RESUMEN
Scedosporium fungi are found in various natural and host-associated environments, including the lungs of cystic fibrosis patients. However, their role in infection development remains underexplored. Here the attachment of conidia of a virulent S. aurantiacum strain WM 06.482 onto the human lung epithelial A549 cells in vitro was visualized using microscopy to examine the initial steps of infection. We showed that 75-80% of fungal conidia were bound to the A549 cells within four hours of co-incubation, and started to produce germ tubes. The germinating conidia seemed to invade the cells through the intercellular space, no intracellular uptake of fungal conidia by the airway epithelial cells after conidial attachment. Transcriptomic analysis of the A549 cells revealed that the up-regulated genes were mainly associated with cell repair and inflammatory processes indicating a protective response against S. aurantiacum infection. Network analysis of the differentially expressed genes showed activation of the innate immune system (NF-kB pathway) leading to the release of pro-inflammatory cytokines. We believe this is the first report showing the transcriptomic response of human alveolar epithelial cells exposed to S. aurantiacum conidia paving a way for better understanding of the mechanism of the infection process.
Asunto(s)
Células Epiteliales/metabolismo , Perfilación de la Expresión Génica/métodos , Regulación Neoplásica de la Expresión Génica , Pulmón/metabolismo , Scedosporium/crecimiento & desarrollo , Células A549 , Células Epiteliales/microbiología , Células Epiteliales/ultraestructura , Ontología de Genes , Redes Reguladoras de Genes , Interacciones Huésped-Patógeno , Humanos , Pulmón/microbiología , Pulmón/patología , Microscopía Confocal , Microscopía Electrónica de Rastreo , Scedosporium/patogenicidad , Scedosporium/ultraestructura , Esporas Fúngicas/crecimiento & desarrollo , Esporas Fúngicas/patogenicidad , Esporas Fúngicas/ultraestructura , VirulenciaRESUMEN
BACKGROUND: Scedosporium apiospermum is a ubiquitous, emerging and multidrug-resistant fungal pathogen with still rather unknown virulence mechanisms. OBJECTIVES/METHODS: The cellular basis of the in vitro interaction between fungi and host cells/tissues is the determinant factor for the development of a successful in vivo infection. Herein, we evaluated the interaction of S. apiospermum conidia with lung epithelial (A549), lung fibroblast (MRC-5) and RAW 264.7 macrophages by light and scanning/transmission electron microscopy. FINDINGS: After 4 h of fungi-host cell contact, the percentage of infected mammalian cells and the number of fungi per infected cell was measured by light microscopy, and the following association indexes were calculated for A549, MRC-5 and macrophage cells: 73.2 ± 25.9, 69.7 ± 22.5 and 59.7 ± 11.1, respectively. Both conidia and germinated conidia were regularly observed interacting with the evaluated cells, with a higher prevalence of non-germinated conidia. Interestingly, nests of germinated conidia were evidenced at the surface of lung cells by scanning electron microscopy. Some germination projections and hyphae were seen penetrating/evading the mammalian cells. Furthermore, internalised conidia were seen within vacuoles as visualised by transmission electron microscopy. MAIN CONCLUSIONS: The present study contributes to a better understanding of S. apiospermum pathogenesis by demonstrating the first steps of the infection process of this opportunistic fungus.
Asunto(s)
Células Epiteliales/microbiología , Pulmón/microbiología , Macrófagos/microbiología , Scedosporium/ultraestructura , Esporas Fúngicas/ultraestructura , Células Epiteliales/ultraestructura , Humanos , Pulmón/ultraestructura , Macrófagos/ultraestructura , Microscopía Electrónica de Rastreo , Scedosporium/fisiología , Esporas Fúngicas/fisiologíaRESUMEN
BACKGROUND Scedosporium/Lomentospora species are opportunistic mould pathogens, presenting notable antifungal resistance. OBJECTIVES/METHODS We analysed the conidia and germinated conidia of S. apiospermum (Sap), S. aurantiacum (Sau), S. minutisporum (Smi) and L. prolificans (Lpr) by scanning electron microscopy and exposition of surface molecules by fluorescence microscopy. FINDINGS Conidia of Sap, Smi and Sau had oval, ellipsoidal and cylindrical shape, respectively, with several irregularities surrounding all surface areas, whereas Lpr conidia were rounded with a smooth surface. The germination of Sap occurred at the conidial bottom, while Smi and Sau germination primarily occurred at the centre of the conidial cell, and Lpr germination initiated at any part of the conidial surface. The staining of N-acetylglucosamine-containing molecules by fluorescein-labelled WGA primarily occurred during the germination of all studied fungi and in the conidial scars, which is the primary location of germination. Calcofluor white, which recognises the polysaccharide chitin, strongly stained the conidial cells and, to a lesser extent, the germination. Both mannose-rich glycoconjugates (evidenced by fluoresceinated-ConA) and cell wall externally located polypeptides presented distinct surface locations and expression according to both morphotypes and fungal species. In contrast, sialic acid and galactose-containing structures were not detected at fungal surfaces. MAIN CONCLUSIONS The present study demonstrated the differential production/exposition of surface molecules on distinct morphotypes of Scedosporium/Lomentospora species.
