Nouvelles voies métaboliques essentielles pour la biosynthèse de la paroi des champignons – REMODELE

A major characteristic of all yeast and moulds is the presence of a cell wall that surrounds the cell of these eukaryotic microorganisms. For many years the cell wall was considered as an inert skeleton. It is now known that the cell wall is an extremely dynamic structure that controls polarized fungal growth. Moreover, the cell wall has both protective and aggressive functions in fungal life. Protective, since it is the first barrier in contact with the environment most often hostile encountered by the fungus. Removal or weakening of this barrier leads indeed to fungal death in absence of osmotic protection. Aggressive since all exchanges between the fungal cell and its environment rely upon a functional and permeable cell wall. In plant and mammalian pathogens, the cell wall is continuously in contact with the host and acts as a sieve and a reservoir for molecules such as enzymes or toxins that play an active role during infection. It also provides force to penetrate any insoluble material that it has to colonize to grow or survive The components of the central skeleton of the fungal cell wall are chitin and '1,3 glucans. They are separately synthesized as linear polysaccharides by the polysaccharide synthases at the plasma membrane and extruded in the cell wall space. In the cell wall, they are modified. Major modifications are the branching of the '1,3 glucans and the cross linking between chitin and glucan. This polysaccharide remodeling confers rigidity to the cell wall and resistance to turgor pressure. This branched glucan-chitin is the structural core of the cell wall common to all fungal species. The rigidification of the cell wall structure is thus an essential event in fungal biology. The nature of the enzymes and regulators that are responsible for the branching and cross-linking of the structural cell wall polysaccharides, is however unknown. Their identification will be the major research objective of this application. We recently developed in our laboratory a very accurate analytical methodology that allows to quantify the different branching points between the constitutive polysaccharides. In addition, we are able to produce in vitro using permeabilized yeasts branched and cross-linked polysaccharides. This is the first time that such linkages are produced in vitro. These data are at the basis of the development of this grant. We have in hands for the first time in science, a methodology that will allow a biochemical and genetical analysis of the biosynthetic activities responsible for the branching and cross linking of the cell wall ' (1,3) glucan and a way to understand the rigidification of the cell wall. All studies will be undertaken with the model yeast Saccharomyces cerevisiae. Major tasks of the grant are: The structural characterization and quantification of all branched or cross linked oligosaccharides in the yeast cell wall in wild type and mutant strains identified through a comparative genomic analysis. The biochemical characterization of the b1,3-glucan remodelling activities in yeast The biochemical purification of the transglycosidases or their identification through the use of the mutant libraries To reach these objectives we have set up a consortium of 2 laboratories that have different but complementary competences. The coordinating partner is the Unit of Aspergillus at the Institut Pasteur (Paris ) headed by JP Latgé, that has a renowned expertise in fungal cell wall structural chemistry and enzymology and Aspergillus molecular biology. The second partner is Muriel Delepierre that directs the CNRS/Institu Pasteur NMR of Biomolecules unit at the Institut Pasteur (Paris). This Unit has shown that NMR is a very efficient and unique tool to investigate the structural chemistry of complex oligosacharides. Our consortium will benefit from a long lasting collaboration with the Unit Architecture et Fonction des Macromolécules Biologiques of the CNRS (Marseille) (B. Henrissat) who will be subcontracted to help us in the in silico analysis of the putative transglycosidases. Looking for remodelling enzymes is the Graal of all mycologists studying cell wall synthesis. Our consortium is the only one tackling to date in the world the identification of remodelling cell wall enzymes by the conjunction of both carbohydrate and protein biochemical and genetical methodologies. This is a major strength of our consortium. Moreover, all partners have worked and published together, a strong indication of their capacity to work collaboratively. In addition to their role in controlling a key event in cell biology that is polarized hyphal growth, these enzymes will be appropriate targets for the development of new drugs because: (1) the fungal cell wall is required for fungal cell integrity and; (2) polysaccharidic components of the cell wall are unique to fungi.