Summary of Study ST004270

This data is available at the NIH Common Fund's National Metabolomics Data Repository (NMDR) website, the Metabolomics Workbench, https://www.metabolomicsworkbench.org, where it has been assigned Project ID PR002696. The data can be accessed directly via it's Project DOI: 10.21228/M85K1P This work is supported by NIH grant, U2C- DK119886.

See: https://www.metabolomicsworkbench.org/about/howtocite.php

This study contains a large results data set and is not available in the mwTab file. It is only available for download via FTP as data file(s) here.

Study ID	ST004270
Study Title	Amino Acid Decarboxylation Preserves Salmonella Fitness During Phagocyte-Derived Oxidative Stress
Study Summary	Successful establishment of infection by non-typhoidal Salmonella depends upon its ability to resist the antimicrobial defenses of the host innate immune response. To withstand the membrane depolarization that potentiates the killing activity of reactive oxygen species (ROS) produced by the phagocyte NADPH oxidase, Salmonella employs metabolic adaptations that maintain intracellular pH homeostasis and membrane energetics. Here, we identify amino acid decarboxylation as a critical determinant of Salmonella virulence and resistance to the oxidative pressures within the host environment. The proton-consuming decarboxylation of L-arginine preserves intracellular ∆pH and enhances Salmonella survival against the bactericidal effects of ROS, while downstream polyamine biosynthesis aids in bacterial recovery following ROS exposure. Polyamines alone cannot substitute for the immediate, protective impact of proton 26 consuming decarboxylation during oxidative stress killing. Specifically, we show that Salmonella relies on the combined activity of the inducible arginine AdiA and ornithine SpeF decarboxylases for resistance to oxidative stress, and that this activity is essential for Salmonella virulence during systemic infection. Together, amino acid decarboxylation and polyamine biosynthesis play complementary, but distinct roles in Salmonella adaptation to phagocyte-derived oxidative stress, providing a new framework for understanding how amino acid catabolism influences bacterial survival in the host
Institute	University of Colorado School of Medicine
Last Name	Stephenson
First Name	Daniel
Address	Research 1 South L18-1303 12801 E. 17th Ave., Aurora, Colorado, 80045, USA
Email	daniel.stephenson@cuanschutz.edu
Phone	303-724-3339
Submit Date	2025-09-24
Raw Data Available	Yes
Raw Data File Type(s)	mzXML, raw(Thermo)
Analysis Type Detail	LC-MS
Release Date	2025-10-31
Release Version	1

Select appropriate tab below to view additional metadata details:

Project:

Project ID:	PR002696
Project DOI:	doi: 10.21228/M85K1P
Project Title:	Amino Acid Decarboxylation Preserves Salmonella Fitness During Phagocyte-Derived Oxidative Stress
Project Summary:	Successful establishment of infection by non-typhoidal Salmonella depends upon its ability to resist the antimicrobial defenses of the host innate immune response. To withstand the membrane depolarization that potentiates the killing activity of reactive oxygen species (ROS) produced by the phagocyte NADPH oxidase, Salmonella employs metabolic adaptations that maintain intracellular pH homeostasis and membrane energetics. Here, we identify amino acid decarboxylation as a critical determinant of Salmonella virulence and resistance to the oxidative pressures within the host environment. The proton-consuming decarboxylation of L-arginine preserves intracellular ∆pH and enhances Salmonella survival against the bactericidal effects of ROS, while downstream polyamine biosynthesis aids in bacterial recovery following ROS exposure. Polyamines alone cannot substitute for the immediate, protective impact of proton 26 consuming decarboxylation during oxidative stress killing. Specifically, we show that Salmonella relies on the combined activity of the inducible arginine AdiA and ornithine SpeF decarboxylases for resistance to oxidative stress, and that this activity is essential for Salmonella virulence during systemic infection. Together, amino acid decarboxylation and polyamine biosynthesis play complementary, but distinct roles in Salmonella adaptation to phagocyte-derived oxidative stress, providing a new framework for understanding how amino acid catabolism influences bacterial survival in the host
Institute:	University of Colorado School of Medicine
Department:	Immunology and Microbiology
Last Name:	Stephenson
First Name:	Daniel
Address:	Research 1 South L18-1303 12801 E. 17th Ave., Aurora, Colorado, 80045, USA
Email:	daniel.stephenson@cuanschutz.edu
Phone:	303-724-3339

Subject:

Subject ID:	SU004423
Subject Type:	Bacteria
Subject Species:	Salmonella enterica
Taxonomy ID:	28901

Factors:

