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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.
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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.
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Microdomínios da Membrana , Micoses , Animais , Membrana Celular , Glicoesfingolipídeos , Fagocitose , Receptores de Reconhecimento de PadrãoRESUMO
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.
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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.
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Aspergillus fumigatus/fisiologia , Vesículas Extracelulares/fisiologia , Proteômica , Protoplastos/fisiologia , Parede Celular/metabolismo , Vesículas Extracelulares/ultraestrutura , Proteínas Fúngicas/metabolismo , Microscopia Eletrônica de Varredura , Microscopia Eletrônica de Transmissão , Microscopia de Fluorescência , Biogênese de Organelas , Protoplastos/ultraestruturaRESUMO
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.
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Golgi reassembly and stacking protein (GRASP) is required for polysaccharide secretion and virulence in Cryptococcus neoformans. In fungal species, extracellular vesicles (EVs) participate in the export of polysaccharides, proteins and RNA. In the present work, we investigated if EV-mediated RNA export is functionally connected with GRASP in C. neoformans using a graspΔ mutant. Since GRASP-mediated unconventional secretion involves autophagosome formation in yeast, we included the atg7Δ mutant with defective autophagic mechanisms in our analysis. All fungal strains exported EVs but deletion of GRASP or ATG7 profoundly affected vesicular dimensions. The mRNA content of the graspΔ EVs differed substantially from that of the other two strains. The transcripts associated to the endoplasmic reticulum were highly abundant transcripts in graspΔ EVs. Among non-coding RNAs (ncRNAs), tRNA fragments were the most abundant in both mutant EVs but graspΔ EVs alone concentrated 22 exclusive sequences. In general, our results showed that the EV RNA content from atg7Δ and WT were more related than the RNA content of graspΔ, suggesting that GRASP, but not the autophagy regulator Atg7, is involved in the EV export of RNA. This is a previously unknown function for a key regulator of unconventional secretion in eukaryotic cells.
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Flippases are responsible for the asymmetric distribution of phospholipids in biological membranes. In the encapsulated fungal pathogen Cryptococcus neoformans, the putative flippase Apt1 is an important regulator of polysaccharide secretion and pathogenesis in mice by unknown mechanisms. In this study, we analyzed the role of C. neoformans Apt1 in intracellular membrane architecture and synthesis of polysaccharide and lipids. Analysis of wild type (WT), apt1Δ (mutant) and apt1Δ::APT1 (complemented) strains by transmission electron microscopy revealed that deletion of APT1 resulted in the formation of irregular vacuoles. Disorganization of vacuolar membranes in apt1Δ cells was accompanied by a significant increase in the amounts of intra-vacuolar and pigment-containing vesicles. Quantitative immunogold labeling of C. neoformans cells with a monoclonal antibody raised to a major capsular component suggested impaired polysaccharide synthesis. APT1 deletion also affected synthesis of phosphatidylserine, phosphatidylethanolamine, inositolphosphoryl ceramide, glucosylceramide and ergosterylglycoside. These results reveal novel functions of Apt1 and are in agreement with the notion that this putative flippase plays an important role in the physiology of C. neoformans.
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Cryptococcus neoformans/genética , Proteínas Fúngicas/genética , Membranas Intracelulares/metabolismo , Lipídeos/biossíntese , Animais , Membrana Celular/genética , Membrana Celular/metabolismo , Cryptococcus neoformans/metabolismo , Cryptococcus neoformans/patogenicidade , Membranas Intracelulares/química , Lipídeos/genética , Camundongos , Polissacarídeos/biossíntese , VirulênciaRESUMO
Cryptococcus neoformans is an environmental fungus that can cause lethal meningoencephalitis in immunocompromised individuals. The mechanisms by which environmental microbes become pathogenic to mammals are still obscure, but different studies suggest that fungal virulence evolved from selection imposed by environmental predators. The soil-living Acanthamoeba castellanii is a well-known predator of C. neoformans. In this work, we evaluated the participation of C. neoformans virulence-associated structures in the interaction of fungal cells with A. castellanii. Fungal extracellular vesicles (EVs) and the polysaccharide glucuronoxylomannan (GXM) were internalized by A. castellanii with no impact on the viability of amoebal cells. EVs, but not free GXM, modulated antifungal properties of A. castellanii by inducing enhanced yeast survival. Phagocytosis of C. neoformans by amoebal cells and the pathogenic potential in a Galleria mellonella model were not affected by EVs, but previous interactions with A. castellanii rendered fungal cells more efficient in killing this invertebrate host. This observation was apparently associated with marked amoeba-induced changes in surface architecture and increased resistance to both oxygen- and nitrogen-derived molecular species. Our results indicate that multiple components with the potential to impact pathogenesis are involved in C. neoformans environmental interactions.
