Modifications de la paroi au cours de la maturation et de la germination des conidies de Scedosporium boydii

Les espèces du complexe Scedosporium apiospermum sont des agents pathogènes émergents qui se situent au deuxième rang parmi les champignons filamenteux rencontrés au cours de la mucoviscidose. Ils sont omniprésents et particulièrement rencontrés dans les zones polluées. En  dépit  de  leur  importance  clinique,  nos  connaissances  sur  leur  biologie  moléculaire  et  leur physiologie restent limitées.  Chez  les  champignons,  la  paroi  constitue  un  bouclier  protecteur  face  à  des  conditions environnementales  défavorables,  et  joue  un  rôle  essentiel  dans  la  pathogénicité.  Ici,  nous avons  étudié  les  changements  dynamiques  de  la  paroi  des  conidies  de  S.  boydii,  l’une  des deux espèces majeures de ce complexe avec S. apiospermum, avec pour objectif d\u27identifier des facteurs de virulence potentiels. En utilisant une large variété de techniques, allant de la microscopie électronique à balayage ou à transmission à l’analyse protéomique des protéines à ancre glycosylphosphatidylinositol (GPI) en passant par la microélectrophorèse et la partition de  phase,  la  cytométrie  en  flux,  la  microscopie  de  force  atomique,  la  résonance paramagnétique  électronique,  ou  encore  des  techniques  moléculaires,  nous  avons  mis  en évidence  diverses  modifications  qui  se  produisent  dans  la  paroi  pendant  la  maturation  et  la germination  des  conidies  de  S.  boydii  et  nous  avons  identifié  la  DHN-mélanine  ainsi  qu\u27un nombre important de protéines à ancre GPI.  Enfin,  nous  avons  fourni  la  première  séquence  complète  du  génome  de  S.  apiospermum  qui appuierait  les  différents  domaines  de  la  recherche  sur  ces  champignons  que  ce  soit  pour l’étude des mécanismes pathogènes ou pour des applications biotechnologiques

What’s new on physiology and virulence factors of Scedosporium apiospermum ?

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Cell wall modifications during conidial maturation of the human pathogenic fungus Pseudallescheria boydii

This is the final version of the article. Available from the publisher via the DOI in this record.Progress in extending the life expectancy of cystic fibrosis (CF) patients remains jeopardized by the increasing incidence of fungal respiratory infections. Pseudallescheria boydii (P. boydii), an emerging pathogen of humans, is a filamentous fungus frequently isolated from the respiratory secretions of CF patients. It is commonly believed that infection by this fungus occurs through inhalation of airborne conidia, but the mechanisms allowing the adherence of Pseudallescheria to the host epithelial cells and its escape from the host immune defenses remain largely unknown. Given that the cell wall orchestrates all these processes, we were interested in studying its dynamic changes in conidia as function of the age of cultures. We found that the surface hydrophobicity and electronegative charge of conidia increased with the age of culture. Melanin that can influence the cell surface properties, was extracted from conidia and estimated using UV-visible spectrophotometry. Cells were also directly examined and compared using electron paramagnetic resonance (EPR) that determines the production of free radicals. Consistent with the increased amount of melanin, the EPR signal intensity decreased suggesting polymerization of melanin. These results were confirmed by flow cytometry after studying the effect of melanin polymerization on the surface accessibility of mannose-containing glycoconjugates to fluorescent concanavalin A. In the absence of melanin, conidia showed a marked increase in fluorescence intensity as the age of culture increased. Using atomic force microscopy, we were unable to find rodlet-forming hydrophobins, molecules that can also affect conidial surface properties. In conclusion, the changes in surface properties and biochemical composition of the conidial wall with the age of culture highlight the process of conidial maturation. Mannose-containing glycoconjugates that are involved in immune recognition, are progressively masked by polymerization of melanin, an antioxidant that is commonly thought to allow fungal escape from the host immune defenses.The study was funded by “Région Pays de la Loire” in the frame of “Myco-AFM” research program). BED was supported by the Dutch Virgo Consortium (FES0908, NGI 050-060-452) and CAPES/BRASIL. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

Cell wall changes during maturation of conidia in Pseudallescheria boydii

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Cell surface properties of two differently virulent strains of Acinetobacter baumannii isolated from a patient

The aim of this study was to unravel, by focusing on cell surface properties, the underlying virulence factors contributing to the difference in the pathogenicity observed in two Acinetobacter baumannii strains isolated from the same patient. The two strains were phenotypically different: (i) a mucoid strain (AB-M), highly virulent in a mouse model of pneumonia, and (ii) a nonmucoid strain (AB-NM), moderately virulent in the same model. The study of the cell surface properties included the microbial adhesion to solvents method, the measurement of the electrophoretic mobility of bacteria, the analysis of biofilm formation by calcofluor white staining, the adherence to silicone catheters, and scanning electron microscopy. The AB-NM strain was more hydrophobic, more adherent to silicone catheters, and produced more biofilm than the AB-M strain. Scanning electron microscopy showed bacterial cells with a rough surface and the formation of large cell clusters for AB-NM whereas the AB-M strain had a smooth surfa..., Le but de cette etude était d’étudier, en se focalisant sur les propriétés de surface des bactéries, des caractères potentiellement impliqués dans la différence de pouvoir pathogène de deux souches d’Acinetobacter baumannii isolées chez un même patient. Ces souches étaient phénotypiquement différentes : une souche mucoïde (AB-M) caractérisée par un pouvoir pathogène élevé dans un modèle expérimental murin de pneumopathie, et une souche non mucoïde (AB-NM), plus faiblement pathogène dans le même modèle. L’étude des propriétés de surface a utilisé la méthode d’adhésion des bactéries aux solvants, la mesure de la mobilité électrophorétique des bactéries, l’analyse de la formation de biofilm par marquage au calcofluor, l’adhésion à des cathéters en silicone, et la microscopie électronique à balayage. La souche AB-NM est apparue plus hydrophobe, plus adhérente aux cathéters, et plus productrice de biofilm que la souche AB-M. L’observation en microscopie électronique à balayage a montré des bactéries à surface ..

