Summary of Study ST000083

This data is available at the NIH Common Fund's National Metabolomics Data Repository (NMDR) website, the Metabolomics Workbench,, 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.


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Study IDST000083
Study TitleA Multi-Omic View of Host-Pathogen-Commensal Interplay in Salmonella-Mediated Intestinal Infection
Study TypeTimecourse of Infection
Study SummaryThe 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. Typhimurium’s 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.
Pacific Northwest National Laboratory
DepartmentBiological Separation and Mass Spectrometry
Last NameMetz
First NameThomas
Submit Date2014-06-25
Num Groups4
Total Subjects30
Raw Data AvailableYes
Raw Data File Type(s)cdf, d
Uploaded File Size268 M
Analysis Type DetailGC-MS
Release Date2014-07-30
Release Version1
Thomas Metz Thomas Metz application/zip

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Project ID:PR000075
Project DOI:doi: 10.21228/M86K5H
Project Title:Systems Biology for EnteroPathogens
Project Type:MS analysis
Institute:Pacific Northwest National Laboratory
Department:Biological Separation and Mass Spectrometry
Last Name:Joshua
First Name:Adkins


Subject ID:SU000102
Subject Type:Animal
Subject Species:Mus musculus
Taxonomy ID:10090
Genotype Strain:129/SvJ
Age Or Age Range:6- to 8-week-old
Animal Animal Supplier:Jackson Laboratories,Bar Harbor, ME
Animal Housing:specific pathogen-free conditions in filter-top cages
Animal Feed:food provided ad libitum
Animal Water:sterile water
Species Group:Mammal


Subject type: Animal; Subject species: Mus musculus (Factor headings shown in green)

mb_sample_id local_sample_id Infection Experimental Group Harvest Day
SA004254SBEP_Microbiome.002Control Control Group 1 1
SA004253SBEP_Microbiome.001Control Control Group 1 -1
SA004255SBEP_Microbiome.005Control Control Group 1 10
SA004256SBEP_Microbiome.006Control Control Group 1 14
SA004257SBEP_Microbiome.007Control Control Group 1 21
SA004258SBEP_Microbiome.008Control Control Group 1 28
SA004259SBEP_Microbiome.003Control Control Group 1 3
SA004260SBEP_Microbiome.004Control Control Group 1 6
SA004262SBEP_Microbiome.010Control Control Group 2 1
SA004261SBEP_Microbiome.009Control Control Group 2 -1
SA004263SBEP_Microbiome.013Control Control Group 2 10
SA004264SBEP_Microbiome.014Control Control Group 2 14
SA004265SBEP_Microbiome.015Control Control Group 2 21
SA004266SBEP_Microbiome.016Control Control Group 2 28
SA004267SBEP_Microbiome.011Control Control Group 2 3
SA004268SBEP_Microbiome.012Control Control Group 2 6
SA004270SBEP_Microbiome.018Infected Experimental 1 1
SA004269SBEP_Microbiome.017Infected Experimental 1 -1
SA004271SBEP_Microbiome.021Infected Experimental 1 10
SA004272SBEP_Microbiome.022Infected Experimental 1 14
SA004273SBEP_Microbiome.023Infected Experimental 1 21
SA004274SBEP_Microbiome.024Infected Experimental 1 28
SA004275SBEP_Microbiome.019Infected Experimental 1 3
SA004276SBEP_Microbiome.020Infected Experimental 1 6
SA004278SBEP_Microbiome.026Infected Experimental 2 1
SA004277SBEP_Microbiome.025Infected Experimental 2 -1
SA004279SBEP_Microbiome.029Infected Experimental 2 10
SA004280SBEP_Microbiome.030Infected Experimental 2 14
SA004281SBEP_Microbiome.027Infected Experimental 2 3
SA004282SBEP_Microbiome.028Infected Experimental 2 6
Showing results 1 to 30 of 30


