MSc Thesis defense
Descrição do Produto
Modulators of the microbiota (antibiotics, probiotics, etc.)
Genetic background, immune system, hormones
Diet, nutritional state, Stress
What is the role of formate in the adaptation response of Pseudomonas fluorescens to combatting oxidative stress?
Can formate be utilized in lieu of NADH/NADPH?
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Control H2O2
Control H2O2
What is the role of formate in the adaptation response of Pseudomonas fluorescens to combatting oxidative stress?
Can formate be utilized in lieu of NADH/NADPH?
Microbiota
Host
Environmental factors
Formate
NAD+ + 2H+
NADH + H+
2
-0.32
NADP+ + 2H+
NADPH + H+
2
-0.32
CO2 + H+
HCOO-
2
-0.42
½ reaction
½ reaction
# electrons
E'0(V)
Can formate replace NADH/NADPH
Application
Formate metabolism is unique to bacteria, so formate producing enzymes could be targeted for pharmaceutical therapy in pathogenic bacteria (labelled experiment).
Special Thanks
Questions?
Draco dormeins ninquam titilandus
METHODS
Glycine metabolism…
Citrate + Glycine media
Citrate + Glycine media + H2O2
Control
Stress
Spectrophotometric
assays
Blue Native Polyacrylamide Gel electrophoresis
(BNPAGE)
High performance liquid chromatography
(HPLC)
Alhasawi, et al., (2015). Microbiological Research 171: 26-31
Monitoring growth
Na2HPO4 (6g), KH2PO4 (3g), 15 mM glycine (1.2g), MgSO4.7H2O (0.2g), and 19 mM citrate (4g) with a pH of 6.8 + trace elements
Na2HPO4 (6g), KH2PO4 (3g), 15 mM glycine (1.2g), MgSO4.7H2O (0.2g), and 19 mM citrate (4g) with a pH of 6.8 + trace elements + H2O2
Harvesting cells
Culture
Spent fluid
(set aside)
Pellets
(cells)
Pellets
(cells)
Pellets
(cells)
Supernatant
(discarded)
Supernatant
(discarded)
Centrifuge 10,000 x g, 15 min
Resuspended in 0.85%NaOH
Centrifuge 10,000 x g, 15 min
Resuspended in 0.85%NaOH
Centrifuge 10,000 x g, 15 min
Regulation experiments
Control culture
Control Cell pellet
3 X Centrifuge 10,000 x g, 15 min
With NaOH resuspensions
H2O2 culture
H2O2 Cell pellet
Divide aliquots
Isolate and prep Control cell pellet
Control cell pellet in H2O2
Grown for 8 hours
Isolate and prepare regulation Control H2O2 cell pellet
Isolate and prep H2O2 cell pellet
H2O2 Cell pellet in control media
Isolate and prepare regulation H2O2 Control cell pellet
Divide aliquots
Future directions
Confirm utility of formate in cell survival.
Monitor metabolism under:
Different carbon sources
Different stress
Conclusion
Formate assists with the antioxidative defense mechanism:
Increases NADPH
Increased NADH
Used as reducing factor in lieu of NADH (FRD)
Research question
Research question
Experiments
Cultures
Cells
Spent media
Metabolomic analysis
(HPLCs, etc.)
cCFE
sCFE
Metabolic flux
Functional proteomics
Metabolomic analysis
(HPLCs, etc.)
