FUNGWALL - The fungal cell wall as a target for antifungal therapies

EC contribution: : € 2.895.000
Duration: : 36 months
Starting date: : 01/01/2005
Funding scheme: : Specific Targeted Research Project
Keywords: : fungi, mycosis, aspergillosis, candidiasis, antifungals, resistance, glucan, chitin, mannan, cell wall
Contract/Grant agreement number: : LSHB-CT-2004-511952
Project web-site: : -

Background:

Fungal pathogens represent the major eukaryotic agents of serious infection in European countries. Infections due to Candida albicans and Aspergillus fumigatus are the most common and clinically important pathogens and were therefore the focus of this project. The repertoire of available antifungal chemotherapeutic agents is inadequate to treat life-threatening infections that are characterised by morbidities that exceed in Europe those due to most important parasitic, bacterial and viral diseases. Therefore there is an urgent need to generate new, efficacious, non-toxic compounds with broad-spectrum antifungal activity. The fungal wall is the skeleton and armour of the fungus but potentially also its Achilles heel since the major components are absent from mammals and can be specifically targeted with compounds that do not affect human metabolism. We have focused our programme on the core cell wall structure that is common to all fungal pathogens and is essential for their growth and viability. The enzymes and reactions associated with the synthesis and the remodelling of these major cell wall polysaccharides as well as the sensing mechanisms important in cell wall integrity and remoulding during morphogenetic processes will be investigated. The challenge for FUNGWALL was to investigate mechanisms of fungal cell wall synthesis in order to identify new antifungal targets to control human fungal infections in Europe.

Aim:

The cell wall of pathogenic fungi is a good target for the development of new drugs for the following reasons: (1) The fungal cell wall is required for fungal cell integrity and is essential for fungal growth and for virulence; (2) Polysaccharidic components of the cell wall are unique to fungi and consequently, putative inhibitors of the biosynthetic pathways responsible for cell wall construction can be potent antifungals, as shown by the recent launch by big Pharmas of drugs inhibiting ß1-3 glucan synthesis.

The objectives of FUNGWALL are centered on the assembly of the cell wall polysaccharide skeleton. The enzymes and reactions associated with chitin, glucan and mannan synthesis, glucan cross-linking and branching will be investigated. New post genomic approaches will enable us to first revisit old targets, define novel targets and to screen for novel compounds that disrupt the integrity of the cell wall with the goal of identifying new generation antifungals that target fungal cell wall biosynthesis. These studies will use primarily the 2 main fungal pathogens in Europe, Candida albicans and Aspergillus fumigatus.

To identify cell wall targets, 4 work packages were defined:

WP1 studies chitin; in WP1, the assembly and modelling of the cell wall chitin will be revisited using a multidisciplinary approach to develop and validate new inhibition assays for the discovery of the first chitin synthesis and chitinase inhibitors to be efficient in vivo against fungal cells.
WP2 will revisit ß1-3 glucan synthesis and propose a new post genomic approach to better understand the inhibitory role of echinocandins; in addition, this WP will investigate for the first time endo ß1-3 glucanase as a putative target.
Branching and cross-linking between chitin and ß1-3 glucan is required in the formation of a resistant skeleton of the cell wall. Linkages between these 2 polysaccharides have been shown to be essential to construct the fibrillar core of the fungal cell wall. Accordingly, enzymes and regulators involved in the biosynthesis of this core structure will be key target molecules. At this time the nature of the enzymes that are responsible for the branching and cross-linking of the structural cell wall polysaccharides. Their identification will be a major research objective of this application and will form the WP3 of this STREP.
Sensing wall modifications and damage and restoring cell wall integrity require a functional network of active sensors and signalling mechanisms. O-mannosylation plays an essential role in maintaining the integrity of these sensing mechanisms. WP4 will study mannosylation in fungi with the objective to identify essential pathways and proteins that become inactive in absence of post translational mannosylation.

Expected and obtained results:

Bringing together the leading laboratories in Europe working on cell wall analysis in fungi, the 36-month FUNGWALL project led to the following achievements:

The validity of chitin synthesis as a legitimate target for antifungal chemotherapy was underlined by the discovery of strong synergistic combinations of anti-chitin and anti-glucan inhibitors.
Chitin hydrolysis was validated as target for antifungal chemotherapy and novel inhibitors of these enzymes were discovered and characterised following crystallisation of various chitinases.
Complete mutant collections of all chitin synthase genes was achieved and characterised for the two major human pathogens Candida albicans and Aspergillus fumigatus and has allowed for the development of new screens for chitin synthase inhibitors.
The mode of action of aminocandin was elucidated though various genomic strategies.
A new ß1,3 glucan binding domain has been characterized and shown to be essential in yeast morphogenesis.
Two new transglycosidase activities have been discovered and shown to be involved in branching of ß1,3 glucans and the synthesis of ß1,6 glucans.
A list of genes with putative cell wall polysaccharide remodelling activity has been identified bio-informatically and validated through various genomic and biochemical approaches.
Several methodologies to analyse carbohydrate-protein interactions have been developed.
The signals determining O-mannosylation have been identified.
A list of all O-mannosylated proteins of S. cerevisiae was established and their role in sensing the environment analysed.
The essentiality of O-mannosylation in yeasts and moulds was underlined.
Two major international conferences on cell wall have been organized.

As regards the state-of-the-art, the achievements of the project have placed Europe in a leading position in the world for the analysis of fungal cell wall. The coupling of biochemical and genetic methodologies was extremely synergetic to tackle this problem and have given a unique flavour to FUNGWALL. A close contract between the different members of the STREP will now continue and will lead to new scientific developments in the area.

In terms of impact of the project on its industry or research sector, major achievements have resulted from the new multidisciplinary genomic and post genomic approaches undertaken. Among them:

A. Fumigatus arrays have been produced in view to analyse combination of drugs.
Molecular characterization of fungal endo ?1,3 glucanase activity has led to a screen for glucanase inhibitors.
Synthetically lethal technology has been applied to transglycosidase candidates and identified new interactive partners.
Methodologies for biochemical HTS of transglycosidase have been developed.
A comprehensive list of cell wall proteins has been produced and analysed in silico and in vivo.

Finally, several new drug targets were identified during the course of FUNGWALL:

chitinases and endo ß1,3 glucanases;
new transglycosidases remodelling ? glucans;
O-mannosyltransferases.

Most strains have been engineered which could be very useful tools in the search for new drugs.

For example, chitin synthase mutants are now available for looking for specific antifungal drugs in both Candida and Aspergillus. Although new drug targets were identified and old ones successfully revisited, we have assisted during the course of this STREP to a reduction in the research and developments efforts put by the pharmaceutical industry in the antifungal business. The chances to see new drugs developed by these companies in the near future are very limited. To palliate this, we have also embarked in a combination therapy strategy that has been based on the research of this STREP on fungal cell wall biosynthesis. It will lead to promising results in the management of patients in Europe suffering from systematic fungal infections. This may lead to new formulation of existing antifungal drugs, a very promising development for SMES.

Coordinator:

Prof. Jean-Paul Latge
Unité des Aspergillus
Institut Pasteur
75724 Paris, France
Tel. +33 01406135 19
jplatge@pasteur.fr

European Commission

FUNGWALL - The fungal cell wall as a target for antifungal therapies

Background:

Aim:

Expected and obtained results:

Coordinator:

Partners: