the eight putative chitin synthase genes (CHS) have been previously generated. However, it is still unclear how deletion of chitin synthase genes affects the cryptococcal capsule. Since the connections between chitin metabolism and capsular polysaccharides in C. neoformans are numerous, we analyzed the effects of deletion of CHS genes on capsular and capsule-related structures of C. neoformans. CHS deletion affected capsular morphology in multiple ways, as determined by scanning electron microscopy and immunofluorescence analysis. Molecular diameter, serological reactivity and export of capsular polysaccharide were also affected in most of the chsΔ mutants, but the most prominent alterations were observed in the chs3Δ strain. C. neoformans cells lacking CHS genes also had altered formation of extracellular vesicles and variable chitinase activity under stress conditions. These results reveal previously unknown functions of CHS genes that greatly impact the physiology of C. neoformans.

Lipid flippases of the P4-ATPase family are ATP-driven transporters that translocate lipids from the exoplasmic to the cytosolic leaflet of biological membranes. In the encapsulated fungal pathogen Cryptococcus neoformans, the P4-ATPase Apt1p is an important regulator of polysaccharide secretion and pathogenesis, but its biochemical characterization is lacking. Phylogenetic analysis revealed that Apt1p belongs to the subclade of P4A-ATPases characterized by the common requirement for a β-subunit. Using heterologous expression in S. cerevisiae, we demonstrate that Apt1p forms a heterodimeric complex with the C. neoformans Cdc50 protein. This association is required for both localization and activity of the transporter complex. Lipid flippase activity of the heterodimeric complex was assessed by complementation tests and uptake assays employing fluorescent lipids and revealed a broad substrate specificity, including several phospholipids, the alkylphospholipid miltefosine, and the glycolipids glucosyl- and galactosylceramide. Our results suggest that transbilayer lipid transport in C. neoformans is finely regulated to promote fungal virulence, which reinforces the potential of Apt1p as a target for antifungal drug development.

The protein Galectin-3 modulates host immunity and plays roles during infections. Here, Almeida et al. show that this protein contributes to host defence against infection with the fungal pathogen Cryptococcus neoformans by inhibiting fungal growth and inducing lysis of fungal extracellular vesicles.

Chromoblastomycosis (CBM) is a chronic subcutaneous mycosis caused by traumatic implantation of many species of black fungi. Due to the refractoriness of some cases and common recurrence of CBM, a more effective and less time-consuming treatment is mandatory. The aim of this study was to identify compounds with in vitro antifungal activity in the Pathogen Box® compound collection against different CBM agents. Synergism of these compounds with drugs currently used to treat CBM was also assessed. An initial screening of the drugs present in this collection at 1 μM was performed with a Fonsecaea pedrosoi clinical strain according to the EUCAST protocol. The compounds with activity against this fungus were also tested against other seven etiologic agents of CBM (Cladophialophora carrionii, Phialophora verrucosa, Exophiala jeanselmei, Exophiala dermatitidis, Fonsecaea monophora, Fonsecaea nubica, and Rhinocladiella similis) at concentrations ranging from 0.039 to 10 μM. The analysis of potential synergism of these compounds with itraconazole and terbinafine was performed by the checkerboard method. Eight compounds inhibited more than 60% of the F. pedrosoi growth: difenoconazole, bitertanol, iodoquinol, azoxystrobin, MMV688179, MMV021013, trifloxystrobin, and auranofin. Iodoquinol produced the lowest MIC values (1.25-2.5 μM) and MMV688179 showed MICs that were higher than all compounds tested (5 - >10 μM). When auranofin and itraconazole were tested in combination, a synergistic interaction (FICI = 0.37) was observed against the C. carrionii isolate. Toxicity analysis revealed that MMV021013 showed high selectivity indices (SI ≥ 10) against the fungi tested. In summary, auranofin, iodoquinol, and MMV021013 were identified as promising compounds to be tested in CBM models of infection.

ABSTRACT The release of extracellular vesicles (EVs) by fungi is a fundamental cellular process. EVs carry several biomolecules, including pigments, proteins, enzymes, lipids, nucleic acids, and carbohydrates, and are involved in physiological and pathological processes. EVs may play a pivotal role in the establishment of fungal infections, as they can interact with the host immune system to elicit multiple outcomes. It has been observed that, depending on the fungal pathogen, EVs can exacerbate or attenuate fungal infections. The study of the interaction between fungal EVs and the host immune system and understanding of the mechanisms that regulate those interactions might be useful for the development of new adjuvants as well as the improvement of protective immune responses against infectious or noninfectious diseases. In this review, we describe the immunomodulatory properties of EVs produced by pathogenic fungi and discuss their potential as adjuvants for prophylactic or therapeutic strategies.

