MetaboLightsapplication/xmlftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS14740/m_MTBLS14740_LC-MS_negative_reverse-phase_v2_maf.tsvftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS14740/i_Investigation.txtftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS14740/s_MTBLS14740.txtftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS14740/a_MTBLS14740_LC-MS_negative_reverse-phase.txtprimary200OKftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS14740<p>Metabolite peaks were identified using Metabolomics Analysis and Visualization Engine (MAVEN) using a previously established standards list (68). AGC target set to 1e6, and maximum IT at 40 ms.</p>MetaboLightsPublicLiquid Chromatography MS - negative - reverse-phase<p>Metabolite quantification was performed with 10 μL of each sample injected. Samples were run through a ACQUITY UPLC® BEH C18 column with a 25-minute gradient with Solvent A [97% water, 3% methanol, 10 mM tributylamine (TBA), 9.8 mM acetic acid, pH 8.2] and Solvent B [100% methanol]. The gradient cycle consisted of 5% Solvent B for 2.5 minutes, increasing gradually to 95% Solvent B at 17 minutes. It was then held at 95% Solvent B until 19.5 minutes. At 20.5 minutes, it returned to 5% Solvent B over 0.5 minutes and held at 5% Solvent B for the remaining 4 minutes of the cycle.</p>Glutathione is a host-derived cysteine source for Clostridioides difficile.Eashant ThusooUniversity of Wisconsin-MadisonWhole Organismmass spectrometry assay<p>C. difficile 630Δerm perRWT [WT] was back diluted 1:50 in 20 mL of BDM +/- 25 mM GSH. Cultures were harvested anaerobically, at mid-log by depositing 5 mL of culture by vacuum filtration onto a 0.2 µm nylon membrane (47 mm diameter). The filter membrane was then placed in a small petri dish that contained 1.5 mL of cold extraction solvent which consisted of 20:20:10 v/v/v acetonitrile, methanol, and water. The petri dish was gently swirled. The filter was turned over, and the petri dish was gently swirled to maximize metabolite extraction. Extraction contents were transferred to a 1.5 mL Eppendorf tube and stored at −80°C. Prior to LC-MS analysis, samples were centrifuged at 21,000xg at 4°C for 10 min, normalized to OD600, and dried using N2 gas. Samples were then resuspended into 70 μL of HPLC grade water and centrifuged at 21,000xg at 4°C for 10 min to remove all particulates. 50 μL of the supernatant from each sample was transferred into a HPLC vial</p>Clostridioides difficile 70-100-2010https://www.ebi.ac.uk/metabolights/MTBLS14740Eashant Thusoo. University of Wisconsin-Madison. thusoo@wisc.edu.Anna Gregory. University of Wisconsin-Madison. algregory2@wisc.edu.Andrew Hryckowian. University of Wisconsin–Madison. hryckowian@medicine.wisc.edu.<p>Metabolite peaks were identified using Metabolomics Analysis and Visualization Engine (MAVEN) using a previously established standards list (68). AGC target set to 1e6, and maximum IT at 40 ms.</p>Biological replicatethusoo@wisc.edu<p>C. difficile 630Δerm perRWT [WT] was back diluted 1:50 in 20 mL of BDM +/- 25 mM GSH. Cultures were harvested anaerobically, at mid-log by depositing 5 mL of culture by vacuum filtration onto a 0.2 µm nylon membrane (47 mm diameter). The filter membrane was then placed in a small petri dish that contained 1.5 mL of cold extraction solvent which consisted of 20:20:10 v/v/v acetonitrile, methanol, and water. The petri dish was gently swirled. The filter was turned over, and the petri dish was gently swirled to maximize metabolite extraction. Extraction contents were transferred to a 1.5 mL Eppendorf tube and stored at −80°C. Prior to LC-MS analysis, samples were centrifuged at 21,000xg at 4°C for 10 min, normalized to OD600, and dried using N2 gas. Samples were then resuspended into 70 μL of HPLC grade water and centrifuged at 21,000xg at 4°C for 10 min to remove all particulates. 50 μL of the supernatant from each sample was transferred into a HPLC vial</p>Metabolomicsultra-performance liquid chromatography-mass spectrometryMetabolomicscentroid spectrumuntargeted analysisThermo Scientific ExactiveWhole Organismexperimental sampledata-independent acquisitionuntargeted metabolite profilingThermo Scientific Vanquish UHPLC SystemLC-MSClostridioides difficile 70-100-2010mzML formatultra-performance liquid chromatography-mass spectrometryMetabolomicscentroid spectrumuntargeted analysisThermo Scientific ExactiveWhole Organismexperimental sampledata-independent acquisitionuntargeted metabolite profilingThermo Scientific Vanquish UHPLC SystemLC-MSClostridioides difficile 70-100-2010mzML format<p>Metabolite quantification was performed with 10 μL of each sample injected. Samples were run through a ACQUITY UPLC® BEH C18 column with a 25-minute gradient with Solvent A [97% water, 3% methanol, 10 mM tributylamine (TBA), 9.8 mM acetic acid, pH 8.2] and Solvent B [100% methanol]. The gradient cycle consisted of 5% Solvent B for 2.5 minutes, increasing gradually to 95% Solvent B at 17 minutes. It was then held at 95% Solvent B until 19.5 minutes. At 20.5 minutes, it returned to 5% Solvent B over 0.5 minutes and held at 5% Solvent B for the remaining 4 minutes of the cycle. Ions were generated by heated electrospray ionization (HESI; negative ion mode) and quantified by a hybrid quadrupole high-resolution mass spectrometer (Q Exactive Orbitrap, Thermo Scientific). The MS scans consisted of full MS scanning in negative mode from m/z from time 0–18 min.</p>falseGlutathione is a host-derived cysteine source for Clostridioides difficileClostridioides difficile is the leading cause of nosocomial diarrhea, afflicting approximately half a million people each year in the USA, burdening both individuals’ quality of life and the healthcare system. In the gastrointestinal (GI) tract, C. difficile must acquire essential nutrients to colonize, establish infection, and persist in a polymicrobial environment. C. difficile is a cysteine auxotroph and the GI tract contains low levels of cysteine, highlighting gaps in our understanding of how C. difficile acquires this amino acid during infection. One possible source of cysteine for C. difficile during infection is glutathione (GSH), a tripeptide thiol found in mammalian cells. Our data indicate that C. difficile encodes two functionally redundant enzymes (UgaA and UgbB) that allow it to use GSH as a source of cysteine. Using murine models of C. difficile infection, we show that C. difficile uses its toxins to increase available GSH in the GI tract during infection. Finally, we show that the ability to utilize GSH gives wild-type C. difficile a fitness advantage over a GSH-deficient mutant in vivo. These findings establish GSH metabolism by C. difficile as a virulence-linked metabolic program and as a possibly impactful target for future therapeutic development.2026-06-102026-06-10MTBLS14740