Asunto(s)
Membrana Celular/ultraestructura , Scedosporium/ultraestructura , Esporas Fúngicas/ultraestructura , Diferenciación Celular , Microscopía Electrónica de Rastreo , Microscopía Fluorescente , Scedosporium/crecimiento & desarrollo , Esporas Fúngicas/fisiologíaRESUMEN
The filamentous fungus Lomentospora (Scedosporium) prolificans is an emerging opportunistic pathogen associated with fatal infections in patients with disturbed immune function. Unfortunately, conventional therapies are hardly of any use against this fungus due to its intrinsic resistance. Therefore, we performed an integrated study of the L. prolificans responses to the first option to treat these mycoses, namely voriconazole, with the aim of unveiling mechanisms involved in the resistance to this compound. To do that, we used a wide range of techniques, including fluorescence and electron microscopy to study morphological alterations, ion chromatography to measure changes in cell-wall carbohydrate composition, and proteomics-based techniques to identify the proteins differentially expressed under the presence of the drug. Significantly, we showed drastic changes occurring in cell shape after voriconazole exposure, L. prolificans hyphae being shorter and wider than under control conditions. Interestingly, we proved that the architecture and carbohydrate composition of the cell wall had been modified in the presence of the drug. Specifically, L. prolificans constructed a more complex organelle with a higher presence of glucans and mannans. In addition to this, we identified several differentially expressed proteins, including Srp1 and heat shock protein 70 (Hsp70), as the most overexpressed under voriconazole-induced stress conditions. The mechanisms described in this study, which may be directly related to L. prolificans antifungal resistance or tolerance, could be used as targets to improve existing therapies or to develop new ones in order to successfully eliminate these mycoses.
Asunto(s)
Antifúngicos/farmacología , Scedosporium/efectos de los fármacos , Scedosporium/metabolismo , Voriconazol/farmacología , Microscopía Electrónica , Microscopía Fluorescente , Proteómica , Scedosporium/ultraestructuraRESUMEN
Reported herein is the ability of Scedosporium apiospermum, S. aurantiacum, S. minutisporum and Lomentospora prolificans conidia to adhere, differentiate into hyphae and form biofilms on both polystyrene and lung epithelial cells. To different degrees, all of the fungi adhered to polystyrene after 4 h, with a predominance of those with germinated conidia. Prolonged fungi-polystyrene contact resulted in the formation of a monolayer of intertwined mycelia, which was identified as a typical biofilm structure due to the presence of a viable mycelial biomass, extracellular matrix and enhanced antifungal resistance. Ultrastructural details were revealed by SEM and CLSM, showing the dense compaction of the mycelial biomass and the presence of channels within the organized biofilm. A similar biofilm structure was observed following the co-culture of each fungus with A549 cells, revealing a mycelial trap covering all of the lung epithelial monolayer. Collectively, these results highlight the potential for biofilm formation by these clinically relevant fungal pathogens.