ABSTRACT Eukaryotic cells, including fungi, release extracellular vesicles (EVs). These lipid bilayered compartments play essential roles in cellular communication and pathogenesis. EV composition is complex and includes proteins, glycans, pigments, and RNA. RNAs with putative roles in pathogenesis have been described in EVs produced by fungi. Here we describe the RNA content in EVs produced by the G186AR and G217B strains of Histoplasma capsulatum, an important human-pathogenic fungal pathogen. A total of 124 mRNAs were identified in both strains. In this set of RNA classes, 93 transcripts were enriched in EVs from the G217B strain, whereas 31 were enriched in EVs produced by the G186AR strain. This result suggests that there are important strain-specific properties in the mRNA composition of fungal EVs. We also identified short fragments (25 to 40 nucleotides in length) that were strain specific, with a greater number identified in EVs produced by the G217B strain. Remarkably, the most highly enriched processes were stress responses and translation. Half of these fragments aligned to the reverse strand of the transcript, suggesting the occurrence of microRNA (miRNA)-like molecules in fungal EVs. We also compared the transcriptome profiles of H. capsulatum with the RNA composition of EVs, and no correlation was observed. Taking the results together, our study provided information about the RNA molecules present in H. capsulatum EVs and about the differences in composition between the strains. In addition, we found no correlation between the most highly expressed transcripts in the cell and their presence in the EVs, reinforcing the idea that the RNAs were directed to the EVs by a regulated mechanism. IMPORTANCE Extracellular vesicles (EVs) play important roles in cellular communication and pathogenesis. The RNA molecules in EVs have been implicated in a variety of processes. EV-associated RNA classes have recently been described in pathogenic fungi; however, only a few reports of studies describing the RNAs in fungal EVs are available. Improved knowledge of EV-associated RNA will contribute to the understanding of their role during infection. In this study, we described the RNA content in EVs produced by two isolates of Histoplasma capsulatum. Our results add this important pathogen to the current short list of fungal species with the ability to use EVs for the extracellular release of RNA.

ABSTRACT Regular protocols for the isolation of fungal extracellular vesicles (EVs) are time-consuming, hard to reproduce, and produce low yields. In an attempt to improve the protocols used for EV isolation, we explored a model of vesicle production after growth of Cryptococcus gattii and Cryptococcus neoformans on solid media. Nanoparticle tracking analysis in combination with transmission electron microscopy revealed that C. gattii and C. neoformans produced EVs in solid media. The properties of cryptococcal vesicles varied according to the culture medium used and the EV-producing species. EV detection was reproduced with an acapsular mutant of C. neoformans, as well as with isolates of Candida albicans, Histoplasma capsulatum, and Saccharomyces cerevisiae. Cryptococcal EVs produced in solid media were biologically active and contained regular vesicular components, including the major polysaccharide glucuronoxylomannan (GXM) and RNA. Since the protocol had higher yields and was much faster than the regular methods used for the isolation of fungal EVs, we asked if it would be applicable to address fundamental questions related to cryptococcal secretion. On the basis that polysaccharide export in Cryptococcus requires highly organized membrane traffic culminating with EV release, we analyzed the participation of a putative scramblase (Aim25; CNBG_3981) in EV-mediated GXM export and capsule formation in C. gattii. EVs from a C. gattii aim25Δ strain differed from those obtained from wild-type (WT) cells in physical-chemical properties and cargo. In a model of surface coating of an acapsular cryptococcal strain with vesicular GXM, EVs obtained from the aim25Δ mutant were more efficiently used as a source of capsular polysaccharides. Lack of the Aim25 scramblase resulted in disorganized membranes and increased capsular dimensions. These results associate the description of a novel protocol for the isolation of fungal EVs with the identification of a previously unknown regulator of polysaccharide release. IMPORTANCE Extracellular vesicles (EVs) are fundamental components of the physiology of cells from all kingdoms. In pathogenic fungi, they participate in important mechanisms of transfer of antifungal resistance and virulence, as well as in immune stimulation and prion transmission. However, studies on the functions of fungal EVs are still limited by the lack of efficient methods for isolation of these compartments. In this study, we developed an alternative protocol for isolation of fungal EVs and demonstrated an application of this new methodology in the study of the physiology of the fungal pathogen Cryptococcus gattii. Our results describe a fast and reliable method for the study of fungal EVs and reveal the participation of scramblase, a phospholipid-translocating enzyme, in secretory processes of C. gattii.

