Summary of Study ST002433
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 PR001566. The data can be accessed directly via it's Project DOI: 10.21228/M88M5G 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 | ST002433 |
| Study Title | Elucidating dynamic anaerobe metabolism with HRMAS 13C NMR and genome-scale modeling |
| Study Summary | Anaerobic microbial metabolism drives critical functions within global ecosystems, host-microbiota interactions, and industrial applications, yet remains ill-defined. Here we advance a versatile approach to elaborate cellular metabolism in obligate anaerobes using the pathogen Clostridioides difficile, an amino acid and carbohydrate-fermenting Clostridia. High-Resolution Magic Angle Spinning (HRMAS) Nuclear Magnetic Resonance (NMR) spectroscopy of C. difficile, grown with fermentable 13C substrates, informed dynamic flux balance analysis (dFBA) of the pathogen’s genome-scale metabolism. Analyses identified dynamic recruitment of oxidative and supporting reductive pathways, with integration of high-flux amino acid and glycolytic metabolism at alanine’s biosynthesis to support efficient energy generation, nitrogen handling, and biomass generation. Model predictions informed an approach leveraging the sensitivity of 13C NMR spectroscopy to simultaneously track cellular carbon and nitrogen flow from [U-13C]glucose and [15N]leucine, confirming the formation of [13C,15N]alanine. Findings identify metabolic strategies used by C. difficile to support its rapid colonization and expansion in gut ecosystems. |
| Institute | Brigham and Women's Hospital |
| Department | Pathology |
| Laboratory | Bry Lab, Massachusetts Host-Microbiome Center; Cheng Lab, Massachusetts General Hospital |
| Last Name | Pavao |
| First Name | Aidan |
| Address | 221 Longwood Ave, EBRC-411, Boston, MA, 02115, USA |
| apavao2@bwh.harvard.edu | |
| Phone | 617-525-7184 |
| Submit Date | 2023-01-05 |
| Num Groups | 8 |
| Total Subjects | 18 |
| Raw Data Available | Yes |
| Raw Data File Type(s) | fid |
| Analysis Type Detail | NMR |
| Release Date | 2023-01-20 |
| Release Version | 1 |
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Project:
| Project ID: | PR001566 |
| Project DOI: | doi: 10.21228/M88M5G |
| Project Title: | Elucidating dynamic anaerobe metabolism with HRMAS 13C NMR and genome-scale modeling |
| Project Summary: | Anaerobic microbial metabolism drives critical functions within global ecosystems, host-microbiota interactions, and industrial applications, yet remains ill-defined. Here we advance a versatile approach to elaborate cellular metabolism in obligate anaerobes using the pathogen Clostridioides difficile, an amino acid and carbohydrate-fermenting Clostridia. High-Resolution Magic Angle Spinning (HRMAS) Nuclear Magnetic Resonance (NMR) spectroscopy of C. difficile, grown with fermentable 13C substrates, informed dynamic flux balance analysis (dFBA) of the pathogen’s genome-scale metabolism. Analyses identified dynamic recruitment of oxidative and supporting reductive pathways, with integration of high-flux amino acid and glycolytic metabolism at alanine’s biosynthesis to support efficient energy generation, nitrogen handling, and biomass generation. Model predictions informed an approach leveraging the sensitivity of 13C NMR spectroscopy to simultaneously track cellular carbon and nitrogen flow from [U-13C]glucose and [15N]leucine, confirming the formation of [13C,15N]alanine. Findings identify metabolic strategies used by C. difficile to support its rapid colonization and expansion in gut ecosystems. |
| Institute: | Brigham and Women's Hospital |
| Department: | Pathology |
| Laboratory: | Bry Lab (Massachusetts Host-Microbiome Center, BWH) and Cheng Lab (Massachusetts General Hospital) |
| Last Name: | Pavao |
| First Name: | Aidan |
| Address: | 221 Longwood Ave, EBRC-411, Boston, MA, 02115, USA |
| Email: | apavao2@bwh.harvard.edu |
| Phone: | 617-525-7184 |
| Funding Source: | NIH R01AI153653, R03AI174158, P30DK056338, S10OD023406, R21CA243255, and R01AG070257; BWH Precision Medicine Institute; MGH A. A. Martinos Center for Biomedical Imaging |
| Publications: | https://doi.org/10.1038/s41589-023-01275-9 |