Asunto(s)
Ascomicetos/fisiología , Biopelículas/crecimiento & desarrollo , Células Epiteliales/microbiología , Hifa/crecimiento & desarrollo , Poliestirenos , Scedosporium/fisiología , Células A549 , Ascomicetos/ultraestructura , Adhesión Bacteriana , Biomasa , Humanos , Microscopía Confocal , Microscopía Electrónica de Rastreo , Poliestirenos/química , Scedosporium/ultraestructuraRESUMEN
Ultrastructural features of conidia, lateral walls of aerial and submerged hyphal cells, and of septal pore apparatus of Scedosporium apiospermum, S. boydii, Pseudallescheria angusta and Scedosporium aurantiacum were studied. Submerged hyphal cells possessed a thick extracellular matrix. Crystalline satellites accessory to the septal pore apparatus were revealed. Fundamental ultrastructural features appeared to be heterogeneous at low taxonomic levels. The closely interrelated members of the S. apiospermum complex showed quantitative ultrastructural variability, but the unambiguously different species S. aurantiacum deviated qualitatively by markers of conidial wall structure, Woronin bodies morphology and presence/absence of crystalline satellites of the septal pore apparatus.
Asunto(s)
Biodiversidad , Scedosporium/ultraestructura , Esporas Fúngicas/ultraestructura , Scedosporium/clasificación , Scedosporium/aislamiento & purificación , Esporas Fúngicas/clasificación , Esporas Fúngicas/aislamiento & purificaciónRESUMEN
Scedosporium boydii is a pathogenic filamentous fungus that causes a wide range of human infections, notably respiratory infections in patients with cystic fibrosis. The development of new therapeutic strategies targeting S. boydii necessitates a better understanding of the physiology of this fungus and the identification of new molecular targets. In this work, we studied the conidium-to-germ tube transition using a variety of techniques including scanning and transmission electron microscopy, atomic force microscopy, two-phase partitioning, microelectrophoresis and cationized ferritin labeling, chemical force spectroscopy, lectin labeling, and nanoLC-MS/MS for cell wall GPI-anchored protein analysis. We demonstrated that the cell wall undergoes structural changes with germination accompanied with a lower hydrophobicity, electrostatic charge and binding capacity to cationized ferritin. Changes during germination also included a higher accessibility of some cell wall polysaccharides to lectins and less CH3/CH3 interactions (hydrophobic adhesion forces mainly due to glycoproteins). We also extracted and identified 20 GPI-anchored proteins from the cell wall of S. boydii, among which one was detected only in the conidial wall extract and 12 only in the mycelial wall extract. The identified sequences belonged to protein families involved in virulence in other fungi like Gelp/Gasp, Crhp, Bglp/Bgtp families and a superoxide dismutase. These results highlighted the cell wall remodeling during germination in S. boydii with the identification of a substantial number of cell wall GPI-anchored conidial or hyphal specific proteins, which provides a basis to investigate the role of these molecules in the host-pathogen interaction and fungal virulence.
Asunto(s)
Pared Celular/química , Proteínas Fúngicas/genética , Proteínas Ligadas a GPI/genética , Regulación Fúngica de la Expresión Génica , Scedosporium/genética , Esporas Fúngicas/genética , Secuencia de Aminoácidos , Pared Celular/metabolismo , Pared Celular/ultraestructura , Ferritinas/genética , Ferritinas/metabolismo , Polisacáridos Fúngicos/química , Polisacáridos Fúngicos/metabolismo , Proteínas Fúngicas/metabolismo , Proteínas Ligadas a GPI/aislamiento & purificación , Proteínas Ligadas a GPI/metabolismo , Glicosilfosfatidilinositoles/química , Glicosilfosfatidilinositoles/metabolismo , Interacciones Hidrofóbicas e Hidrofílicas , Lectinas/química , Lectinas/metabolismo , Anotación de Secuencia Molecular , Datos de Secuencia Molecular , Micelio/genética , Micelio/crecimiento & desarrollo , Micelio/metabolismo , Micelio/ultraestructura , Unión Proteica , Scedosporium/crecimiento & desarrollo , Scedosporium/metabolismo , Scedosporium/ultraestructura , Esporas Fúngicas/crecimiento & desarrollo , Esporas Fúngicas/metabolismo , Esporas Fúngicas/ultraestructura , Electricidad Estática , Superóxido Dismutasa/genética , Superóxido Dismutasa/metabolismoRESUMEN
The majority of mycoses which lead to mycotic tumors in patients without any predisposing underlying disease are either caused by Cryptococcus gattii and C. neoformans or by dematiaceous fungi which include Cladophialophora bantiana, Ramichloridium mackenziei, Exophiala and Fonsecaea species. The detection of hyphae in granuloma in the brain should lead to screening for pigmented fungi, which are recognized best in hematoxylin eosin (HE) or sometimes also in periodic acid-Schiff (PAS) stained sections. In patients who survive a near drowning accident and those who develop brain abscesses, scedosporiosis should always be considered as a possible infection.
