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Research program: Systems Biology of the Living Cell 1Subprogram: Molecular Biology & Microbial Food Safety
2
Swammerdam Institute for Life Sciences
Molecular Biology and Microbial Food Safetytheme: Stress response in bio(medical) systems
research approach Systems Biology
Quantitative systems analysis of stress induced biochemical and physiological cellular responses (bioenergetics of temperature, pH & antimicrobial stress)
Drug induced mitochondrial dysfunction (C. elegans)
Environmental stress induced response on physiology (S. cerevisiae and C. albicans)
Microbial stress: sporulation and antibiotic resistance.
3Swammerdam Institute for Life Sciences
The role of mitochondria in temperature stress:Dynamic control of yeast respiratory efficiency
Why? To prevent irreversible damage? ROS?
30oC 37oC
38oC 38oC→30oC
Postmus et al., Microbiology 2011
4Swammerdam Institute for Life Sciences
Systems biology answering the why
Aim:
Systems biology of ROS and ageing: Gertien Smits, Hans v/d Spek
Integrated approach C. elegans & S. cerevisiae
Integration of quantitative biochemistry, physiology, molecular biology and cell biology in quantitative, time resolved models of
stress and age induced ROS production, ROS damage induction, and cellular responses
Collaborators: Chris de Koster, Natal van Riel, Takenori Yamamoto, Ron Wanders, Peter Reiss, Frank Baas
Ageing: developmental drift or ROS induced damage
New researcher in the group: McGillarvy fellow Yelena Budovskaya (+PhD)
Budovskaya et al., 2008. Cell
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Swammerdam Institute for Life Sciences
Molecular Biology and Microbial Food Safetytheme: Stress response in bio(medical) systems
research approach Systems Biology
Drug induced mitochondrial dysfunction (C. elegans)
Environmental stress induced response on physiology (S. cerevisiae and C. albicans)
Microbial stress: sporulation and antibiotic resistance.
“Omics” approaches to understand adaptation to pH, temperature and antimicrobial stress (and to prevent it):Frans Klis, Gertien SmitsStanley Brul, Benno ter Kuile with collaborations:De Koster, Manders, Rep.RUMC, VWA, AMC
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Molecular Biology and Microbial Food Safetytheme: Stress response in biomedical systems
the role of the intracellular pH
nucleus7.0 vacuole
5.6
ATPADP
ATPADP
Vma1
ATPADP
Vma1
ATPADP
late endosome
early endosome
peroxisomes?
mitochondrialmatrix7.2-5
mitochondrialintermembrane
space?
endoplasmicreticulum
7.0
Golginetwork
secretoryvesicle
5.2 H+
H+
H+
H+
H+
Na+/K+
K+
H+
Kha1
Vnx1
Na+/K+
H+
Nha1Nha1
Vma1
Pma1
K+
Trk1,2Trk1,2
cytosol7.0
Na+/K+
H+
Nhx1
Vma1
ATPADP
Vma1
ATPADP
6.7
6.0
nucleus7.0
ADP
ATP
H+
ETCH+
H+
F1FOATPase
H+
Pi
ATP
ADP
PiC
AAC
Orij et al., BBA 2011
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Regulation of yeast lipid biosynthesis by pHcyt
Young,….Orij…..Smits and Loewen. 2010. Science (329)
Growth No Growth
Young et al.,Science 2010
8Swammerdam Institute for Life Sciences
Intracellular pH: a second messenger for cellular decision making?
