Transcript EUROCELLWALL – QKL3-2000

EUROCELLWALL – QOL3-2000-01537
Title:
Exploiting
yeast cell wall
for high
throughput
screening of
antimicrobial
agents
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 and Company
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)
EUROCELLWALL – QKL3-2000-01537
Motivation
Title:
Exploiting
yeast cell wall
for high
throughput
screening of
antimicrobial
agents
The cell wall:
unique structure in microorganism
identical between non-pathogen and pathogen
ideal target for antimicrobial molecules
EUROCELLWALL – QKL3-2000-01537
A scheme of the structure of the
yeast cell wall
What Sis well known?
S
 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
Mannoprotein
40%
GPI Anchor
1-3 glucan
50%
Transmembrane
Protein
1-6 glucan
8%
Chitin
2%
EUROCELLWALL – QKL3-2000-01537
The project workplan
target gene
Title
Exploiting yeast
cell wall for high
throughout
screening of
antimicrobial
agents
Leave for
academic research
no
yes
Function
Phenotype of the deletants:
Essential? cell wall defective?
growth defect...
Structure-function
catalytic domain
Expression/purification
no
Academic interests
publication
yes
Design an assay for
HTS (large screening)
Assay
to be patented
HTS phases for identify molecules with
antifungal activities
EUROCELLWALL - QKL3-2000-01537
Title
Exploiting yeast
cell wall for high
throughout
screening of
antimicrobial
agents
Objectives
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.
EUROCELLWALL – QKL3-2000-01537
Title
Exploiting yeast
cell wall for high
throughout
screening of
antimicrobial
agents
WP
WP
WP
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)
The
workpackage
P8:
UsalII-Spain
(Pr F Del Rey)
P1: Insa
–France (Pr (Dr
François)
P10:
Aventis-France
Zundel)
P3:UsalI-Spain (Dr Roncero)
P5:Biosearch
(Dr Carrano)
1: Setting-up
HTSSa
assays
with enzymes involved in
P6;cell
UniAbdn-Uk
(Dr Gooday
& Dr Gow) pathway
the
wall
remodelling
and cross-linking
P1:
Insa
–France
(Pr
François)
P10: Aventis-France (Dr Zundel)
P2:Unimi-Italy (Dr Popolo)
UCM-Spain (Dr Arroyo)
2: P4:
Setting-up HTS assays from molecular and
P7:Uni-Regens -Ger (Pr Tanner)
biochemical
characterisation
chitin pathway
P8: Usal II –Spain
(Pr F DelofRey)
P9; DKFZ-Ger (Dr Hoheisel)
partners
3: All
Identify
new targets
for HTS(Pr
through
the
coordinator:
INSA-DGBA
François)
characterisation of the cell wall integrity pathway
by combined genomic and proteomic analysis.
WP 4: project management
EUROCELLWALL – QKL3-2000-01537
Management
WP1
Title
Exploiting yeast
cell wall for high
throughout
screening of
antimicrobial
agents
Partner n°
WP
coordinator
coordinator
2
4
5
6
7
8
10
2
WP3
1
3
5
6
10
1
2
4
7
8
9
3
4
Meeting/contact
period
Every
6
months
Frequent
contact
WP4
1+
administrative officer
- Biosearch It. (P5)
- Aventis (P10)
- Partner 7 (chairman)
Advisory
committee
Exploitation
plans
WP2
confidential
Every
year
publications
Patents/ Exploitations
EUROCELLWALL – QKL3-2000-01537
9 Milestones
 M1: O-mannosylation assay
Title
 M2: Engineered strains in chitin synthesis pathway
Exploiting yeast  M3: In vitro assay of chitin
cell wall for high
 M4: Characterize enzymes/activity of cross-linking and remodelling
throughout
pathway
screening of
M5: Assay for cross-linking/remodelling enzymes
antimicrobial
agents
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)
EUROCELLWALL – QKL3-2000-01537
WP3: Identify new targets from a genomic and proteomic analysis
of the cell wall compensatory mechanism
Mannoproteins (40%)
mnn9
Outer
cell wall
 (1,6) glucans (8%)
kre6
 (1,3) glucans (50%)
fks1
knr4
Chitin (2%)
Plasma
membrane
Inner
cell wall
Glycophospholipid-anchored
surface protein
gas1
By partner 1, 4, 7, 9 (+ 2 and 5)
EUROCELLWALL – QKL3-2000-01537
% of the cell wall
80
WHAT KIND OF GENES ARE INVOLVED IN THE
COMPENSATORY MECHANISM ?
60
Glucose (Glucan)
40
Mannose (Mannoprotein)
Glucosamine (Chitin)
20
0
wt
mnn9
kre6
knr4
wt
fks1
 mutations/agressions cause a cell wall reorganisation
this may even results in a strenghtening of the wall !
EUROCELLWALL – QKL3-2000-01537
(work of partners 1, 7 and 9)
11 cm
cDNA labelled with 33P
PCR product of
yeast ORFs
22 cm
 700 µm
The 6200 Open Reading Frames (ORF) of
Saccharomyces cerevisiae on polypropylene
filter.
EUROCELLWALL – QKL3-2000-01537
Classification and hierarchical
clustering analysis
(work by partners 1 & 9)
up regulated
down regulated
unchanged
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
80 genes
45 genes
Mutants
gas1
mnn9
fks1
knr4
kre6
EUROCELLWALL – QKL3-2000-01537
 What was found from this Bio-informatic analysis?
gene
promotor
-700bp
Cis-factor
-1 ATG
sequences
distribution
function
Rlm1p binding-site
STRE
HSE / HSTF
GCR1
NTAWWWWTAG
CCCCT
GAANNTCC
RGCTTCCWC
54/80
54/80
54/80
48/80
Cell wall
Reponse to stress
Reponse to stress
Carbon Metabolism
PHO4
MATA1
STUAP1/SOK2
NNNCACGTKNGN
TGAGTANNT
NWWCGCGWNM
43/80
52/80
57/80
Pi Metabolism
Morphogenesis
Cell cycle
CAGCCTC
31/80
?
new consensus
EUROCELLWALL – QKL3-2000-01537
 Biological validation of the Bio-informatic analysis?
wt
Promotor analysis of up-regulated genes
independently
msn2

