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EUROCELLWALL – QOL3-2000-01537

EUROCELLWALL – QOL3-2000-01537. Acronym: EUROCELLWALL Project number: QLK3-2000-01537 EC contribution : 1,454,008 Euros Duration : 36 months Starting date Nov 1 st , 2000 Contract type : Shared cost 10 partners including: a SME and Company. Title : Exploiting yeast cell wall

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EUROCELLWALL – QOL3-2000-01537

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  1. EUROCELLWALL – QOL3-2000-01537 Acronym: EUROCELLWALL Project number: QLK3-2000-01537 EC contribution: 1,454,008 Euros Duration: 36 months Starting date Nov 1st, 2000 Contract type: Shared cost 10 partners including: a SME andCompany Title: Exploiting yeast cell wall for high throughput screening of antimicrobial agents Key Action n° 3: The Cell factory  Research Programm 3.3: New biological and biotechnological processes and products for agro-industry, agri-food and high value added chemicals www.insa-tlse.fr/gba/eurocellwall.html(under construction) www.dkfz-heidelberg.de/funct_genome/ (arrays data)

  2. The cell wall: unique structure in microorganism identical between non-pathogen and pathogen ideal target for antimicrobial molecules EUROCELLWALL – QKL3-2000-01537 Motivation Title: Exploiting yeast cell wall for high throughput screening of antimicrobial agents

  3. What is well known? • its composition • it is a modular structure (cross-linkage and remodelling) • there are >100 genes for cell wall construction • Why slow progress in developing novel antifungals? •  incomplete knowledge of assembly mechanism •  poor biochemistry and enzymology of the cell wall •  lack of well-characterized targets S S Transmembrane Protein Mannoprotein 40% GPI Anchor • 1-3 glucan 50% • 1-6 glucan 8% Chitin 2% EUROCELLWALL – QKL3-2000-01537 A scheme of the structure of the yeast cell wall

  4. EUROCELLWALL – QKL3-2000-01537 The project workplan target gene Title Exploiting yeast cell wall for high throughout screening of antimicrobial agents no Phenotype of the deletants: Essential? cell wall defective? growth defect... Leave for academic research yes Structure-function catalytic domain Expression/purification Function no Academic interests publication Design an assay for HTS (large screening) yes Assay to be patented HTS phases for identify molecules with antifungal activities

  5. EUROCELLWALL - QKL3-2000-01537 Objectives Title Exploiting yeast cell wall for high throughout screening of antimicrobial agents 1-Convert molecular data into manageable cell wall targets. 2-Generate novel assays from identified cell wall targets for high throughput screening of a collection of molecules with potential antifungal activities. 3- Identify novel cell wall targets by use of global (and integrated) genomic (microarrays) and proteomic (2D –gel, LC-MS) technologies.

  6. P2:Unimi-Italy (Dr Popolo) P4:UCM-Spain (Pr Arroyo) P5: Biosearch sa (Dr Carrano) P6; UniAbdn-Uk (Pr Gooday-Gow) P7: Uni-Regens-Ger (Pr W Tanner) P8: UsalII-Spain (Pr F Del Rey) P10: Aventis-France (Dr Zundel) P1: Insa –France (Pr François) P3:UsalI-Spain (Dr Roncero) P5:Biosearch Sa (Dr Carrano) P6; UniAbdn-Uk (Dr Gooday & Dr Gow) P10: Aventis-France (Dr Zundel) P1: Insa –France (Pr François) P2:Unimi-Italy (Dr Popolo) P4: UCM-Spain (Dr Arroyo) P7:Uni-Regens -Ger (Pr Tanner) P8: Usal II –Spain (Pr F Del Rey) P9; DKFZ-Ger (Dr Hoheisel) All partners coordinator: INSA-DGBA (Pr François) EUROCELLWALL – QKL3-2000-01537 The workpackage Title Exploiting yeast cell wall for high throughout screening of antimicrobial agents WP 1:Setting-up HTS assays with enzymes involved in the cell wall remodelling and cross-linking pathway WP 2:Setting-up HTS assays from molecular and biochemical characterisation of chitin pathway WP 3:Identify new targets for HTS through the characterisation of the cell wall integrity pathway by combined genomic and proteomic analysis. WP 4:project management

  7. WP coordinator Advisory committee Exploitation plans EUROCELLWALL – QKL3-2000-01537 Management Meeting/contact period WP1 WP2 WP3 Title Exploiting yeast cell wall for high throughout screening of antimicrobial agents 2 4 5 6 7 8 10 1 3 5 6 10 1 2 4 Partner n° Every 6 months 7 8 9 2 3 4 Frequent contact WP4 1 + administrative officer coordinator - Biosearch It. (P5) Every year - Aventis (P10) - Partner 7 (chairman) confidential publications Patents/ Exploitations

