Summary of Study ST000085
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 PR000075. The data can be accessed directly via it's Project DOI: 10.21228/M86K5H This work is supported by NIH grant, U2C- DK119886.
See: https://www.metabolomicsworkbench.org/about/howtocite.php
| Study ID | ST000085 |
| Study Title | Salmonella Modulates Metabolism during Growth under Conditions that Induce Expression of Virulence Genes |
| Study Type | growth conditions, timecourse |
| Study Summary | Salmonella enterica serovar Typhimurium (S. Typhimurium) is a facultative pathogen that uses complex mechanisms to invade and proliferate within mammalian host cells. To investigate possible contributions of metabolic processes to virulence in S. Typhimurium grown under conditions known to induce expression of virulence genes, we used a metabolomics-driven systems biology approach coupled with genome scale modeling. First, we identified distinct metabolite profiles associated with bacteria grown in either rich or virulence-inducing media and report the most comprehensive coverage of the S. Typhimurium metabolome to date. Second, we applied an omics-informed genome scale modeling analysis of the functional consequences of adaptive alterations in S. Typhimurium metabolism during growth under our conditions. Modeling efforts highlighted a decreased cellular capability to both produce and utilize intracellular amino acids during stationary phase culture in virulence conditions, despite significant abundance increases for these molecules as observed by our metabolomics measurements. Furthermore, analyses of omics data in the context of the metabolic model indicated rewiring of the metabolic network to support pathways associated with virulence. For example, cellular concentrations of polyamines were perturbed, as well as the predicted capacity for secretion and uptake. |
| Institute | Pacific Northwest National Laboratory |
| Department | Biological Separation and Mass Spectrometry |
| Last Name | Metz |
| First Name | Thomas |
| thomas.metz@pnnl.gov | |
| Submit Date | 2014-06-25 |
| Num Groups | 3 |
| Total Subjects | 18 |
| Raw Data Available | Yes |
| Raw Data File Type(s) | cdf, d |
| Uploaded File Size | 245 M |
| Analysis Type Detail | GC-MS |
| Release Date | 2014-08-08 |
| Release Version | 1 |
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Sample Preparation:
| Sampleprep ID: | SP000100 |
| Sampleprep Summary: | Cell pellets resuspended in ammonium bicarbonate, lysed via bead beating, extracted with four volumes of chilled (-20°C) chloroform/methanol (2:1, v/v) |
| Sampleprep Protocol Comments: | Cell pellets were stored at -80°C prior to thawing and were subsequently resuspended in an appropriate volume of 100 mM ammonium bicarbonate according to their wet weight to compensate for any differences in cell numbers. The cells were lysed by bead-beating, and the lysates were transferred into new tubes. Subsequently, 100 µL aliquots of cell lysates were extracted with four volumes of chilled (-20°C) chloroform/methanol (2:1, v/v), and the aqueous phases after centrifugation were transferred to glass vials and dried in vacuo (SpeedVac; Thermo Scientific, Waltham, MA). All samples were kept at -80°C prior to chemical derivatization for GC-MS analysis. Dried metabolite extracts were briefly dried again to remove any residual water prior to chemical derivatization. To protect carbonyl groups and reduce the number of tautomeric isomers, 20 µL of methoxyamine in pyridine (30 mg/mL) were added to each sample, followed by incubation at 37°C with generous shaking for 90 min. To derivatize hydroxyl and amine groups to trimethylsilyated (TMS) forms, 80 µL of N-methyl-N-(trimethylsilyl)trifluoroacetamide (MSTFA) with 1% trimethylchlorosilane (TMCS) were added to each vial, followed by incubation at 37°C with shaking for 30 min. The samples were allowed to cool to room temperature and were then analyzed by GC-MS in random order. For technical replicates, each of the derivatized samples was split into two different vials and analyzed separately. |
| Processing Method: | Lysed via bead-beating |
| Extraction Method: | four volumes of chilled (-20°C) chloroform/methanol (2:1, v/v), and the aqueous phases after centrifugation were transferred to glass vials and dried in vacuo (SpeedVac; Thermo Scientific, Waltham, MA) |
| Extract Concentration Dilution: | chloroform/methanol (2:1, v/v) |
| Extract Enrichment: | dried in vacuo |
| Extract Storage: | dried in vacuo, stored at -80° C |
| Sample Resuspension: | 20 µL of methoxyamine in pyridine (30 mg/mL) |
| Sample Derivatization: | 20 µL of methoxyamine in pyridine (30 mg/mL), 80 µL of N-methyl-N-(trimethylsilyl)trifluoroacetamide (MSTFA) with 1% trimethylchlorosilane (TMCS) |
