Spore Germination and Outgrowth Inhibition. Stanley Brul, Wishwash - PowerPoint PPT Presentation

The Bacterial Spore Proteome; Identifying Targets for Spore Germination and Outgrowth Inhibition. Stanley Brul, Wishwash Abhyankar, Rachna Pandey, Johan van Beilen, Norbert Vischer, Erik Manders, Alex Ter Beek, Leo de Koning and Chris de Koster Van Leeuwenhoek Centre for Advanced Microscopy, Mass Spectrometry of Biomacromolecules & Molecular Biology & Microbial Food Safety Swammerdam Institute for Life Sciences University of Amsterdam IAFP 2016 Athens

Genomes & proteomes are accessible (see Mol. Biol. of the Cell 2015) IAFP 2016 Athens 2

Spore physiology & proteomics GeLC-MS/MS study of spore IM germination receptors & associated targets Study of the effect of growth conditions on spore outer layers Study of the effect of sporulation temperature on the spore coat proteome and stress resistance 3 IAFP 2016 Athens

Bacillus subtilis studies (genomes available) Study of the effect of growth conditions on spore outer layers and stress resistance B. subtilis PY79 Kamphorst et al., 2016; Manuscript under preperation 4 IAFP 2016 Athens

Study aims Spores cultured on solid agar medium possess higher thermal resistance than spores cultured in a liquid medium. How does the sporulation environment affect spore resistance? Comparison of the outer layer protein levels of spores cultures on solid and liquid medium using metabolic 15 N labeling. Kamphorst et al., 2016; Manuscript under preperation 5 IAFP 2016 Athens

Relative quantification approach TS agar plate Growth at 37 ° C till OD 600 ~ 0.3-0.4 14 N spores made on solid 2xSG agar  Growth at 37 ° C till Growth at 37 ° C till OD 600 ~ 0.3-0.4 OD 600 ~ 0.3-0.4 medium plates. 15 N 14 N TSB liquid MOPS 2xSG medium liquid 15 N spores made in 15 NH 4 Cl supplied liquid  medium medium Growth at 37 ° C till MOPS buffered liquid medium. max. dilution reaches OD 600 ~ 0.3-0.4 14 N 15 N MOPS 2xSG liquid Sporulation allowed for 120 hrs. (5 liquid medium  medium Growth at 37 ° C days). till 14 N OD 600 ~ 0.3-0.4 15 N MOPS 2xSG liquid Agar Sporulation was induced by glucose medium  plates starvation and cell crowding. 5 day incubation at 37 ° C 5 day incubation at 37 ° C Mixing 14 N & 15 N spores Mixing based on OD 600 .  (1:1 mixing based on OD 600 ) Spore coat isolation & SDS extraction Reduction, Alkylation & Trypsin digestion LC-FT-ICR-MS/MS analysis & Kamphorst et al., 2016; Manuscript under preperation 14 N/ 15 N ratio calculations IAFP 2016 Athens

Observations of spore crops 14 N spores 15 N spores 14 N 15 N Kamphorst et al., 2016; Manuscript under preperation 7 IAFP 2016 Athens

Inner Coat Outer Coat & Crust Up regulated in liquid medium 14 N 15 N Up regulated in solid medium Kamphorst et al., 2016; Manuscript under preperation 8 IAFP 2016 Athens

Spore morphology Electron micrographs of 14 N & 15 N spores 14 N (solid medium) 15 N (liquid medium) Thickness of Outer coat layer varies significantly in the two spore populations! Kamphorst et al., 2016; Manuscript under preperation Inner coat layers possibly more cross-linked 9 IAFP 2016 Athens

Summary Spores cultured in the liquid medium have significant thicker outer  coat than spores cultured on the solid medium. Spores cultured in the liquid medium have a significant different coat  protein composition compared to the spores cultured on solid medium. The large variation over the replica’s in the 14 N/ 15 N peptide ratios for  certain proteins suggest variations in cross-linking between these coat proteins . Kamphorst et al., 2016; Manuscript under preperation 10 IAFP 2016 Athens

Possible cross-link targets in the coat Inner coat Outer coat Cortex 30% of the coat proteins are resistent to extraction SpoIVA Peptidoglycan Cross-linking of coat proteins? Abhyankar et al., 2015. Food Microbiology IAFP 2016 Athens 11

Spore physiology & Proteomics Study of the effect of sporulation temperature on the spore coat proteome B. weihenstephanensis WSBC10204 Psychrotolerant spore former (genome was unavailable) 12 IAFP 2016 Athens

The newest genome of B. weihenstephanensis IAFP 2016 Athens 13

Method Tryptic Soy Broth liquid medium Overnight, 30 ° C Minimal Sporulation medium 12 ° C 30 ° C 10 days 5 days Spore harvest Spore coat isolation & protein extraction Reduction, Alkylation & Trypsin digestion LC-FT-ICR-MS/MS Stelder et al., 2016; Manuscript under review analysis 14 IAFP 2016 Athens