| Contributors: | Aidan Pavao, Brintha Girinathan, Johann Peltier, Pamela Altamirano Silva, Bruno Dupuy, Isabella H. Muti, Craig Malloy, Leo L. Cheng, Lynn Bry |
Subject:
| Subject ID: | SU002522 |
| Subject Type: | Bacteria |
| Subject Species: | Clostridioides difficile |
| Taxonomy ID: | NCBI:txid1496 |
| Genotype Strain: | ATCC 43255 delPaLoc |
Factors:
Subject type: Bacteria; Subject species: Clostridioides difficile (Factor headings shown in green)
| mb_sample_id | local_sample_id | Condition |
|---|---|---|
| SA243357 | Data8_13CGlc2 | 13C-Glucose |
| SA243358 | Data9_13CGlc3 | 13C-Glucose |
| SA243359 | Data12_13CGlc_endpt3 | 13C-Glucose |
| SA243360 | Data7_13CGlc1 | 13C-Glucose |
| SA243361 | Data10_13CGlc_endpt1 | 13C-Glucose |
| SA243362 | Data11_13CGlc_endpt2 | 13C-Glucose |
| SA243363 | Data19_13CGlc_standards | 13C-Glucose, 13C-Acetate, 13C-Alanine, 13C-Ethanol, 13C-Butyrate |
| SA243364 | Data15_13CGlc_15NLeu_endpt3 | 13C-Glucose, 15N-Leucine |
| SA243365 | Data16_13CGlc_15NLeu_stack | 13C-Glucose, 15N-Leucine |
| SA243366 | Data13_13CGlc_15NLeu_endpt1 | 13C-Glucose, 15N-Leucine |
| SA243367 | Data14_13CGlc_15NLeu_endpt2 | 13C-Glucose, 15N-Leucine |
| SA243368 | Data5_13CLeu2 | 13C-Leucine |
| SA243369 | Data4_13CLeu1 | 13C-Leucine |
| SA243370 | Data6_13CLeu3 | 13C-Leucine |
| SA243371 | Data2_13CPro2 | 13C-Proline |
| SA243372 | Data3_13CPro3 | 13C-Proline |
| SA243373 | Data1_13CPro1 | 13C-Proline |
| SA243374 | Data20_2D | 13C-Proline, 13C-Leucine, 13C-Glucose |
| SA243376 | Data18_13CPro-Se | 13C-Proline, no Selenium source |
| SA243375 | Data17_13CPro+Se | 13C-Proline, Sodium Selenite |
| Showing results 1 to 20 of 20 |
Collection:
| Collection ID: | CO002515 |
| Collection Summary: | For live cell time series, labeled MMM with 10% D2O was inoculated with 100,000 vegetative Clostridioides difficile ATCC43255 delPaLoc cells in a Kel-F HRMAS rotor insert under anaerobic atmosphere. The sample was spun at 3600 Hz at 37°C in a 4mm zirconia rotor and successive spectra were acquired over 36+ hours. For static culture supernatants, labeled MMM was inoculated with 100,000 vegetative Clostridioides difficile ATCC43255 delPaLoc cells in an anaerobic chamber and incubated at 37°C for 48 hours. Cultures were centrifuged and supernatants collected and lyophilized, then resuspended in D2O prior to NMR spectra acquisition. |
| Sample Type: | Bacterial cells |
| Volumeoramount Collected: | 30 µL |
| Collection Vials: | Kel-F inserts for 4mm MAS rotor |
| Collection Tube Temp: | 37°C |
Treatment:
| Treatment ID: | TR002534 |
| Treatment Summary: | For live cell time series, cells were grown in MMM with 10% D2O and one or more labeled substrates (L-[U-13C]Proline, L-[U-13C]Leucine, [U-13C]Glucose, or both [U-13C]Glucose and L-[15N]Leucine). For culture supernatants, cells were grown in MMM with [U-13C]Glucose, with or without L-[15N]Leucine. For the selenium perturbation time series, cells were grown in MMM with 10% L-[U-13C]Proline, with or without 100µM sodium selenite. |
| Treatment Compound: | L-[U-13C]Proline, L-[U-13C]Leucine, [U-13C]Glucose, L-[15N]Leucine, sodium selenite |
| Cell Media: | C. difficile Modified Minimal Medium (MMM) with 100µM sodium selenite and 10% D2O |
| Cell Envir Cond: | anaerobic, 37°C |
Sample Preparation:
| Sampleprep ID: | SP002528 |
| Sampleprep Summary: | For live cell NMR time series, samples were added to the HRMAS rotor neat. For culture supernatants, lyophilized samples were resuspended in D2O added to the NMR rotor neat. |
Analysis:
| Analysis ID: | AN003963 |
| Laboratory Name: | Cheng Lab |
| Analysis Type: | NMR |
| Acquisition Parameters File: | acqus |
| Software Version: | TopSpin 3.6.2 |
| Operator Name: | Leo L Cheng |
| Processing Parameters File: | pdata 1 procs |
| Data Format: | Bruker |
| Num Factors: | 8 |
| Num Metabolites: | 4 |
| Units: | proportion of Alanine |
NMR:
| NMR ID: | NM000262 |
| Analysis ID: | AN003963 |
| Instrument Name: | Bruker Avance III HD |
| Instrument Type: | FT-NMR |
| NMR Experiment Type: | Other |
| NMR Comments: | Experiments collected 1D 1H, 1D 13C, 2D 1H, 2D 13C, and 2D 1H-13C data. |
| Spectrometer Frequency: | 600 MHz |
| NMR Solvent: | C. difficile Modified Minimal Medium (MMM) with 100µM sodium selenite and 10% D2O |
| Temperature: | 37 |
