Summary of Study ST003608

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 PR002231. The data can be accessed directly via it's Project DOI: 10.21228/M87R79 This work is supported by NIH grant, U2C- DK119886.

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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.

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Study IDST003608
Study TitleGlobal metabolomics to compare metabolic profiles of live vs heat-killed thymidine-auxotrophe E. Coli
Study SummaryGlobal metabolomics of live and heat-killed thymidine-auxotrophe Escherichia Coli K12
Institute
University of Colorado Anschutz Medical Campus
Last NameHaines
First NameJulie
Address12801 E 17th Ave, Room 1303, Aurora, Colorado, 80045, USA
Emailjulie.haines@cuanschutz.edu
Phone3037243339
Submit Date2024-11-26
Raw Data AvailableYes
Raw Data File Type(s)raw(Thermo)
Analysis Type DetailLC-MS
Release Date2024-12-27
Release Version1
Julie Haines Julie Haines
https://dx.doi.org/10.21228/M87R79
ftp://www.metabolomicsworkbench.org/Studies/ application/zip

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Project:

Project ID:PR002231
Project DOI:doi: 10.21228/M87R79
Project Title:Macrophages recycle phagocytosed bacteria to fuel immunometabolic responses
Project Summary:Macrophages specialize in phagocytosis, a cellular process that eliminates extracellular matter, including microbes, through internalization and degradation. Despite the critical role of phagocytosis during bacterial infection, the fate of phagocytosed microbial cargo and its impact on host cell is poorly understood. Here, we reveal that ingested bacteria constitute an alternative nutrient source that skews immunometabolic host responses. Tracing stable isotope-labelled bacteria, we found that phagolysosomal degradation of bacteria provides carbon atoms and amino acids that are recycled into various metabolic pathways, including glutathione and itaconate biosynthesis, and satisfy macrophage bioenergetic needs. Metabolic recycling of microbially-derived nutrients is regulated by the nutrient sensing mTORC1 and intricately tied to microbial viability. Dead bacteria, as opposed to live ones, are enriched in cyclic- adenosine monophosphate (AMP), sustain the cellular AMP pool and subsequently activate AMP protein kinase (AMPK) to inhibit mTORC1. Consequently, killed bacteria strongly fuel metabolic recycling and support macrophage survival, but elicit decreased reactive oxygen species (ROS) production and a reduced IL-1β secretion compared to viable bacteria. These results reveal a novel insight into the fate of engulfed microbes and highlights a microbial viability-associated metabolite that triggers host metabolic and immune responses. Our findings hold promise for shaping immunometabolic intervention in various immune-related pathologies.
Institute:University of Colorado Anschutz Medical Campus
Laboratory:Lab of Angelo D'Alessandro in collaboration with lab of Johan Garaude (INSERM, Fr)
Last Name:Haines
First Name:Julie
Address:12801 E 17th Ave, Room 1303, Aurora, Colorado, 80045, USA
Email:julie.haines@cuanschutz.edu
Phone:3037243339

Subject:

Subject ID:SU003737
Subject Type:Cultured cells
Subject Species:Escherichia coli
Gender:Not applicable

Factors:

Subject type: Cultured cells; Subject species: Escherichia coli (Factor headings shown in green)

mb_sample_id local_sample_id E coli status Sample source
SA392759AA83eC-4_r11-dead E coli cells
SA392760AA83eC-5_r12-dead E coli cells
SA392761AA83eC-6_r6-dead E coli cells
SA392762AA83eC-1_r5-live E coli cells
SA392763AA83eC-2_r8-live E coli cells
SA392764AA83eC-3_r1-live E coli cells
Showing results 1 to 6 of 6

Collection:

Collection ID:CO003730
Collection Summary:1E9 Bacteria were washed with cold PBS, frozen as dry cell pellet, and stored at -80˚C until processing.
Sample Type:Bacterial cells

Treatment:

Treatment ID:TR003746
Treatment Summary:Preparation of viable and killed bacteria: ThyA- E. coli were grown overnight with shaking in LB supplemented with thymidine (500 ug/ml) and trimethoprim (50 ug/ml), diluted 1/40, and grown until log-phase [optical density at 600 nm (OD600) of 0.8-1.2]. Bacteria were washed with PBS and subjected to heat-killing by re-suspension in PBS and subsequently incubation at 60˚C for 60-90 min. Efficient killing was confirmed by overnight plating on LB-agar plates.