Temperature dependence of identified proteins Stelder et al., 2016; Manuscript under review Non-spore associated proteins Proteins with known Proteins with spore association 14 18 9 unknown function 8 27 10 3 17 3 12 ° C 30 ° C 15 IAFP 2016 Athens

B. Weihenstephanensis data indicate arginase as 12ºC target 12 ºC 16 IAFP 2016 Athens

Spores physiology & Proteomics GeLC-MS/MS study of spore IM germination receptors & associated targets B. subtilis PY79 Zheng et al., 2016 17 IAFP 2016 Athens

Method Proteomic analysis of the purified B. subtilis spore inner membrane Zheng et al., 2016 18 IAFP 2016 Athens

IM protein identification Zheng et al., 2016 19 IAFP 2016 Athens

Some specific proteins identified:  Enzymes involved in Coenzyme A synthesis viz. PanB, PanC, PanE, CoaBC, CoaE and IlvD.  CoA reported to be di-sulfide cross-linked to proteins in B. megaterium spores Setlow & Setlow, 1977  Role of CoA as a modulator of metabolism in germinating spores is worth studying.  SpeA & SpeE involved in biosynthesis of Spermidine (polyamine) were identified.  Spermidine produced in germinating spores of B. megaterium . What is its role there? Taken from Hobley et al. 2012; established by Setlow, 1974) 20 IAFP 2016 Athens

HTrC & YpeB are found in the inner membrane and may be targets to interfere with the basis of germination triggering SleB and thus cortex and coat lysis (Zheng et al. 2016, Journal Proteome Res.; see also Bernhards et al. J. Bact. 2015; Meany et al. Anaerobe 2015 ) Ca 2+ DPA HtcR YpeB CORTEX ? ? SleB Coat CwlJ (hydrolases) Germinants IAFP 2016 Athens 21

Swarge et al., Manuscript under preparation Further developments 1: ‘ One pot’ sample processing method Spores in AmBiC buffer for time resolved spore proteomics Bead beating + Urea+ DTT Disrupted  Recent developments in the studies of sporulation spores and germination processes have brought forward Addition of Acetonitrile Alkylation with the need of comprehensive time lapse spore Iodoacetamide proteome analyses. LysC Double digestion of proteins Trypsin  In order to enable monitoring of protein levels during sporulation and germination we have developed a ‘ one pot ’ spore processing method for mass spectrometric analysis of proteins from all ZIC-HILIC peptide spore layers. The method is applicable to Bacillus pre-fractionation subtilis , B. cereus and Clostridium difficile . FT-ICR-MS/MS 22 IAFP 2016 Athens

Further developments 2:  Nowadays using 'omics' tools molecular mechanistic data can be gathered to answer questions pertaining to predictive molecular modelling of microbial behavior.  Accurate single cell germination and outgrowth data is needed for building accurate models (use of reporter proteins)! Papers Single spore & cell analysis using Spore-Tracker Pandey et al., 2013, 2015 & Manuscript under review New: Cooperativity of SpoVA channel gating introduces spore memory for germination stimuli 23 IAFP 2016 Athens

Spore-tracker allows single spore data analysis (Pandey et al. 2013, 2015) IAFP 2016 Athens 24

Observation of germination and outgrowth Heterogenity of wt and sorbic acid stressed B.subtilis spores Control 3 mM Potassium Sorbate IAFP 2016 Athens 25

pHi measurements in single bacteria with improved (I)pHluorin possible Three “pH probes ” using three promoters at the amyE locus to drive IpHluorin:  P ptsG → vegetative cell specific (growth on glucose)  P spoIIA → mothercell specific  P sspE → forespore specific Van Beilen & Brul (2013) P ptsG -IpHluorin P spoIIA -IpHluorin P sspE -IpHluorin IAFP 2016 Athens 26

Lineage tracing allows identification of single cell heterogeneity in microcolonies under acid stress Phase contrast 390 nm 470 nm IAFP 2016 Athens 27

Conclusions  Live-imaging of intracellular pH using IpHluorin in Bacillus subtilis is possible in vegetative cells (and now germinating / outgrowing spores).  Heterogeneity in stress response to potassium acetate and sorbate (food preservatives) exists at the microcolony level.  Lineage tracing shows similar heterogeneity in stress response in individual cells of micro-colonies upon (acetate) acid stress. IAFP 2016 Athens 28

Further developments 2:  Nowadays using 'omics' tools molecular mechanistic data can be gathered to answer questions pertaining to predictive molecular modelling of microbial behavior.  Accurate single cell germination and outgrowth data is needed for building accurate models (use of reporter proteins)! New: Cooperativity of SpoVA channel gating introduces spore memory for germination stimuli 29 IAFP 2016 Athens