t (hours)
0
0.1
0.2
0.3
0.4
0.5
4.5 5 5.5 6 6.5 7 7.5
-1.5
-1
-0.5
0
0.5
1
0 2 4 6 8 10 12 14 16
6
6.2
6.4
6.6
6.8
7
7.2
0 2 4 6 8 10 12 14 16
pHc
gro
wth
rat
ep
Hc
ln (
OD
600
cor)
pH 5.0
pH 4.5
pH 4.0
pH 3.5
pH 3.0
pH 2.5
pH 2.0
0
0.04
0.08
0.12
0.16
0.2
6.8 7 7.2 7.4pHcyt
6.8 6.9 7.0 7.1 7.2 7.3 7.46.8 6.9 7.0 7.1 7.2 7.3 7.4
fra
ctio
n
wild type
mutants
wild type
mutants
Mutants Primary screen Rescreening Total found % Identified
Table 1 Screen statistics
pHcyt low
pHcyt high
19
8
57
99
76
107
84
93
Mutants Primary screen Rescreening Total found % Identified
Table 1 Screen statistics
pHcyt low
pHcyt high
19
8
57
99
76
107
84
93
0 5 10 15
*
***
***
*
*
*
**
**
*
**
*
cellular nitrogen compound metabolic process
cellular nitrogen compound metabolic process
mitochondrion organizationmitochondrion organization
regulation of intracellular pHregulation of intracellular pH
tRNA aminoacylationtRNA aminoacylation
structural constituent of ribosomestructural constituent of ribosome
hydrogen ion transmembrane transporter activity
hydrogen ion transmembrane transporter activity
aminoacyl-tRNA ligase activityaminoacyl-tRNA ligase activity
mitochondrial inner membranemitochondrial inner membrane
nucleoplasmnucleoplasm
mitochondrial large ribosomal subunitmitochondrial large ribosomal subunit
proton-transportingV-type ATPase complex
proton-transportingV-type ATPase complex
proton-transporting two-sector ATPase complex, catalytic domain
proton-transporting two-sector ATPase complex, catalytic domain
**
component
function
process
component
function
process
fold enrichmentfold enrichment
Orij et al., submitted
9Swammerdam Institute for Life Sciences
Intracellular pH: a second messenger for cellular decision making?
Orij et al., submitted
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The role of the wall proteome of Candida albicans in mucosal and systemic infections
1 cm
pH 4
pH 7 invasive
Before washing After washing
pH 7
pH 4
pH 7
pH 4
Sosinska et al. Microbiology 2010; Sorgo et al. Euk. Cell 2011; Klis et al. Future Microbiology, 2011
11Swammerdam Institute for Life Sciences
-6.00
-4.00
-2.00
0.00
2.00
4.00
6.00
8.00
10.00
So
d5
Ph
r1
Hy
r1
Als
3
Als
1Ih
d1/
Pg
a36
Rb
t5C
rh1
1
So
d4
Als
5
Ch
t2
Ssr
1
To
s1M
p65
/Scw
1Y
wp
1/P
ga
24
Pg
a4
Utr
2
Pir
1E
cm33
Rh
d3
/Pg
a29
Als
4
Ph
r2
Lo
g2
rati
os
Quantitation of the adaptations in the wall proteome of Candida albicans in biomats grown at pH 7 and 4
Goals● Determine wall protein levels during infection-related stress conditions using MS-based quantitation (FT-MS) ● Identify wall protein-based vaccine targets and potential diagnostic proteins
Collaborations● Chris de Koster (MS), Eric Manders, Martijn Rep (Fusarium oxysporum) ● Mihai Netea, Univ Nijmegen (vaccine targets, diagnostics)
Heilmann et al., Microbiology 2011
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Bacterial spore formers are of prime concern to microbial food stability:
• Spores from the genus Bacillus are extremely stress resistant (heat!)
• This allows surviving spores to germinate and grow causing food spoilage
Brul et al., Food Microbiology 2011
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Johan van Beilen (PhDst)
Genetic Metabolic Diseases (AMC):
Prof. Ron Wanders
Weak Organic Acid Stress: Membrane & Intracellular pH
Elucidating the function of RodZ in weak organic acid resistance in B. subtilis.
