Summary of Study ST000083
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 | ST000083 |
| Study Title | A Multi-Omic View of Host-Pathogen-Commensal Interplay in Salmonella-Mediated Intestinal Infection |
| Study Type | Timecourse of Infection |
| Study Summary | The potential for commensal microorganisms indigenous to a host (the microbiome or microbiota) to alter infection outcome by influencing host-pathogen interplay is largely unknown. We used a multi-omics systems approach, incorporating proteomics, metabolomics, glycomics, and metagenomics, to explore the molecular interplay between the murine host, the pathogen Salmonella enterica serovar Typhimurium (S. Typhimurium), and commensal gut microorganisms during intestinal infection with S. Typhimurium. We find proteomic evidence that S. Typhimurium thrives within the infected 129/SvJ mouse gut without antibiotic pre-treatment, inducing inflammation and disrupting the intestinal microbiome (e.g., suppressing Bacteroidetes and Firmicutes while promoting growth of Salmonella and Enterococcus). Alteration of the host microbiome population structure was highly correlated with gut environmental changes, including the accumulation of metabolites normally consumed by commensal microbiota. Finally, the less characterized phase of S. Typhimuriums lifecycle was investigated, and both proteomic and glycomic evidence suggests S. Typhimurium may take advantage of increased fucose moieties to metabolize fucose while growing in the gut. The application of multiple omics measurements to Salmonella-induced intestinal inflammation provides insights into complex molecular strategies employed during pathogenesis between host, pathogen, and the microbiome. |
| 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 | 4 |
| Total Subjects | 30 |
| Raw Data Available | Yes |
| Raw Data File Type(s) | cdf, d |
| Uploaded File Size | 268 M |
| Analysis Type Detail | GC-MS |
| Release Date | 2014-07-30 |
| Release Version | 1 |
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Sample Preparation:
| Sampleprep ID: | SP000098 |
| Sampleprep Summary: | Feces thawed, buffer added, vortexed, filtered and centrifuged after which supernatant subjected to further centrifugation and chemical derivatization |
| Sampleprep Protocol Comments: | After thawing, 150 mM ammonium bicarbonate buffer was added to the sample (between 1-2.5 ml based upon starting weight; volumes were recorded and used for downstream normalization), which was subsequently vortexed to disrupt fecal pellets. The resulting slurry was filtered through a 70 mm sieve to separate and remove large debris (mostly undigested food particles). Filtrate was centrifuged (900 x g for 10 min), and the protein-rich pellet thought to contain cellular material was retained as P1. The supernatant was centrifuged to further clarify the sample (15,000 x g for 10 min). The pellet was retained as P2 and the supernatant retained as SN2. All chemicals and reagents used in metabolomics analyses were purchased from Sigma-Aldrich (St. Louis, MO), except for ammonium bicarbonate (Merck, Darmstadt, Germany), mixture of fatty acid methyl esters (FAMEs) and deuterated myristic acid (Agilent Technologies, Santa Clara, CA). Deionized and purified water was used to prepare buffer and standard solutions (Nanopure Infinity ultrapure water system, Barnstead, Newton, WA). SN2 samples (see Fecal sample preparation) were transferred to 0.6 ml microcentrifuge tubes, and water soluble metabolites were extracted with four volumes of chilled (-20° C) chloroform: methanol mixture (2:1). After separating the two phases via centrifugation (12,000 x g, 5 min), the upper aqueous layers were transferred to glass vials and dried under a vacuum concentrator (SpeedVac; Thermo Scientific, Waltham, MA). All extracted metabolites were subjected to chemical derivatization to enhance their stability and volatility during GC-MS analysis. Methoxyamine in pyridine (30 mg/ml) was added to each dried sample, and incubated at 37° C with shaking for 90 min to protect carbonyl groups and reduce the number of tautomeric peaks. N-methyl-N- (trimethylsilyl) trifluoroacetamide (MSTFA) with 1% trimethylchlorosilane (TMCS) was then added, followed by incubation at 37° C with shaking for 30 min to transform hydroxyl and amine groups to trimethylsilyated (TMS) forms. The samples were then allowed to cool to room temperature and were analyzed using gas chromatography (GC)-MS. |
| Processing Method: | Homogenization, Filtration, Centrifugation |
| Extraction Method: | SN2 samples (see Fecal sample preparation) were transferred to 0.6 ml microcentrifuge tubes, and water soluble metabolites were extracted with four volumes of chilled (-20° C) chloroform: methanol mixture (2:1). After separating the two phases via centrifugation (12,000 x g, 5 min), the upper aqueous layers were transferred to glass vials and dried under a vacuum concentrator (SpeedVac; Thermo Scientific, Waltham, MA). |
| Extract Concentration Dilution: | chloroform: methanol mixture (2:1) |
| Extract Enrichment: | dried under a vacuum concentrator |
| Extract Storage: | dried under a vacuum concentrator |
| Sample Resuspension: | Methoxyamine in pyridine (30 mg/ml) |
| Sample Derivatization: | Methoxyamine in pyridine (30 mg/ml), N-methyl-N- (trimethylsilyl) trifluoroacetamide (MSTFA) with 1% trimethylchlorosilane (TMCS) |