Asunto(s)
Encefalopatías/patología , Infecciones Fúngicas del Sistema Nervioso Central/patología , Inmunocompetencia , Basidiomycota/clasificación , Basidiomycota/ultraestructura , Encéfalo/microbiología , Encéfalo/patología , Encefalopatías/inmunología , Encefalopatías/microbiología , Infecciones Fúngicas del Sistema Nervioso Central/inmunología , Infecciones Fúngicas del Sistema Nervioso Central/microbiología , Feohifomicosis Cerebral/inmunología , Feohifomicosis Cerebral/microbiología , Feohifomicosis Cerebral/patología , Cryptococcus gattii/clasificación , Cryptococcus gattii/ultraestructura , Diagnóstico Diferencial , Hongos/clasificación , Hongos/aislamiento & purificación , Meningitis Criptocócica/inmunología , Meningitis Criptocócica/microbiología , Meningitis Criptocócica/patología , Técnicas de Tipificación Micológica , Scedosporium/clasificación , Scedosporium/ultraestructuraRESUMEN
The main purpose of the present paper is to establish the connection between phylogenetic and morphological data and ecological features of strains of Pseudallescheria, Petriella, and Scedosporium. For the phylogenetic analysis sequences of the ITS region and the large subunit (partial sequences) of the rDNA were used. Cultural characteristics were observed on MEA 2 % and Weitzman-Silva Hutner Agar. Results showed, that three major groups could be differentiated, corresponding to Pseudallescheria, Petriella and S. prolificans. Among Petriella species only Pe. setifera is reasonably delimited. Pe. musispora was found to be synonymous with Pe. setifera. S. prolificans proved to be a homogenous species on the basis of ITS-sequences. Morphologically, Pseudallescheria and Petriella are distinguished by ostiolate vs non-ostiolate ascomata, a bipartition reflected also in ITS sequence data. We hypothesise a secondary loss of the ostiole of Pseudallescheria due to its ecological preferences. Infraspecific grouping within the highly variable species P. boydii is consistent for at least one clade in the ITS tree. The evolution of lineages with increased virulence within P. boydii is discussed.
Asunto(s)
Infecciones Oportunistas/microbiología , Pseudallescheria/genética , Scedosporium/genética , Secuencia de Bases , ADN de Hongos/química , ADN de Hongos/genética , ADN Intergénico/química , ADN Intergénico/genética , Ecosistema , Humanos , Datos de Secuencia Molecular , Filogenia , Reacción en Cadena de la Polimerasa , Pseudallescheria/crecimiento & desarrollo , Pseudallescheria/ultraestructura , ARN Ribosómico/química , ARN Ribosómico/genética , Scedosporium/crecimiento & desarrollo , Scedosporium/ultraestructura , Alineación de SecuenciaRESUMEN
A case of endobronchitis by Scedosporium apiospermum in a child with cystic fibrosis is presented. The bronchial aspirate's cytology showed the presence of a large amount of septated-dichotomized hyphae. The bronchial aspirate's culture showed the presence of Scedosporium apiospermum in a pure culture of three consecutive samples. The scanning electron microscopy study of the mucosal surface revealed scarce mycelia with the presence of abundant conidiae. The transmission electron microscopy of the mucosa revealed inflammatory infiltrates constituted by macrophages, polymorphonuclear leukocytes, a lot of dichotomized mycelia and macrophages with hyphae and conidiae within the phagosomes. The patient was treated with amphotericin B and itraconazole.
