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  Director : d’Enfert, Christophe (denfert@pasteur.fr)


  abstract

 

We study several biological processes in fungi of the genus Aspergillus and Candida with the aim of developing new strategies for the control of fungal growth. Our work focuses on the identification of antifungal targets through the detailed study of a critical process in the life cycle of filamentous fungi, namely, the germination of spores, or through an exhaustive search of essential genes in the human fungal pathogen Aspergillus fumigatus. Also we take advantage of genomic tools in order to study biofilm formation by the major fungal pathogen of humans, Candida albicans.



  report

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Filamentous fungi of the genus Aspergillus have been widely used for biotechnological processes but are also responsible for human disease. Spore germination is a key developmental step in the fungal life cycle and is critical for the establishment of the fungus in a new environmental niche. Our aim is to define the molecular and biochemical events that are involved during the early stages of spore germination. In particular, we wish to characterize the signaling pathways mediating the recognition of environmental signals necessary for germination and their transduction towards the resumption of various metabolic processes that are necessary for germination.

Our work has resulted in the demonstration that cAMP signaling is involved in the early stages of spore germination in the model filamentous fungus Aspergillus nidulans. Indeed, cAMP synthesis, mediated by the CyaA adenylate cyclase, and the PkaA catalytic subunit of the cAMP-dependent protein kinase are both necessary for the response of the spore to an external carbon source and the timely formation of a germ tube (Collaboration N. Keller, USA). A second Ser/Thr kinase (SchA) related to mammalian PKB is also involved in the control of spore germination. Finally, our results show that signalling mediated by the RasA GTPase which is homologous to the human ras protooncogene controls germination independently of cAMP signalling.

Present research is aimed at identifying the mechanisms of activation of adenylate cyclase and the targets of cAMP during spore germination. In this regard, we are interested in heterotrimeric G proteins and G protein coupled receptors. We have recently shown that only one of the three A. nidulans Ga subunits, namely GanB, is involved in the early stages of spore germination (Collaboration K.-Y. Yahng, South Korea). A study of the physical interaction of GanB with adenylate cyclase is being conducted. The sequencing of A. nidulans ESTs (Expressed Sequence Tags) that has been performed in collaboration with the Genomics Platform ( PT1 of the Institut Pasteur Génopole ) will now allow the development DNA-arrays which will be used to study in greater details A. nidulans strains with defect in cAMP signaling.

Systematic identification of essential genes in the fungal pathogen of humans, Aspergillus fumigatus (A. Firon and C. d'Enfert)

Aspergillus fumigatus is the causative agent of invasive aspergillosis, which represents the second cause of death resulting from fungal infections in hospitals. Invasive aspergillosis is associated with a high mortality. This is mostly due to the poor sensitivity of the currently available diagnostic tests and the sole use of amphotericin B therapy, which has deleterious side effects. Analysis of candidate virulence factors by reverse genetics has not been successful for the understanding of the pathogenic processes. We have therefore focused our interest towards the development of novel molecular tools that could be used to identify through insertional mutagenesis genes that are essential for growth of A. fumigatus under laboratory conditions and/or during invasion of the host and may hence serve as antifungal targets. This program is performed in collaboration with Bayer CropScience ( http://www.bayercropscience.com ).

A. fumigatus strains have been constructed that can be used to test whether a gene is essential for growth under laboratory conditions. We have used this system to develop a screen for A. fumigatus essential genes using insertional mutagenesis. This has been achieved using the impala transposable element from Fusarium oxysporum and has lead to the identification in a preliminary screen of 29 new genetic loci which are essential for the growth of A. fumigatus. The genes that we have identified are involved in a wide variety of cellular processes and some are specific to A. fumigatus. These results suggest that a systematic search for A. fumigatus genes can now be undertaken in order to establish a compendium of antifungal targets for this pathogen.

Candida albicans: genomics, biofilms and epidemiology (S. Aubert, M.-E. Bougnoux, F. Cottier, S. Garcia-Sanchez, P. Knechtle et C. d'Enfert)

Candida albicans is currently the main fungal pathogen of humans. In particular, C. albicans is responsible for systemic infections in patients, which are severely immuno-compromized and receive broad-spectrum antibiotherapy. Systemic candidiasis are associated with a high mortality despite the availability of antifungal treatments. A better management of C. albicans infections will require an understanding of their epidemiology and physio-pathology as well as the development of new drugs. Our research is therefore interested in the epidemiology of C. albicans infections and in the study of processes related to pathogenicity through genomic approaches.

Whole-genome transcriptional analysis appears as a powerful tool to gain a better understanding of gene function and of the mechanisms that are involved during the adaptation of an organism to various conditions, in particular host-pathogen interaction. Through our participation in the French Fungal Infection Network and the European Galar Fungail network we have undertaken the development DNA-arrays for transcript profiling in C. albicans. Through this work we have established a C. albicans genomics database, CandidaDB and produced whole genome DNA-arrays in collaboration with Eurogentec ( http://www.eurogentec.com ).

We have initiated in the framework of a Programme Transversal de Recherche with the Molecular Mycology Unit and the Genetics of Biofilms Laboratory a study of the molecular mechanisms that govern biofilm formation by C. albicans. Biofilms are three-dimensional associations of microorganisms. C. albicans biofilms are more resistant to antifungals than planktonic cells and might be responsible for sequels that occur following antifungal treatment of systemic candidiasis. We have established several models of biofilm, which have been analyzed by transcript profiling. On this basis, several genes have been selected for further study with the aim to evaluate their contribution to the formation and physiology of C. albicans biofilms.

We have established a new typing methodology for C. albicans which is based on Multi Locus Sequence Typing (MLST). This method involves the sequencing of six independent loci which show intra-specific variations. This method is highly reproducible, has a high discriminatory power and is currently used to study collections of C. albicans clinical and commensal isolates in collaboration with the Genomics Platform (PT1) of the Institut Pasteur Génopole.

Photo 1 : Model of activation of spore germination and cAMP signalling in A. nidulans

Photo 2 : Biofilm of Candida albicans formed on a plastic surface (Scanning Electron Microscopy; collaboration E. Arbeille, Université de Tours)

Keywords: Aspergillus nidulans, Aspergillus fumigatus, Candida albicans, cAMP, kinase, signal transduction, germination, spore, conidia, antifungal, target, transposon, DNA chip, DNA array, biofilm, epidemiology, Multi-Locus Sequence Typing, genomics, transcriptome, transcript profiling



  publications

puce Publications of the unit on Pasteur's references database


  personnel

  Office staff Researchers Scientific trainees Other personnel
  Thepaut, Sylvana (sthepaut@pasteur.fr) Bougnoux, Marie-Elisabeth, Université Paris Ile de France Ouest (Assistant Professor, clinician,bougnoux@pasteur.fr)

d’Enfert, Christophe, Institut Pasteur (Associate Professor,denfert@pasteur.fr)

Cottier, Fabien (PhD student,fcottier@pasteur.fr)

Eisman-Redondo, Blanca (PhD student, Madrid University)

Firon, Arnaud (PhD student)

Garcia-Sanchez, Susana (post-doctoral fellow,sgarcia@pasteur.fr)

Knechtle, Philipp (post-doctoral fellow,knechtle@pasteur.fr)

Lafon, Anne (PhD student,alafon@pasteur.fr)

Aubert, Sylvie (technician,syaubert@pasteur.fr)

Chaveroche, Marie-Kim (technician,mkchaver@pasteur.fr)


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