Gene Disruption in Scedosporium aurantiacum: Proof of Concept with the Disruption of SODC Gene Encoding a Cytosolic Cu,Zn-Superoxide Dismutase

Scedosporium species are opportunistic pathogens responsible for a large variety of infections in humans. An increasing occurrence was observed in patients with underlying conditions such as immunosuppression or cystic fibrosis. Indeed, the genus Scedosporium ranks the second among the filamentous fungi colonizing the respiratory tracts of the CF patients. To date, there is very scarce information on the pathogenic mechanisms, at least in part because of the limited genetic tools available. In the present study, we successfully developed an efficient transformation and targeted gene disruption approach on the species Scedosporium aurantiacum. The disruption cassette was constructed using double-joint PCR procedure, and resistance to hygromycin B as the selection marker. This proof of concept was performed on the functional gene SODC encoding the Cu,Zn-superoxide dismutase. Disruption of the SODC gene improved susceptibility of the fungus to oxidative stress. This technical advance should open new research areas and help to better understand the biology of Scedosporium species

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

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Spontaneous self-assembly of SC3 hydrophobins into nanorods in aqueous solution

Hydrophobins are small surface active proteins secreted by filamentous fungi. Because of their ability to self-assemble at hydrophilic–hydrophobic interfaces, hydrophobins play a key role in fungal growth and development. In the present work, the organization in aqueous solution of SC3 hydrophobins from the fungus Schizophyllum commune was assessed using Dynamic Light Scattering, Atomic Force Microscopy and fluorescence spectroscopy. These complementary approaches have demonstrated that SC3 hydrophobins are able not only to spontaneously self-assemble at the air–water interface but also in pure water. AFM experiments evidenced that hydrophobins self-assemble in solution into nanorods. Fluorescence assays with thioflavin T allowed establishing that the mechanism governing SC3 hydrophobin self-assembly into nanorods involves β-sheet stacking. SC3 assembly was shown to be strongly influenced by ionic strength and solution pH. The presence of a very low ionic strength significantly favoured the protein self-assembly but a further increase of ions in solution disrupted the protein assembly. It was assessed that solution pH had a significant effect on the SC3 hydrophobins organization. In peculiar, the self-assembly process was considerably reduced at acidic pH. Our findings demonstrate that the self-assembly of SC3 hydrophobins into nanorods of well-defined length can be directly controlled in solution. Such control allows opening the way for the development of new smart self-assembled structures for targeted applications

Comparison of multiple methods for the study of the molecular epidemiology of Aspergillus fumigatus

Date du colloque&nbsp;: 01/2008</p

Draft Genome Sequence of the Human-Pathogenic Fungus Scedosporium boydii

The opportunistic fungal pathogen Scedosporium boydii is the most common Scedosporium species in French patients with cystic fibrosis. Here we present the first genome report for S. boydii, providing a resource which may enable the elucidation of the pathogenic mechanisms in this species

Laser nephelometry applied in an automated microplate system to study filamentous fungus growth.

By contrast with photometry (i.e., the measurement of light transmitted through a particle suspension), nephelometry is a direct method of measuring light scattered by particles in suspension. Since the scattered light intensity is directly proportional to the suspended particle concentration, nephelometry is a promising method for recording microbial growth and especially for studying filamentous fungi, which cannot be efficiently investigated through spectrophotometric assays. We describe herein for the first time a filamentous fungi–tailored procedure based on microscale liquid cultivation and automated nephelometric recording of growth, followed by extraction of relevant variables (lag time and growth rate) from the obtained growth curves. This microplate reader technique is applicable for the evaluation of antifungal activity and for large-scale phenotypic profiling

Organization of Patient Management and Fungal Epidemiology in Cystic Fibrosis

The achievement of a better life for cystic fibrosis (CF) patients is mainly caused by a better management and infection control over the last three decades. Herein, we want to summarize the cornerstones for an effective management of CF patients and to give an overview of the knowledge about the fungal epidemiology in this clinical context in Europe. Data from a retrospective analysis encompassing 66,616 samples from 3235 CF patients followed-up in 9 CF centers from different European countries are shown

Retrospective analysis of microorganisms isolated from cystic fibrosis patients in Southern Italy, 2002-2010.