Subject type: Bacteria; Subject species: Salmonella enterica (Factor headings shown in green)

mb_sample_id	local_sample_id	Treatment	Sample source
SA497754	DS2-098-012	MOPS-glucose 400 uM H2O2	Salmonella
SA497755	DS2-098-011	MOPS-glucose 400 uM H2O2	Salmonella
SA497756	DS2-098-013	MOPS-glucose 400 uM H2O2	Salmonella
SA497757	DS2-098-014	MOPS-glucose 400 uM H2O2	Salmonella
SA497758	DS2-098-015	MOPS-glucose 400 uM H2O2	Salmonella
SA497759	DS2-098-001	MOPS-glucose	Salmonella
SA497760	DS2-098-002	MOPS-glucose	Salmonella
SA497761	DS2-098-003	MOPS-glucose	Salmonella
SA497762	DS2-098-004	MOPS-glucose	Salmonella
SA497763	DS2-098-005	MOPS-glucose	Salmonella

Showing results 1 to 10 of 10

Showing results 1 to 10 of 10

Collection:

Collection ID:	CO004416
Collection Summary:	Salmonella grown overnight in LB broth at 37°C were diluted to 5 × 10⁷ CFU/mL in MOPS–glucose minimal medium and incubated aerobically at 37°C for 4 h. Cultures were divided and either treated with or without 400 µM H₂O₂ for 30 min. Cells were harvested by centrifugation at 8,000 × g for 5 min, and pellets were flash-frozen and stored at –80°C prior to extraction.
Sample Type:	Bacterial cells

Treatment:

Treatment ID:	TR004432
Treatment Summary:	Cultures were routinely started from either a small amount of -80°C DMSO stock or a single colony, which was inoculated into the appropriate liquid growth medium and grown overnight (16 to 20 h) aerobically at 37°C with shaking. Where indicated, either LB (Lysogeny broth), EG (Essential salts + glucose) minimal media [1.7 mM MgSO4, 9.5 mM citric acid, 57.4 mM K2HPO4,16.7 mM H5NNaO4P, 0.4% D-Glucose; pH 7.0 unless otherwise noted], or MOPS (Morpholino propanesulfonic acid) minimal media [40 mM MOPS buffer, 4 mM tricine, 0.4% D glucose, 2mM K2HPO4, 10 µM FeSO4·7H2O, 9.5 mM NH4Cl, 276 µM K2SO4, 500 nM CaCl2, 50 mM NaCl, 525 µM MgCl2, 2.9 nM (NH4)6Mo7O24·4H2O, 400 nM H3BO3, 30 nM CoCl2, 9.6 nM CuSO4, 80.8 nM MnCl2, and 9.74 nM ZnSO4; pH 7.2] were used. The initial pH of all media formulations supplemented with amino acids and/or putrescine was measured and confirmed to 25 match that of the unsupplemented base medium (pH 7.0). For H2O2 survival, qRT-PCR, and intracellular pH assays, overnight cultures were grown in EG minimal media supplemented with 80 µg/mL of the appropriate amino acid(s) for the catabolic pathway of interest (i.e. L-arginine for ∆adiA, ∆speA, ∆adiA∆speA, and ∆speB; L-ornithine for ∆speF, ∆speC, and ∆speF∆speC; and L arginine + L-ornithine for ∆adiA∆speF, ∆speA∆speC, and ∆adiA∆speA∆speF∆speC). As appropriate, penicillin, chloramphenicol, or kanamycin was added at final concentrations of 250, 20, or 50 µg/mL, respectively. To determine CFU, bacterial cultures were serially diluted 10-fold in phosphate-buffered saline (PBS) and spread onto LB agar plates followed by growth at 37°C for 16 h. Salmonella grown overnight in LB broth at 37°C were diluted to 5 × 10⁷ CFU/mL in MOPS–glucose minimal medium and incubated aerobically with shaking at 37°C for 4 h. Cultures were then split and either left untreated or 400 µM H2O2 was directly added to the treatment samples. Both untreated and treated samples were then incubated for 30 min at 37°C with shaking. Cells were then harvested by centrifugation at 8,000 × g for 5 min, and pellets were flash frozen and stored at –80°C prior to extraction.

Treatment ID:

TR004432

Treatment Summary:

