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Research program: Systems Biology of the Living Cell 1

Research program: Systems Biology of the Living Cell 1 Subprogram: Molecular Biology & Microbial Food Safety. Molecular Biology and Microbial Food Safety theme: Stress response in bio(medical) systems research approach Systems Biology. Drug induced mitochondrial dysfunction ( C. elegans ).

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Research program: Systems Biology of the Living Cell 1

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  1. Research program: Systems Biology of the Living Cell 1 Subprogram: Molecular Biology & Microbial Food Safety

  2. Molecular Biology and Microbial Food Safetytheme: Stress response in bio(medical) systemsresearch approach Systems Biology Drug induced mitochondrial dysfunction (C. elegans) Environmental stress induced response on physiology (S. cerevisiae and C. albicans) Quantitative systems analysis of stress induced biochemical and physiological cellular responses (bioenergetics of temperature, pH & antimicrobial stress) Microbial stress: sporulation and antibiotic resistance. Swammerdam Institute for Life Sciences

  3. The role of mitochondria in temperature stress:Dynamic control of yeast respiratory efficiency 30oC 37oC 38oC 38oC→30oC Why? To prevent irreversible damage? ROS? Swammerdam Institute for Life Sciences Postmus et al., Microbiology 2011

  4. 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) Swammerdam Institute for Life Sciences Budovskaya et al., 2008. Cell

  5. 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) “Omics” approaches to understand adaptation to pH, temperature and antimicrobialstress (and to prevent it): Frans Klis, Gertien Smits Stanley Brul, Benno ter Kuile with collaborations: De Koster, Manders, Rep. RUMC, VWA, AMC Microbial stress: sporulation and antibiotic resistance. Swammerdam Institute for Life Sciences

  6. Molecular Biology and Microbial Food Safetytheme: Stress response in biomedical systemsthe role of the intracellular pH Orij et al., BBA 2011

  7. Regulation of yeast lipid biosynthesis by pHcyt Young,….Orij…..Smits and Loewen. 2010. Science (329) Growth No Growth Young et al.,Science 2010

  8. 0.2 0.16 0.12 fraction 0.08 wild type wild type 0.04 mutants mutants 0 6.9 6.9 7.0 7.0 7.1 7.1 7.2 7.2 7.3 7.3 7.4 7.4 6.8 6.8 6.8 7 7.2 7.4 pH cyt Table 1 Screen statistics Table 1 Screen statistics Mutants Mutants % Identified % Identified Primary screen Primary screen Rescreening Rescreening Total found Total found pH pH low low 57 57 84 84 19 19 76 76 cyt cyt pHc pH pH high high 8 8 99 99 107 107 93 93 cyt cyt proton proton - - transporting two transporting two - - sector sector ** ATPase complex, catalytic domain ATPase complex, catalytic domain proton proton - - transporting transporting * V V - - type ATPase complex type ATPase complex ** mitochondrial large ribosomal subunit mitochondrial large ribosomal subunit nucleoplasm nucleoplasm *** mitochondrial inner membrane mitochondrial inner membrane * aminoacyl aminoacyl - - tRNA ligase activity tRNA ligase activity * hydrogen ion transmembrane hydrogen ion transmembrane ** transporter activity transporter activity ** structural constituent of ribosome structural constituent of ribosome * tRNA aminoacylation tRNA aminoacylation * regulation of intracellular pH regulation of intracellular pH component component ln (OD600 cor) *** function function mitochondrion organization mitochondrion organization process process cellular nitrogen compound cellular nitrogen compound * metabolic process metabolic process 0 5 10 15 fold enrichment fold enrichment growth rate pHc pH 5.0 pH 4.5 pH 4.0 pH 3.5 pH 3.0 pH 2.5 pH 2.0 Intracellular pH: a second messenger for cellular decision making? t (hours) Swammerdam Institute for Life Sciences Orij et al., submitted

  9. Intracellular pH: a second messenger for cellular decision making? Swammerdam Institute for Life Sciences Orij et al., submitted

  10. The role of the wall proteome of Candida albicans in mucosal and systemic infections Before washing After washing pH 7 invasive pH 7 pH 4 pH 7 pH 4 pH 4 Sosinska et al. Microbiology 2010; Sorgo et al. Euk. Cell 2011; Klis et al. Future Microbiology, 2011

  11. 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) Swammerdam Institute for Life Sciences Heilmann et al., Microbiology 2011

  12. 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

  13. Weak Organic Acid Stress: Membrane & Intracellular pH Johan van Beilen (PhDst) In vivo pH meter: pHulorin Elucidating the function of RodZ in weak organic acid resistance in B. subtilis. Genetic Metabolic Diseases (AMC): Prof. Ron Wanders Ter Beek and Brul, Curr. Opin Biotech. 2010

  14. Problem of spore germination & outgrowth: Heterogeneity! Time-resolved single cell analysis Single-cell live imaging of heterogeneous germination and outgrowth of Bacillus subtilis (cells &) spores Germination Heat treatment Time-resolved single spore germination & outgrowth analysis Centre for Advanced Microscopy: Dr. Erik Manders Rachna Pandey (ERASMUS MUNDUS PhDst)

  15. Reduction & alkylation of disulfide bridges Spore coat isolation & SDS extraction 96 hours Trypsin digestion Harvesting of spores Desalting TSB MOPS MOPS MOPS dilutions pre-culture Sporulation medium medium LC-ESI-MS/MS MASCOT analysis The spore coat proteome of Bacilli Wishwas Abhyankar (ERASMUS MUNDUS, PhDst) Mass Spectrometry of Biomacromolecules: Prof. Chris de Koster Abhyankar et al., Proteomics 2011

  16. Time (minutes) 0 5 10 15 20 25 30 40 50 60 70 80 90 100 110 120 130 Targeted Inhibition of Bacterial Spores (TIBS) Goal: To find targets that prevent spore germination & outgrowth under conditions of (mild) preservation Alex Ter Beek (PD) • 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 Microarray Department: Dr. Timo Breit Ter Beek et al., Food Microbiology 2011

  17. 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 Swammerdam Institute for Life Sciences

  18. Molecular Biology and Microbial Food Safety;research focus of our group members Systems Biology of Stress & Ageing Gertien 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 yeasts Gertien Smits (staff), Frans Klis (staff), Stanley Brul (staff), Alice Sorgo (PhD), Clemens Heilmann (PhD), Azmat Ullah (PhD) 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). General Support Marian de Jong and Muus de Haan (both ~1 day a week. Rest teaching & SILS) Swammerdam Institute for Life Sciences

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