ObjectiveThis study aim was to determine the prevalence of microorganisms in the respiratory tract of patients with cystic fibrosis (CF) admitted to the CF Reference Centre in Southern Italy between 2002-2010. Methods Microbiology assessment of samples (sputum and tracheal aspirates) collected from patients with pulmonary exacerbation admitted to hospital was carried out. All patients were registered in a database and clinical and microbiological data were retrospectively analysed. Results Overall, 188 patients were included and a total of 1217 samples were analysed. The most common microorganisms were Staphylococcus aureus (78.7% of the patients) and Pseudomonas aeruginosa (58%), followed by Candida albicans (19.1%), Haemophilus influenzae (13.3%) and Aspergillus fumigatus (9.6%). Conclusion Compared to similar studies performed in other European countries, our microbiological data, especially the low occurrence of filamentous fungi, suggest a specific local epidemiology, probably related to some uncommon CFTR mutations, which are specific to Southern Italy

What can be learned from phenotyping and genotyping analyses of Scedosporium prolificans isolates from diverse origins?

Introduction: Scedosporium prolificans is a filamentous fungus considered as an emerging opportunistic member of the order Microascaceae. This fungus has a broad clinical spectrum and can cause different types of infections: localized colonization in immunocompetent hosts or disseminated mycosis in immunocompromised patients. Moreover, S. prolificans resistance to most antifungal agents has been reported. Compared to the well characterized Scedosporium / Pseudallescheria complex, little is known about the fundamental aspects of S. prolificans biology, pathogenicity and epidemiology. Aim of the study: Our goal was to characterize a large population of S. prolificans strains, isolated from animals, human, or environment samples in different countries (European, USA and Australia). Results &amp; methods: All strains were prospectively collected, and grown at 30# C on Sabouraud’s agar medium with kanamycin for 1 week. DNA was extracted from subcultures using UltraClean Fecal# DNA kit (MoBio, France). To improve the knowledge of this species at the phylogenetic level, we combined phenotypic criteria such as macroscopic and microscopic morphology features, antifungal susceptibilities based on E-test# method, and genotypic characterization using multi-loci approaches (superoxide dismutase, beta-tubulin and internal transcript spacer genes). Phylogenetic trees were constructed with unambiguously CLUSTALW aligned sequences using the neighbour-joining method with Kimura-2 parameter as substitution model and maximum parsimony analysis, using the BioEdit version 7.0.0 and Phylip version 2.0 softwares. Discussion: Among our collection composed of 59 isolates, we identified three macroscopically different morphotypes of S. prolificans and some genetic polymorphisms (1.8–2.2% difference between the analyzed sequences). These low sequence polymorphisms reflected intra-specific genetic variations. Therefore, we hypothesized that S. prolificans might be stable in space, and apparently insensitive to xenical or environmental factors. No correlation between clinical-biological characteristics and genotypic or phenotypic criteria of S. prolificans strains was found. In conclusion, our results supported the current perception of S. prolificans as a unique species and an emerging opportunistic pathogen

Combined antifungal therapy is superior to monotherapy in pulmonary scedosporiosis in cystic fibrosis

Cystic fibrosis (CF) is characterised by chronic airway infection with bacteria and fungi. Infections caused by Scedosporium/Lomentospora species can occur and are difficult to treat. Moulds belonging to the genus Scedosporium/Lomentospora are detected most frequently in respiratory samples of patients with CF, next to Aspergillus spp. Our aim was to define pulmonary fungal infections due to Scedosporium/Lomentospora in CF and to study the antimycotic treatment. In this multicentre study (12 centres; duration January 2008 to December 2014) 31 patients with a lung infection caused by moulds of the genus Scedosporium/Lomentospora were included. 36 courses of antifungal treatment were documented. Scedosporium apiospermum sensu stricto accounted for 48.4% of cases. In 20/31 patients a therapeutic response under antimycotics (median duration 3.9 months) was achieved. Triple and double therapy was significantly more effective compared to monotherapy regarding FEV1, radiology, and symptoms. This data suggests that combined treatment is superior to monotherapy in patients with CF