Collection ID:CO000085
Collection Summary:Feces collected and frozen at seven time points post infection and one time point pre-infection
Collection Protocol Comments:On the day prior to infection (day -1), and at seven time points post-infection, mice from one cage were transferred to a clean cage and fecal samples produced at that time were collected, pooled, and immediately frozen at -80° C. Samples were collected on days -1, 1, 3, 6, 10, 14, 21, and 28.
Sample Type:Feces
Collection Method:Fecal matter collection
Collection Location:mice from one cage were transferred to a clean cage and fecal samples produced
Collection Frequency:days -1, 1, 3, 6, 10, 14, 21, and 28 relative to day of infection (day 0)
Collection Time:days -1, 1, 3, 6, 10, 14, 21, and 28 relative to day of infection (day 0)
Volumeoramount Collected:four to 25 fecal pellets per pooled sample
Storage Conditions:frozen at -80° C


Treatment ID:TR000103
Treatment Summary:Experimental: Infected with 1.6 x 10^8 CFU S. Typhimurium | mouse | Control: treated with equal volume saline solution
Treatment Protocol Comments:A final inoculum of 1.6 x 10^8 CFU S. Typhimurium/mouse was delivered by oral gavage to 10 mice (two cages of five mice each = Salmonella-infected). An equal number of mock-infected animals (two cages of five mice each = control) were administered an equal volume of sterile saline. Our infecting dose (10^8 CFU/mouse) aimed to establish a persistent infection that would ensure observation of S. Typhimurium proteins in downstream analyses.
Treatment:Biotic / Abiotic
Treatment Compound:S. Typhimurium / Saline
Treatment Dose:1.6 x 10^8 CFU S. Typhimurium/mouse / equal volume saline solution
Treatment Vehicle:Saline
Animal Fasting:14 h before orogastric inoculation
Animal Endp Euthanasia:Carbon Dioxide Asphixiation followed by Cervical Dislocaton
Animal Endp Tissue Coll List:Feces
Animal Endp Tissue Proc Method:Homogenization

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)

Combined analysis:

Analysis ID AN000135
Analysis type MS
Chromatography type GC
Chromatography system Agilent 7890A
Column Agilent HP5-MS (30m × 0.25mm, 0.25 um)
MS Type EI
MS instrument type Single quadrupole
MS instrument name Agilent 5975C
Units Peak area


Chromatography ID:CH000095
Chromatography Summary:Agilent 7890A gas chromatograph with a HP-5MS gas chromatography column using Chemstation
Chromatography Comments:Chromatography was carried out on an Agilent 7890A gas chromatograph using the manufacturer's software (Chemstation) and a HP-5MS gas chromatography column (Agilent Technologies, Santa Clara, CA; 30 m x 0.25 mm x 0.25 m film thickness). The sample injection mode was splitless, and 1 L of each sample was injected. The injection port temperature was held at 250 C throughout the analysis. The GC oven was held at 60 C for 1 min after injection, and the temperature was then increased to 325 C by 10 C/min, followed by a 5 min hold at 325 C. The helium gas flow rates for each Experiment were determined by the Agilent Retention Time Locking function based on analysis of deuterated myristic acid and were in the range of 0.450.5 mL/min.
Instrument Name:Agilent 7890A
Column Name:Agilent HP5-MS (30m × 0.25mm, 0.25 um)
Flow Rate:0.450.5 mL/min
Injection Temperature:250 C
Sample Injection:1 L, splitless
Analytical Time:37.5 min
Oven Temperature:60 C for 1 min, then increased to 325 C by 10 C/min, followed by a 5 min hold at 325 C
Sample Syringe Size:10 L
Chromatography Type:GC


MS ID:MS000111
Analysis ID:AN000135
Instrument Name:Agilent 5975C
Instrument Type:Single quadrupole
MS Type:EI
MS Comments:An Agilent GC 7890A coupled with a single quadrupole MSD 5975C (Agilent Technologies, Inc.; Santa Clara, CA, USA) was used, and the samples were blocked and analyzed in random order for each experiment. Data were collected over the mass range 50-550 m/z. A mixture of FAMEs (C8-C28) was analyzed once per day together with the samples for retention index alignment purposes during subsequent data analysis.
Scan Range Moverz:50-550 m/z