  8. EUROCELLWALL – QKL3-2000-01537 9 Milestones  M1: O-mannosylation assay M2: Engineered strains in chitin synthesis pathway M3: In vitro assay of chitin M4: Characterize enzymes/activity of cross-linking and remodelling pathway M5: Assay for cross-linking/remodelling enzymes M6: Cloning of genes from M. grisea (rice pathogen) M7: Novel drug-targets from genomic and proteomic analysis M8: identify Antifungal activities M9: meeting (every 6 months and Progess report (every year) Title Exploiting yeast cell wall for high throughout screening of antimicrobial agents    

  9. Mannoproteins(40%) Outer cell wall  (1,6) glucans(8%)  (1,3) glucans(50%) Inner cell wall Chitin(2%) Glycophospholipid-anchored surface protein gas1 EUROCELLWALL – QKL3-2000-01537 WP3: Identify new targets from a genomic and proteomic analysis of the cell wall compensatory mechanism mnn9 kre6 CELL WALL 150 nm fks1 knr4 Plasma membrane By partner 1, 4, 7, 9 (+ 2 and 5)

  10. WHAT KIND OF GENES ARE INVOLVED IN THE COMPENSATORY MECHANISM ? EUROCELLWALL – QKL3-2000-01537 80 60 Glucose (Glucan) % of the cell wall 40 Mannose (Mannoprotein) Glucosamine (Chitin) 20 0 knr4 wt mnn9 kre6 wt fks1  mutations/agressions cause a cell wall reorganisation this may even results in a strenghtening of the wall !

  11. cDNA labelled with 33P PCR product of yeast ORFs 700 µm EUROCELLWALL – QKL3-2000-01537 (work of partners 1, 7 and 9) 11 cm 22 cm The 6200 Open Reading Frames (ORF) of Saccharomyces cerevisiae on polypropylene filter.

  12. EUROCELLWALL – QKL3-2000-01537 Classification and hierarchical clustering analysis (work by partners 1 & 9) up regulated down regulated unchanged

  13. Mutants gas1 mnn9 fks1 knr4 kre6 80 genes 45 genes EUROCELLWALL – QKL3-2000-01537 (work by partner 1, 4) Bio-informatic analysis of the promoter Clustering: Common regulatory elements in the promoter . Search for cis-motif in promoters Use of algorithms in the literature  Develop novel algorithms

  14. What was found from this Bio-informatic analysis? promotor gene -700bp -1 ATG Cis-factor sequences distribution function Rlm1p binding-site NTAWWWWTAG 54/80 Cell wall STRE CCCCT 54/80 Reponse to stress HSE / HSTF GAANNTCC 54/80 Reponse to stress GCR1 RGCTTCCWC 48/80 Carbon Metabolism PHO4 NNNCACGTKNGN 43/80 Pi Metabolism MATA1 TGAGTANNT 52/80 Morphogenesis STUAP1/SOK2 NWWCGCGWNM 57/80 Cell cycle new consensus CAGCCTC 31/80 ? EUROCELLWALL – QKL3-2000-01537

  15. Promotor analysis of up-regulated genes independently of the cell wall mutation Sequences Sequences Distribution Distribution Function Function Rlm1p Rlm1p - - NTAWWWWTAG NTAWWWWTAG 54/80 54/80 Cell Cell wall wall STRE STRE CCCCT CCCCT 54/80 54/80 Stress Stress HSE / HSTF HSE / HSTF GAANNTCC GAANNTCC 54/80 54/80 Stress Stress GCR1 GCR1 RGCTTCCWC RGCTTCCWC 48/80 48/80 Central Central metabolism metabolism PHO4 PHO4 NNNCACGTKNGN NNNCACGTKNGN 43/80 43/80 wt msn2 msn4 pho4 rlm1 Phosphate Phosphate metabolism metabolism MATA1 MATA1 TGAGTANNT TGAGTANNT 52/80 52/80 Morphogenese Morphogenese Caffeine 4 mM STUAP1/SOK2 STUAP1 NWWCGCGWNM NWWCGCGWNM 57/80 57/80 Cell Cell cycle cycle NIT2 TATCTM 80/80 Nitrogen metabolism Potentiel Consensus wt msn2 msn4 pho4 rlm1 CFW 0.025 mg/ml wt msn2 msn4 pho4 rlm1 SDS 0.05% wt msn2 msn4 pho4 rlm1 Congo Red 0.25 mg/ml  Combinatorial Effect? EUROCELLWALL – QKL3-2000-01537 Biological validation of the Bio-informatic analysis?

  16. EUROCELLWALL – QKL3-2000-01537 Some conclusions & perspectives • very good partnership (training and transfert of Know-how) • on schedule (deliverables and milestones) • be able to find targets (competitiveness) Title Exploiting yeast cell wall for high throughout screening of antimicrobial agents But still huge biochemical works  not able to go over the first HTS screening due to budget limitation! thus open to wide collaborations

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