ABSTRACT Cryptococcus neoformans is an encapsulated fungal pathogen that causes meningoencephalitis. There are no prophylactic tools for cryptococcosis. Previously, our group showed that a C. neoformans mutant lacking the gene encoding sterylglucosidase (Δsgl1) induced protection in both immunocompetent and immunocompromised murine models of cryptococcosis. Since sterylglucosidase catalyzes degradation of sterylglucosides (SGs), accumulation of this glycolipid could be responsible for protective immunity. In this study, we analyzed whether the activity of SGs is sufficient for the protective effect induced by the Δsgl1 strain. We observed that the accumulation of SGs impacted several properties of the main polysaccharide that composes the fungal capsule, glucuronoxylomannan (GXM). We therefore used genetic manipulation to delete the SGL1 gene in the acapsular mutant Δcap59 to generate a double mutant (strain Δcap59/Δsgl1) that was shown to be nonpathogenic and cleared from the lung of mice within 7 days post-intranasal infection. The inflammatory immune response triggered by the Δcap59/Δsgl1 mutant in the lung differed from the response seen with the other strains. The double mutant did not induce protection in a vaccination model, suggesting that SG-related protection requires the main capsular polysaccharide. Finally, GXM-containing extracellular vesicles (EVs) enriched in SGs delayed the acute lethality of Galleria mellonella against C. neoformans infection. These studies highlighted a key role for GXM and SGs in inducing protection against a secondary cryptococcal infection, and, since EVs notoriously contain GXM, these results suggest the potential use of Δsgl1 EVs as a vaccination strategy for cryptococcosis. IMPORTANCE The number of deaths from cryptococcal meningitis is around 180,000 per year. The disease is the second leading cause of mortality among individuals with AIDS. Antifungal treatment is costly and associated with adverse effects and resistance, evidencing the urgency of development of both therapeutic and prophylactic tools. Here we demonstrate the key roles of polysaccharide- and glycolipid-containing structures in a vaccination model to prevent cryptococcosis.

Extracellular vesicles (EVs) are produced by all domains of life. In fungi, these structures were first described in Cryptococcus neoformans and, since then, they were characterized in several pathogenic and non-pathogenic fungal species. Cryptococcal EVs participate in the export of virulence factors that directly impact the Cryptococcus–host interaction. Our knowledge of the biogenesis and pathogenic roles of Cryptococcus EVs is still limited, but recent methodological and scientific advances have improved our understanding of how cryptococcal EVs participate in both physiological and pathogenic events. In this review, we will discuss the importance of cryptococcal EVs, including early historical studies suggesting their existence in Cryptococcus, their putative mechanisms of biogenesis, methods of isolation, and possible roles in the interaction with host cells.

In the past few years, fungal diseases caused estimated over 1.6 million deaths annually and over one billion people suffer from severe fungal diseases (Brown et al., 2012; Anonymous, 2017b). Public health surveillance of fungal diseases is generally not compulsory, suggesting that most estimates are conservative (Casadevall, 2017; Anonymous, 2017a). Fungal disease can also damage plants and crops, causing major losses in agricultural activities and food production (Savary et al., 2012). Animal pathogenic fungi are threatening bats, amphibians and reptiles with extinction (Casadevall, 2017). It is estimated that fungi are the highest threat for animal-host and plant-host species, representing the major cause (approximately 65%) of pathogen-driven host loss (Fisher et al., 2012). In this complex scenario, it is now clear that the global warming and accompanying climate changes have resulted in increased incidence of many fungal diseases (Garcia-Solache and Casadevall, 2010). On the basis of all these factors, concerns on the occurrence of a pandemic of fungal origin in a near future have been raised (Casadevall, 2017). In this context, to stop forgetting and underestimating fungal diseases is mandatory.

ABSTRACT The roles of ergosterol in the regulation of membrane fluidity and structure, as well its role as a target for the activity of antifungals, have been known for decades. Two recent studies, however, demonstrated that ergosterol is an immunologically active lipid that induces pyroptosis and that virtually all steps of its biosynthetic process are potential drug targets. In combination, those two reports strongly indicate that the biological relevance of ergosterol is broader than the current literature suggests.