Sample Preparation:

Sampleprep ID:SP003744
Sampleprep Summary:Metabolites from frozen pellets were extracted at 1e9 cells per mL using ice cold 5:3:2 methanol:acetonitrile:water (v/v/v) with vigorous vortexing at 4 degrees C followed by centrifugation as described for 10 min at 18,000 g. Supernatants were maintained at 4°C until analysis that same day.
Processing Storage Conditions:4℃
Extract Storage:-80℃

Combined analysis:

Analysis ID AN005929 AN005930
Analysis type MS MS
Chromatography type Reversed phase Reversed phase
Chromatography system Thermo Vanquish Thermo Vanquish
Column Phenomenex Kinetex C18 (150 x 2.1mm,1.7um) Phenomenex Kinetex C18 (150 x 2.1mm,1.7um)
MS Type ESI ESI
MS instrument type Orbitrap Orbitrap
MS instrument name Thermo Orbitrap Exploris 120 Thermo Orbitrap Exploris 120
Ion Mode NEGATIVE POSITIVE
Units peak area peak area

Chromatography:

Chromatography ID:CH004504
Chromatography Summary:Negative C18
Instrument Name:Thermo Vanquish
Column Name:Phenomenex Kinetex C18 (150 x 2.1mm,1.7um)
Column Temperature:45
Flow Gradient:0-0.5 min 0% B, 0.5-1.1 min 0-100% B, 1.1-2.75 min hold at 100% B, 2.75-3 min 100-0% B, 3-5 min hold at 0% B
Flow Rate:450 uL/min
Sample Injection:10 uL
Solvent A:95% water/5% acetonitrile; 1 mM ammonium acetate
Solvent B:95% acetonitrile/5% water; 1 mM ammonium acetate
Chromatography Type:Reversed phase
  
Chromatography ID:CH004505
Chromatography Summary:Positive C18
Instrument Name:Thermo Vanquish
Column Name:Phenomenex Kinetex C18 (150 x 2.1mm,1.7um)
Column Temperature:45
Flow Gradient:0-0.5 min 5% B, 0.5-1.1 min 5-95% B, 1.1-2.75 min hold at 95% B, 2.75-3 min 95-5% B, 3-5 min hold at 5% B
Flow Rate:450 uL/min
Sample Injection:10 uL
Solvent A:100% water; 0.1% formic acid
Solvent B:100% acetonitrile; 0.1% formic acid
Chromatography Type:Reversed phase

MS:

MS ID:MS005646
Analysis ID:AN005929
Instrument Name:Thermo Orbitrap Exploris 120
Instrument Type:Orbitrap
MS Type:ESI
MS Comments:We use a Thermo Orbitrap Exploris 120. Resolution 120,000, scan range 65-975 m/z, maximum injection time 100 ms, microscans 1, automatic gain control (AGC) detection duration 20 msec, source voltage 2.0 kV, capillary temperature 320 C, vaporizer temp 200 C, and sheath gas 50, auxiliary gas 10, and sweep gas 1 (all nitrogen). Data converted to mzXML using RawConverter. Metabolites were annotated and integrated using Maven in conjunction with the KEGG database.
Ion Mode:NEGATIVE
  
MS ID:MS005647
Analysis ID:AN005930
Instrument Name:Thermo Orbitrap Exploris 120
Instrument Type:Orbitrap
MS Type:ESI
MS Comments:We use a Thermo Orbitrap Exploris 120. Resolution 120,000, scan range 65-975 m/z, maximum injection time 100 ms, microscans 1, automatic gain control (AGC) detection duration 20 msec, source voltage 3.5 kV, capillary temperature 320 C, vaporizer temp 200 C, and sheath gas 50, auxiliary gas 10, and sweep gas 1 (all nitrogen). Data converted to mzXML using RawConverter. Metabolites were annotated and integrated using Maven in conjunction with the KEGG database.
Ion Mode:POSITIVE
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