Cell wall modifications during maturation and germination of the conidia in the opportunistic fungus Scedosporium apiospermum

Introduction: Recent prevention measures led to an increase in life expectancy of cystic fibrosis (CF) patients; however, this progress remained jeopardized by various microbial infections. Scedosporium apiospermum is the second most frequent filamentous fungus found in the respiratory tract of CF patients. Unlike other infectious agents, the pathogenic mechanisms of this fungus are far less studied. We aim through this project to study the life cycle and the cell surface composition in order to set the basis for understanding the adherence mechanisms of S. apiospermum inside a human host. Results: So far, we have studied the cell wall modifications during maturation and germination of spores. Germination was studied in yeast peptone dextrose (YPD) broth and malt medium at different temperatures (20 C, 25 C and 37 C). The highest percentage of germination was obtained in YPD at 37 C, conditions which were selected for further experiments. The comparison of spores collected from 5-, 9- or 14 day-old cultures showed that spores from 9 day-old cultures exhibited the highest percentage of germination, capacity to bind concanavalin A (Con A) and cell-surface hydrophobicity (CSH), and the lowest electrostatic charge. Electron microscopy showed that the cell wall of S. apiospermum conidia is composed of two layers (electron-dense outer layer and electron transparent inner layer). Germination resulted in a thinner and fibrillar outer layer that was occasionally detached upon elongation of the filament. These ultra-structural changes were substantiated with fluorescent microscopy; the hyphal part was intensely labeled by Con-A and wheat germ agglutinin (WGA) in contrast to the mother cell. Furthermore, germination resulted in a remarkable decrease in the surface electronegativity and CSH.   Conclusion: We demonstrated that S. apiospermum spores undergo maturation. Spores isolated from cultures of different age differ in their capacity to germinate, their surface accessibility to Con A and their surface physical properties (CSH and electronegativity). Upon germination, a thinning of the outer cell wall layer is observed, rendering the inner layer more accessible to lectins (Con A and WGA) and resultingin a reduced CSH and electronegative charge. All these results point out that the cell wall in S. apiospermum is a dynamic structure and the variability in its properties and composition might affect its adherence to the host tissues

Draft Genome Sequence of the Pathogenic Fungus Scedosporium apiospermum

This is the final version of the article. Available from the publisher via the DOI in this record.The first genome of one species of the Scedosporium apiospermum complex, responsible for localized to severe disseminated infections according to the immune status of the host, will contribute to a better understanding of the pathogenicity of these fungi and also to the discovery of the mechanisms underlying their low susceptibility to current antifungals.This work was supported by a grant (RF20120600725) from the association Vaincre la Mucoviscidose (France), which is gratefully acknowledged

Photodegradation of the Mycobacterium ulcerans Toxin, Mycolactones: Considerations for Handling and Storage

Background: Mycolactones are toxins secreted by M. ulcerans, the etiological agent of Buruli ulcer. These toxins, which are the main virulence factors of the bacilli, are responsible for skin lesions. Considering their specificity for M. ulcerans and their presence in skin lesions even at early stages, mycolactones are promising candidates for the development of a diagnostic tool for M. ulcerans infection. Stability of purified mycolactones towards light and heat has not yet been investigated, despite the importance of such parameters in the selection of strategies for a diagnosis tool development. In this context, the effects of UV, light and temperature on mycolactone stability and biological activity were studied. Methodology/Principal Findings: To investigate the effect of these physical parameters, mycolactones were exposed to different wavelengths in several solvents and temperatures. Structural changes and biological activity were monitored. Whilst high temperature had no effect on mycolactones, UV irradiation (UV-A, UV-B and UV-C) and sunlight exposure caused a considerable degradation, as revealed by LC-MS and NMR analysis, correlated with a loss of biological activity. Moreover, effect of UVs on mycolactone caused a photodegradation rather than a phototransformation due to the identification of degradation product. Conclusion/Significance: This study demonstrates the high sensitivity of mycolactones to UVs as such it defines instruction

Enzymatic Mechanisms Involved in Evasion of Fungi to the Oxidative Stress: Focus on Scedosporium apiospermum

The airways of patients with cystic fibrosis (CF) are frequently colonized by various filamentous fungi, mainly Aspergillus fumigatus and Scedosporium species. To establish within the respiratory tract and cause an infection, these opportunistic fungi express pathogenic factors allowing adherence to the host tissues, uptake of extracellular iron, or evasion to the host immune response. During the colonization process, inhaled conidia and the subsequent hyphae are exposed to reactive oxygen species (ROS) and reactive nitrogen species (RNS) released by phagocytic cells, which cause in the fungal cells an oxidative stress and a nitrosative stress, respectively. To cope with these constraints, fungal pathogens have developed various mechanisms that protect the fungus against ROS and RNS, including enzymatic antioxidant systems. In this review, we summarize the different works performed on ROS- and RNS-detoxifying enzymes in fungi commonly encountered in the airways of CF patients and highlight their role in pathogenesis of the airway colonization or respiratory infections. The potential of these enzymes as serodiagnostic tools is also emphasized. In addition, taking advantage of the recent availability of the whole genome sequence of S. apiospermum, we identified the various genes encoding ROS- and RNS-detoxifying enzymes, which pave the way for future investigations on the role of these enzymes in pathogenesis of these emerging species since they may constitute new therapeutics targets