Summary of Study ST003609
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.
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.
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| Study ID | ST003609 |
| Study Title | 13C-tracing of central energy metabolism in thymidine-auxotroph E. Coli |
| Study Summary | 13C-tracing metabolomics of live thymidine-auxotroph Escherichia Coli K12 cultured (1h) or not (0h) in RPMI medium. |
| Institute | University of Colorado Anschutz Medical Campus |
| Last Name | Haines |
| First Name | Julie |
| Address | 12801 E 17th Ave, Room 1303, Aurora, Colorado, 80045, USA |
| julie.haines@cuanschutz.edu | |
| Phone | 3037243339 |
| Submit Date | 2024-11-26 |
| Raw Data Available | Yes |
| Raw Data File Type(s) | raw(Thermo) |
| Analysis Type Detail | LC-MS |
| Release Date | 2024-12-27 |
| Release Version | 1 |
Select appropriate tab below to view additional metadata details:
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: | SU003738 |
| 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 | time (h) | Sample source |
|---|---|---|---|
| SA392765 | E. coli T0_1 | 0 | E coli cells |
| SA392766 | E. coli T0_2 | 0 | E coli cells |
| SA392767 | E. coli T0_3 | 0 | E coli cells |
| SA392768 | E. coli T1_1 | 1 | E coli cells |
| SA392769 | E. coli T1_2 | 1 | E coli cells |
| SA392770 | E. coli T1_3 | 1 | E coli cells |
| Showing results 1 to 6 of 6 |
Collection:
| Collection ID: | CO003731 |
| 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: | TR003747 |
| Treatment Summary: | 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]. For labeling of bacteria, 10 ul of an overnight cultured of thyA- E. coli was added to 20 ml of a filtered M9 minimal medium salts (Life Technologies) supplemented with 1 mM thiamine, 1 mM MgSO4, 0.1 M CaCl2, 500 ug/ml thymidine, 50 ug/ml trimethoprim, and 0.5% U-13C glucose (Campro Scientific). Bacteria were grown for 72h then washed with PBS. Bacteria were placed in a 6-well plate containing complete RPMI medium+10%SVF. Plates were centrifuged for 5 min at 1500 rpm. Then half of the bacteria were collected (T0), washed with PBS and frozen at -80C. The other part was incubated at 37C for 1h, washed with PBS and frozen at -80C (T1). |
Sample Preparation:
| Sampleprep ID: | SP003745 |
| Sampleprep Summary: | Metabolites from frozen pellets were extracted at 2e9 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. 50 uL aliquots of supernatants were diluted 1:1 with cold 5:3:2 methanol:acetonitrile:water. Resulting samples were maintained at 4°C until analysis that same day. |
| Processing Storage Conditions: | 4℃ |
| Extract Storage: | -80℃ |
Combined analysis:
| Analysis ID | AN005931 | AN005932 |
|---|---|---|
| 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: | CH004506 |
| 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: | CH004507 |
| 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: | MS005648 |
| Analysis ID: | AN005931 |
| 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: | MS005649 |
| Analysis ID: | AN005932 |
| 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 |
