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

International audienc

Cell wall changes during maturation of conidia in Pseudallescheria boydii

International audienc

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

ARP2 mutation in a brown mutant of Aspergillus fumigatus leads to a loss of competitivity

Aspergillus fumigatus is the major filamentous fungus colonizing the airways of cystic fibrosis patients (CF). It is usually responsible for a chronic colonization and sometimes for chronic respiratory infections. However, it may also cause severe infections in patients undergoing lung transplantation. The prognosis for these infections still remains uncertain and there is an urgent need for the identification of new antifungal targets. Among the fungal components which have been studied, melanin of the conidial wall was confirmed as an important virulence factor, protecting the fungus against the host immune defences. Using isolates deficient in melanin synthesis because of a mutation in the ALB1 (white isolates) or in the ARP2 gene (brown isolate), we showed that melanin is required for correct assembly of the different layers of the conidial wall and, therefore, for the expression of adhesins and other virulence factors at the conidial surface. Mutations in the ALB1 gene have been shown to result in a marked reduction in virulence of the fungus in a mouse model of disseminated aspergillosis, but nothing is known about the role of the ARP2 gene. We therefore focused our attention on the brown isolate IHEM 15998, mutated in the ARP2 gene, which was recovered from respiratory secretions of a CF patient, but not detected in later samples while chronic colonization by a wild-type (WT) strain of A. fumigatus was observed. In vitro experiments were first conducted, which revealed that mutation in the ARP2 gene resulted in a fitness cost when co-cultivated with the WT strain. Likewise, flow cytometry was used to investigate the oxidative burst response of phagocytes co-incubated with the conidia. Compared to the WT strain, stimulation of neutrophils as well as macrophages was higher with the brown isolate. FITC-labeled conidia were also incubated with human cytokine-induced cultured macrophages, afterward phagocytosis was quantified by flow cytometry, which revealed an increased conidial uptake for the brown isolate. Virulence was studied in immunosuppressed (inhalation of conidia) or immunocompetent mice (intravenous inoculation). Conversely to that observed in immunosuppressed mice, mortality in immunocompetent mice was significantly lower with the brown isolate compared to a WT strain. Together, these results confirmed that the partial deficiency in melanin biosynthesis observed in the ARP2 mutant isolate could be responsible for its lack of competitivity

Exploring ultra-low β* values in ATF2 - R&D Programme proposal

We propose to explore the beam sizes and performance of the ATF2 Final Focus System for reduced IP beta functions up to a factor between 2 and 4 below its design. The results will demonstrate the feasibility of the system in a chromaticity regime of interest for CLIC and ILC