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Acanthamoeba castellanii/fisiologia , Cryptococcus neoformans/fisiologia , Interações Microbianas , Animais , Sobrevivência Celular/efeitos dos fármacos , Criptococose/microbiologia , Modelos Animais de Doenças , Lepidópteros , Viabilidade Microbiana , Fagocitose/efeitos dos fármacos , Polissacarídeos/metabolismo , Vesículas Secretórias/metabolismo , Análise de Sobrevida , VirulênciaRESUMO
The global mortality due to cryptococcosis caused by Cryptococcus neoformans or C. gattii is unacceptably high. Currently available therapies are decades old and may be impacted by drug resistance. Therefore, the need for more effective antifungal drugs for cryptococcosis is evident. A number of Cryptococcus virulence factors have been studied in detail, providing crucial information about the fungal biology and putative molecular targets for antifungals. This review focuses on the use of well-described virulence factors of Cryptococcus as potential anticryptococcal agents.
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A gestação gemelar com mola hidatiforme completa que coexiste com feto vivo (GGMC) é uma entidade rara. Embora as recomendações sejam de conduta expectante, são descritas diversas complicações maternas e fetais, como o aumento da incidência de abortamento espontâneo, de parto prematuro, de sangramento vaginal, de pré-eclampsia grave e de doença trofoblástica persistente, entre outras complicações. Neste trabalho, descrevemos a evolução clínica de um caso de GGMC que evoluiu para crise tireotóxica, pré-eclâmpsia grave, interrupção da gestação e necessidade de cuidados intensivos maternos. A necropsia fetal evidenciou feto do sexo feminino, sem malformações aparentes, com alterações placentárias que favorecem cromossomopatia. A avaliação dos restos ovulares evidenciou vilosidades com hiperplasia do trofoblasto e vesículas, achados compatíveis com mola hidatiforme completa. Atualmente, após 15 meses de seguimento, a paciente permanece assintomática e com níveis indetectáveis de gonadotrofina coriônica.
Twin pregnancy with complete hydatidiform mole coexisting with a live fetus is a rare entity, and although the recommendations are expectant management of various maternal and fetal complications are described, such as increasing the number of spontaneous abortion, premature delivery, vaginal bleeding, pre-eclampsia and severe persistent trophoblastic disease, among other complications. In this paper, we describe the clinical course of a case of GGMC who developed thyrotoxic crisis, preeclampsia severe, termination of pregnancy and maternal need for intensive care. Fetal autopsy showed a female fetus with no apparent defects; with placental changes favoring chromosomal disorders. The evaluationof ovular remains showed villi with trophoblastic hyperplasia and vesicles, suggestive of complete mole. Currently, after 15 months of follow up, the patient remains asymptomatic with undetectable levels of chorionic gonadotropin.
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Humanos , Feminino , Gravidez , Adulto , Mola Hidatiforme/complicações , Mola Hidatiforme/diagnóstico , Gravidez de Gêmeos , Neoplasias Uterinas , Aborto Espontâneo , Gonadotropina Coriônica , Diagnóstico Pré-Natal/mortalidade , Pré-EclâmpsiaRESUMO
Flippases are key regulators of membrane asymmetry and secretory mechanisms. Vesicular polysaccharide secretion is essential for the pathogenic mechanisms of Cryptococcus neoformans. On the basis of the observations that flippases are required for polysaccharide secretion in plants and the putative Apt1 flippase is required for cryptococcal virulence, we analyzed the role of this enzyme in polysaccharide release by C. neoformans, using a previously characterized apt1Δ mutant. Mutant and wild-type (WT) cells shared important phenotypic characteristics, including capsule morphology and dimensions, glucuronoxylomannan (GXM) composition, molecular size, and serological properties. The apt1Δ mutant, however, produced extracellular vesicles (EVs) with a lower GXM content and different size distribution in comparison with those of WT cells. Our data also suggested a defective intracellular GXM synthesis in mutant cells, in addition to changes in the architecture of the Golgi apparatus. These findings were correlated with diminished GXM production during in vitro growth, macrophage infection, and lung colonization. This phenotype was associated with decreased survival of the mutant in the lungs of infected mice, reduced induction of interleukin-6 (IL-6) cytokine levels, and inefficacy in colonization of the brain. Taken together, our results indicate that the lack of APT1 caused defects in both GXM synthesis and vesicular export to the extracellular milieu by C. neoformans via processes that are apparently related to the pathogenic mechanisms used by this fungus during animal infection.