In vivo pH meter: pHulorin
Ter Beek and Brul, Curr. Opin Biotech. 2010
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Heat treatment
Problem of spore germination & outgrowth: Heterogeneity!
Single-cell live imaging of heterogeneous germination and outgrowth of Bacillus subtilis (cells &) spores
Rachna Pandey (ERASMUS MUNDUS PhDst)
Centre for Advanced Microscopy:
Dr. Erik Manders
Germination
1
2
3 4
5 67
89
10 11
12
1
2
3 4
56
7
89
10 11
12
Time-resolved single cell analysis
Time-resolved single spore germination & outgrowth analysis
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The spore coat proteome of Bacilli
Wishwas Abhyankar
(ERASMUS MUNDUS, PhDst)
Mass Spectrometry of Biomacromolecules: Prof. Chris de Koster
TSB MOPS MOPS MOPS dilutions pre-culture Sporulation medium medium
96 hours Spore coat isolation
& SDS extraction
Trypsin digestion Desalting
LC-ESI-MS/MS
MASCOT analysis
Harvesting of spores
Reduction &
alkylationof disulfide
bridges
Abhyankar et al., Proteomics 2011
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Targeted Inhibition of Bacterial Spores (TIBS)
Microarray Department:Dr. Timo Breit
Alex Ter Beek (PD)
Goal: To find targets that prevent spore germination & outgrowth under conditions
of (mild) preservation
50 10 15 20 25 30 5040 60 70 80 90 100110120130 Time (minutes)
We need to understand the mechanistic basis of spore germination & outgrowth (in control & stress conditions)
heat weak organic acids (sorbic-, lactic acid)
Expression studies of B. subtilis spore outgrowth
under (mild) stress conditions
Ter Beek et al., Food Microbiology 2011
17Swammerdam Institute for Life Sciences
Adaptations to pH & antimicrobial stress; challenges & collaborations within SILS
Goals Biomedical yeasts● Determine Candida albicans wall protein levels during infection-related stress conditions (important pH perturbation) using MS-based quantitation (FT-MS) ● Identify wall protein-based vaccine targets and potential diagnostic proteins
Collaborations (apart from yeast groups UvA, VU, FINSysB EU)● Chris de Koster (MS), Eric Manders, Martijn Rep (Fusarium oxysporum) ● Mihai Netea, Radboud Univ. Med. Centre (vaccine targets, diagnostics)
Goals bacterial (food)spoilage organisms ● Determine the role of the intracellular pH in regulating cell growth ● Identify targets to interfere with spore germination & growth of Bacilli● Identify the genetic basis (mutations) underlying stress adaptation
Collaborations (apart from the European Spore Conference core)● Chris de Koster (MS), Eric Manders, (Live Imaging), AMC (genome seq.)● TNO (screening antimicrobial compounds), FES partners for spore purification
18Swammerdam Institute for Life Sciences
Molecular Biology and Microbial Food Safety;research focus of our group members
Food spoilage bacteria
Stanley Brul (staff), Benno Ter Kuile (VWA & staff), Jan Smelt (seior scientist) Merijn Schuurmans (post-doc*), Nadine Händel (PhD VWA), Alex Ter Beek (post-doc), Johan van Beilen (PhD), Wishwas Abhyankar (PhD), Rachna Pandey (PhD), NN4 (PhD FES).
Systems Biology of Stress & AgeingGertien Smits (staff), Hans van der Spek (staff), Yelena Budovskaya (staff McGillarvy), R. de Boer (PhD), Rueben Smith (PhD), Marco Lezzerini (PhD McGillarvy), Belinda Koenders (technical support 0,5), Marcel Scholte (supp. 0,4)
Medical yeastsGertien Smits (staff), Frans Klis (staff), Stanley Brul (staff), Alice Sorgo (PhD), Clemens Heilmann (PhD), Azmat Ullah (PhD)
General Support Marian de Jong and Muus de Haan (both ~1 day a week. Rest teaching & SILS)