Asunto(s)
Antifúngicos/uso terapéutico , Bronquitis/microbiología , Fibrosis Quística/complicaciones , Micetoma/microbiología , Scedosporium/aislamiento & purificación , Anfotericina B/uso terapéutico , Bronquios/microbiología , Bronquitis/tratamiento farmacológico , Bronquitis/etiología , Niño , Susceptibilidad a Enfermedades , Quimioterapia Combinada , Femenino , Humanos , Itraconazol/uso terapéutico , Microscopía Electrónica , Micetoma/tratamiento farmacológico , Micetoma/etiología , Mucosa Respiratoria/microbiología , Scedosporium/crecimiento & desarrollo , Scedosporium/ultraestructuraRESUMEN
BACKGROUND: Scedosporium prolificans is a dematiaceous fungus that is known to cause a wide spectrum of infections in humans, bearing a severity and a prognosis that is relationed with the patients immune status. METHODS: A retrospective review was made of the clinical charts of all patients who developed positive S. prolificans cultures in our centre from 1990 to 2000. Isolates were identified by colonial morphology and microscopic features. The in vitro susceptibility was evaluated using the microdilution method according to NCCLS. RESULTS: S. prolificans was isolated in 15 patients. Eight were affected with cystic fibrosis and the isolation of S. prolificans in their airways did not worsen their clinical status. Among the remaining 7 cases there were five leukemic patients with neutropenia and two immunocompetent hosts with cutaneous infection and endocarditis. Four of five neutropenic patients died of sudden sepsis and S. prolificans was isolated from blood cultures made a few days before their death, and the fifth neutropenic case suffered a bilateral pneumonia with improving course probably due to recovery from neutropenia. As to the immunocompetent group the clinical course was good in the cutaneous infection case, but the endocarditis case died four days after the antifungical therapy was started. All the isolates tested were found to be resistant to amphotericin, 5 flucytosine, fluconazole, itraconazole, voriconazole, miconazole and terbinafine. CONCLUSIONS: Scedosporium prolificans is a fungal pathogen that colonizes the airways of patients affected with cystic fibrosis. It can also cause a wide variety of infections, whose severity and prognosis depends on the patients immune status. Due to the resistance of this fungus to antifungal drugs, the therapeutic options are limited. Only with the correction of neutropenia and surgery in local infections in immunocompetent hosts it has been possible to cure these infections.
Asunto(s)
Micetoma/epidemiología , Scedosporium/aislamiento & purificación , Enfermedad Aguda , Adolescente , Adulto , Anciano , Antifúngicos/uso terapéutico , Preescolar , Fibrosis Quística/complicaciones , Susceptibilidad a Enfermedades , Farmacorresistencia Fúngica Múltiple , Endocarditis/etiología , Femenino , Humanos , Inmunocompetencia , Lactante , Recién Nacido , Leucemia Mieloide/complicaciones , Masculino , Persona de Mediana Edad , Micetoma/tratamiento farmacológico , Micetoma/microbiología , Neutropenia/complicaciones , Infecciones Oportunistas/epidemiología , Infecciones Oportunistas/microbiología , Leucemia-Linfoma Linfoblástico de Células Precursoras/complicaciones , Estudios Retrospectivos , Scedosporium/efectos de los fármacos , Scedosporium/ultraestructura , España/epidemiología , Infección de Heridas/microbiologíaRESUMEN
The performance of biofilters inoculated with the fungus Scedosporium apiospermum was evaluated. This fungus was isolated from a biofilter which operated with toluene for more than 6 months. The experiments were performed in a 2.9 L reactor packed with vermiculite or with vermiculite-granular activated carbon as packing material. The initial moisture content of the support and the inlet concentration of toluene were 70% and 6 g/m3, respectively. As the pressure drop increased from 5-40 mm H2O a strong initial growth was observed. Stable operation was maintained for 20 days with a moisture content of 55% and a biomass of 33 mg biomass/g dry support. These conditions were achieved with intermittent addition of culture medium, which permitted a stable elimination capacity (EC) of 100 g/m3(reactor)h without clogging. Pressure drop across the bed and CO2 production were related to toluene elimination. Measurement of toluene, at different levels of the biofilter, showed that the system attained higher local EC (200 g/m3(r)h) at the reactor outlet. These conditions were related to local humidity conditions. When the mineral medium was added periodically before the EC decreases, EC of approximately 258 g/m3(r)h were maintained with removal efficiencies of 98%. Under these conditions the average moisture content was 60% and 41 mg biomass/g dry support was produced. No sporulation was observed. Evaluation of bacterial content and activities showed that the toluene elimination was only due to S. apiospermum catabolism.