Caffeine
of the
cell wall mutationmsn4
4 mM
pho4
rlm1
Promotor
-700 bp
-1 ATG
CFW
0.025 mg/ml
Sequences
Distribution
Rlm1p
-
STRE
HSE / HSTF
GCR1
PHO4
MATA1
STUAP1
STUAP1/SOK2
NIT2
Potentiel Consensus
wt
msn2
msn4
pho4
rlm1
Gene
Function
NTAWWWWTAG
54/80
CCCCT
54/80
GAANNTCC
SDS
RGCTTCCWC
0.05%
54/80
NNNCACGTKNGN
43/80
TGAGTANNT
52/80
NWWCGCGWNM
57/80
Cell cycle
TATCTM
80/80
Nitrogen metabolism
48/80
Congo Red
0.25 mg/ml
Cell wall
Stress
wt
msn2Stress

msn4Central
 metabolism
metabolism
pho4Phosphate

rlm1Morphogenese

wt
msn2
msn4
pho4
rlm1
 Combinatorial Effect?
EUROCELLWALL – QKL3-2000-01537
Some conclusions & perspectives
Title
Exploiting yeast
cell wall for high
throughout
screening of
antimicrobial
agents
very good partnership
(training and transfert of Know-how)
 on schedule (deliverables and milestones)
be able to find targets (competitiveness)
But
 still huge biochemical works
 not able to go over the first HTS screening
due to budget limitation!
thus
open to wide collaborations