Bacterial cell growth & Formate production
Control
500μM H2O2
Formate production in Cfe
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Whole cell experiment
Control
500μM H2O2
Enzymes of central metabolism
Isocitrate lyase
NADH Oxidase
Complex I
Ctrl H2O2
Ctrl H2O2
Ctrl H2O2
Spectrophotometric assay
Ctrl H2O2
NAD-dependent formate dehydrogenase
Ctrl H2O2 H2O2 ctrl
Regulation experiments (FDH-NAD)
133 kDa
66 kDa
Ctrl H2O2
43463.96 ± 1785.24
75398.06 ± 2213.024
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FDH-NAD
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NADP-Dependent formate dehydrogenase
Ctrl H2O2 H2O2 ctrl
Regulation experiments
(FDH-NADP)
Ctrl H2O2
Inhibition with azide
(FDH-NADP)
Ctrl H2O2
FDH-NADP
Formate-dependent fumarate reductase
Ctrl H2O2 Ctrl H2O2 H2O2 ctrl
Formate-dependent FRD
Regulation experiments - FRD
NADH-dependent FRD
Ctrl H2O2
Control
500μM H2O2
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HPLC Reaction
Isolating CFE
Isolated Bacteria
(In cell storage buffer)
Sonicate
(4 x 15s)
Cell free extract
Supernatant
(crude soluble fraction)
Pellets
(Membrane fraction)
Centrifuge 180,000 x g, 180 min
Resuspend in CSB
Supernatant
(Soluble fraction)
Pellets
(Discard debris)
Centrifuge 180,000 x g, 120 min
Centrifuge 10,000 x g, 20 min, remove unbroken cells
Pseudomonas fluorescens ATCC13525
Gram-negative bacteria
Extremely versatile metabolism
Metabolic adaptation strategies
Auger et al., (2014). Biochim Biophys Acta. 1850:43–50
Beriault et al. (2005). Electrophoresis. 26:2892–7.
Bignucolo A, et al., (2013). J Biotechnol. 167:309–15.
Lemire et al., (2014). J Neurosci Res. 92: 464–75.
+ ROS
α- ketoglutarate
Succinate
+ CO2
NADH
NADPH
TCA
ETC
C. deficile infection
Homeostasis
Eloe-Fadrosh & Rasko, (2013). Annu Rev Med. 64: 145-163
Gerritsen, et al., (2011). Genes Nutr. 6(3): 209-240
Xu, et al., (2007). PLoS Biol. 5(7): e156
C
C
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C
C
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"You are not "YOU", alone…"
Nature doi:10.1038/nature.2016.19136
Your body has more microbes than human cells
Bacteria can be both good and bad for you
Alhasawi, et al., (2015). Microbiological Research 171: 26-31
Citrate
Isocitrate
Glyoxylate
Glycine
Formate
Oxalyl-CoA
H2O2
H2O + CO2
ICL
GTA
GDH
AGODH
Glycine metabolism
The role of formate in combatting oxidative stress
Sean C. Thomas
Supervisor: Dr. Omri
Defence Mechanisms
Chemical barriers
Eg. Tears
Mechanical Barriers
Eg. Skin
Reflexes
Eg. Sneeze
Inflammation
Fever
Host immunity.
Kills microbes via:
Phagocytosis (oxidative burst)
Cell mediated immunity
Cytokines
Defense mechanisms
Outline
1. Introduction
2. Thesis objectives
3. Results & Discussion
4. Conclusion & Future works
Sources of ROS
ROS
Fe2+ + H2O2 Fe3+ + OH- + OH.
Fe3+ + O2.- Fe2+ + O2
Phagosome
Excited organic molecules
Mailloux, et al., (2007).PlosOne 2(8): e690
Normal metabolism
HPLCs
The oxidative burst
Bogdan, (2007). Nature immunology 8: 1029-1031
Important role in the immune system
Mechanism of particle/bacterial degradation
ROS detoxification
O2-
H2O2
2H2O
H2O + O2
2GSH
GSSG
NADPH
NADP+
SOD
Catalase
GPx
Glutathione
Reductase
Metabolic shifts?
ROS
Mailloux, et al., (2007).PlosOne 2(8): e690
BNPAGE + confirmation (HPLC)
Han, et al., (2012). Anal Bioanal Chem. 405 (6):1821-31
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Need to have a general activity picture. Would be awesome if I could animate that. Good idea!!!! Show bnpage machine and add gel making pic.
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All experiments
Then regulation experiments
Then BNPAGE and HPLCs
Then
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Methods
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In one study using glycine metabolism we designed experiments to determine how the bacteria modifies it's metabolism
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From excised band in reaction mixture
% increase of succinate
2016-03-20
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% increase in metabolite level
% increase in Formate
Formate in CFE
Arbitrary units (AUC)
Time (hrs)
[protein (mg ml-1 )]
Arbitrary Units (AUC)
From excised band in reaction mixture
% increase in formate
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