Research project C3/019 (Research action C3)
Context
Aspergillus fumigatus is a cosmopolitan saprophytic fungus from soil, present in outdoor as well as in indoor air. It is also at the first rank of opportunistic fungus dangerous for man, causing invasive infections particularly in immunocompromised patients, and colonizing the airway of cystic fibrosis (CF) patients, contributing to the degradation of lung function.
The pathogenicity of the species A. fumigatus is more important than that of other filamentous fungi present in the environment due in a large part to its thermotolerance. It is why numerous potential virulence factors have been extensively studied, and it is now well established that virulence is multifactorial. Understanding the pathogenicity of the species A. fumigatus requires a better knowledge of epidemiology of infections and characterization of the strains at the genetic as well as at the proteic expression level. Moreover, for legal reasons, there is an urgent need to develop a standardized computerizable molecular typing method which could unambiguously establish the relationship between strains from an environmental source and patients strains.
Project description
Previous studies performed (by members of the network) have shown that some genotypes of A. fumigatus are more frequently isolated in patients and that genetic diversity in patients is less important than in the environment. This suggests that even if all the isolates are potentially pathogenic in an receptive host, some strains could be more adapted to the development in man (hostile environment) for this environmental fungus.
Patients with invasive aspergillosis are often contaminated by several different isolates at the same time, these multiple isolates being genetically different. It has also been shown in sequencial isolates (collected at different time) of lung transplant patients a wide diversity of genotypes, as also observed in CF patients recently colonised. However, chronically colonised CF patients presented a more limited number of genotypes and particular genotype common to these patients was evidenced, suggesting a genotype selection with the ageing of colonisation.
The Biomedical collection of fungi and yeast has developed an important sub-collection of A. fumigatus from human and environment origin. At the beginning of the project 1657 isolates of A. fumigatus are available in the collection, 389 of them have already been cited in publications.
Objectives / methodologies
Taking into account the state of the art in the field of the project, experience of the partners, biological material and data available in the BCCM/IHEM collection, we have defined the following objectives and methodologies.
Objective 1
Develop the subcollection of A. fumigatus isolates (in accordance to the need of the other objectives) and gather all the information available in a single database. The BCCM/IHEM catalogue available on http://bccm.belspo.be/db/ is based on criteria common to European network catalogue but not adapted for medical and scientific data.
Objective 2
Previous studies of typing of A. fumigatus are heterogenous and are sometimes difficult to export from one laboratory to another. There is an urgent need to define a "golden standard" method for typing of A. fumigatus. Multilocus sequence typing MLST has emerged as a powerful and portable method initially developed for bacteria. It is recently used for Candida albicans. Indeed its portability is due to the fact that it is a sequencing based method. The objective of the initial project was to design a MLST typing scheme for A. fumigatus. Due to the delay before the beginning of the project, this study has been launched by other teams, in particular by the Dr. Frank Odds, (Professor of Medical Mycology at the Aberdeen Institute of Medical Science). A collaboration has been asked to this team and the application to typing of the sub-collection can be performed more rapidly. Seven genes have been selected by this team (ANX4, BGT1, CAT1, LIP, MAT1-2, SODB and ZRF2) as well as primers for sequencing. This method will be compared to others method for which data and strains are available. Partner 2 will implement MLST and screen sub-collection.
Objective 3
This objective focuses on the identification of virulence factors of the dominant genotypes by the comparison of the different genotypes identified in environmental or clinical samples from CF patients or LT recipients. In this aim, a proteomic approach will be developed using SELDI-TOF technique (surface enhaced laser desorption/ionisation) with different surfaces of chips array (hydrophilic, hydrophobic, metal surfaces affinity). The profiles will be analyzed by Ciphergen system. 2D electrophoresis can in a further part characterize biomarkers of interest.
Another approach will be the study of phenotypic properties involved in virulence such as the adherence capabilities of the conidia. Expression at the surface of the conidia of fungal receptors will be investigated by flow cytometry, and some physical properties such as electrostatic charges and cell surface hydrophobicity will be investigated by microelectrophoresis and two-phase partitioning.
Results/products
A standardized reproducible typing method MLST will be compared to other tools available and help to understand epidemiology of colonisation in cystic fibrosis patients and deep infections.
Hypothesis of genotypic selection has to be verified as well as capabilities of adherence of conidia. These data could be used as a diagnostic tool and guide therapeutic decision facing to airway colonisation by A. fumigatus in CF patients.
SELDI will allow us to characterize for the first time fungal biomarkers of interest useful as diagnostic tool.
The BCCM/IHEM Collection will develop a service of supply of DNA and protein extracts performed under highly standardized conditions.
Interaction between the different partners
Scientific Institute of Public Health - Mycology Section (Host Laboratory of the BCCM/IHEM Collection).
Project coordination. Preservation of isolates, preparation of standardized DNA and proteic extracts. 2D electrophoresis.
Laboratorium voor Farmaceutische Microbiologie - Ghent University
MLST typing
Laboratoire de Parasitologie Mycologie Médicale & Centre d’Investigation Clinique (Grenoble, France)
SELDI analysis, 2D electrophoresis
Laboratoire de Parasitologie Mycologie - Groupe d'Etude et d'Interaction Hôte Parasite
(Angers, France).
Adherence capabilities of conidia
Partners
Scientific Institute of Public Health- Mycology Section
Juliette Wytsmanstreet 14
B-1050 Brussels
Tel + 32 2 642 56 30
Fax +32 2 642 55 19
Dr. Françoise Symoens (f.symoens@iph.fgov.be)
Ghent University
Laboratorium voor Farmaceutische Microbiologie- Universiteit Gent
Harelbekestraat 72
B-9000 Gent
Tel + 32 9 264 80 91
Fax + 32 9 264 81 95
Dr. Tom Coenye (Tom.Coenye@UGent), Dr. Hans Nelis (Hans.Nelis@Ugent.be)
Laboratoire de Parasitologie Mycologie Médicale & Centre d’Investigation Clinique
CHU A Michallon, BP 217 38043 Grenoble
Tel + 33 4 76 76 54 90
Fax + 33 4 76 76 56 60
Dr.Claudine Pinel (CPinel@chu-grenoble.fr), Dr. Renée Grillot (RGrillot@chu-grenoble.fr)
Host Parasite Interaction Study Group (Groupe d’Etude des Interactions Hôte-Parasite)
Laboratoire de Parasitologie-Mycologie
4, rue Larrey F-49033 Angers Cedex 01
Tel+ 33 2 41 35 34 72
Fax + 33 2 41 35 36 16
Dr. Jean-Philippe Bouchara (Jean-Philippe.Bouchara@univ-angers.fr)
Users Committee
Dr. Lucien Noens, Department of Hematology and Bloodbank - Ghent University Hospital
Dr. Bart Gordts AZ Sint Jan AV – Brugge
Dr. Renaat Peleman - Hoofdgeneesheer UZ - Ghent - Vakgroep Inwendige ziekten
Prof. Danielle Swinne, BCCM/IHEM Collection - Scientific Institute of Public Health