Cultures were routinely started from either a small amount of -80°C DMSO stock or a single colony, which was inoculated into the appropriate liquid growth medium and grown overnight (16 to 20 h) aerobically at 37°C with shaking. Where indicated, either LB (Lysogeny broth), EG (Essential salts + glucose) minimal media [1.7 mM MgSO4, 9.5 mM citric acid, 57.4 mM K2HPO4,16.7 mM H5NNaO4P, 0.4% D-Glucose; pH 7.0 unless otherwise noted], or MOPS (Morpholino propanesulfonic acid) minimal media [40 mM MOPS buffer, 4 mM tricine, 0.4% D glucose, 2mM K2HPO4, 10 µM FeSO4·7H2O, 9.5 mM NH4Cl, 276 µM K2SO4, 500 nM CaCl2, 50 mM NaCl, 525 µM MgCl2, 2.9 nM (NH4)6Mo7O24·4H2O, 400 nM H3BO3, 30 nM CoCl2, 9.6 nM CuSO4, 80.8 nM MnCl2, and 9.74 nM ZnSO4; pH 7.2] were used. The initial pH of all media formulations supplemented with amino acids and/or putrescine was measured and confirmed to 25 match that of the unsupplemented base medium (pH 7.0). For H2O2 survival, qRT-PCR, and intracellular pH assays, overnight cultures were grown in EG minimal media supplemented with 80 µg/mL of the appropriate amino acid(s) for the catabolic pathway of interest (i.e. L-arginine for ∆adiA, ∆speA, ∆adiA∆speA, and ∆speB; L-ornithine for ∆speF, ∆speC, and ∆speF∆speC; and L arginine + L-ornithine for ∆adiA∆speF, ∆speA∆speC, and ∆adiA∆speA∆speF∆speC). As appropriate, penicillin, chloramphenicol, or kanamycin was added at final concentrations of 250, 20, or 50 µg/mL, respectively. To determine CFU, bacterial cultures were serially diluted 10-fold in phosphate-buffered saline (PBS) and spread onto LB agar plates followed by growth at 37°C for 16 h. Salmonella grown overnight in LB broth at 37°C were diluted to 5 × 10⁷ CFU/mL in MOPS–glucose minimal medium and incubated aerobically with shaking at 37°C for 4 h. Cultures were then split and either left untreated or 400 µM H2O2 was directly added to the treatment samples. Both untreated and treated samples were then incubated for 30 min at 37°C with shaking. Cells were then harvested by centrifugation at 8,000 × g for 5 min, and pellets were flash frozen and stored at –80°C prior to extraction.

Sample Preparation:

Sampleprep ID:	SP004429
Sampleprep Summary:	Metabolomics were extracted via protein crash. Extraction of metabolomics from cells was as follows: Variable amounts of cold MeOH:ACN:H2O (5:3:2, v:v:v) were added to each sample for a final cell concentration of 6.5e8 CFU/mL. Samples were then vortexed at 4 °C for 30 minutes. Following vortexing, samples were centrifuged at 12700 RPM for 10 minutes at 4 °C and supernatant was transferred to a new autosampler vial for analysis. A portion of extract from each sample was also combined to create a technical mixture, injected throughout the run for quality control.

Sampleprep ID:

SP004429

Sampleprep Summary:

Metabolomics were extracted via protein crash. Extraction of metabolomics from cells was as follows: Variable amounts of cold MeOH:ACN:H2O (5:3:2, v:v:v) were added to each sample for a final cell concentration of 6.5e8 CFU/mL. Samples were then vortexed at 4 °C for 30 minutes. Following vortexing, samples were centrifuged at 12700 RPM for 10 minutes at 4 °C and supernatant was transferred to a new autosampler vial for analysis. A portion of extract from each sample was also combined to create a technical mixture, injected throughout the run for quality control.

Chromatography:

Chromatography ID:	CH005399
Chromatography Summary:	Metabolomics Positive
Chromatography Comments:	The times mentioned in the flow gradient are the specific time the gradient changes in the method. The methods were 5 minutes.
Instrument Name:	Thermo Vanquish
Column Name:	Waters ACQUITY UPLC BEH C18 (100 x 2.1mm,1.7um)
Column Temperature:	45
Flow Gradient:	0 min - 0.45 ml/min - 0% B, 0.5 min - 0.45ml/min - 0% B, 1.1 min - 0.45ml/min - 100% B, 2.75 min - 0.45ml/min - 100% B, 3 min - 0.45ml/min - 0% B, 5min - 0.45ml/min - 0%B
Flow Rate:	0.45mL/min
Solvent A:	100% Water; 0.1% Formic Acid
Solvent B:	100% Acetonitrile; 0.1% Formic Acid
Chromatography Type:	Reversed phase

Chromatography ID:	CH005400
Chromatography Summary:	Metabolomics Negative
Chromatography Comments:	The times mentioned in the flow gradient are the specific time the gradient changes in the method. The methods were 5 minutes.
Instrument Name:	Thermo Vanquish
Column Name:	Waters ACQUITY UPLC BEH C18 (100 x 2.1mm,1.7um)
Column Temperature:	45
Flow Gradient:	0 min - 0.45 ml/min - 0% B, 0.5 min - 0.45ml/min - 0% B, 1.1 min - 0.45ml/min - 100% B, 2.75 min - 0.45ml/min - 100% B, 3 min - 0.45ml/min - 0% B, 5min - 0.45ml/min - 0%B
Flow Rate:	0.45mL/min
Solvent A:	100% Water; 10mM Ammonium Aceate
Solvent B:	50% Methanol/50% Acetonitrile; 10mM Ammonium Acetate
Chromatography Type:	Reversed phase

Analysis:

Analysis ID:	AN007107
Analysis Type:	MS
Chromatography ID:	CH005399
Num Factors:	2
Num Metabolites:	45
Units:	Peak area

Analysis ID:	AN007108
Analysis Type:	MS
Chromatography ID:	CH005400
Num Factors:	2
Num Metabolites:	55
Units:	Peak area