Summary of Study ST001125
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 PR000754. The data can be accessed directly via it's Project DOI: 10.21228/M8709G 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 | ST001125 |
| Study Title | WT and ΔSPT cultures of B. thetaiotaomicron and B. ovatus grown BHI liquid media (part I) |
| Study Summary | Lipid profiling was applied to WT and ΔSPT cultures of B. thetaiotaomicron and B. ovatus grown BHI liquid media revealing a greater variety of Bacteroides-derived sphingolipids than previously recognized, including ceramide phosphoinositol and deoxy-sphingolipids. |
| Institute | Broad Institute of MIT and Harvard |
| Department | Gastrointestinal Unit, Center for the Study of Inflammatory Bowel Disease, University Medical Center Groningen |
| Last Name | Avila-Pacheco |
| First Name | Julian |
| Address | 415 Main Street |
| jravilap@broadinstitute.org | |
| Phone | 617-714-8264 |
| Submit Date | 2019-01-15 |
| Num Groups | 4 |
| Total Subjects | 8 |
| Raw Data Available | Yes |
| Raw Data File Type(s) | raw(Thermo) |
| Analysis Type Detail | LC-MS |
| Release Date | 2019-03-06 |
| Release Version | 1 |
Select appropriate tab below to view additional metadata details:
Project:
| Project ID: | PR000754 |
| Project DOI: | doi: 10.21228/M8709G |
| Project Title: | Bacteroides-derived sphingolipids are critical for maintaining intestinal homeostasis and symbiosis |
| Project Summary: | Sphingolipids are structural membrane components and important eukaryotic signaling molecules. We hypothesized that sphingolipids mediate intestinal health as they were identified as the most upregulated metabolite feature in stool of inflammatory bowel disease (IBD) patients. Commensal Bacteroidetes also produce sphingolipids, but the impact of these metabolites on host pathways is largely uncharacterized. To study Bacteroidetes sphingolipids in intestinal health, we colonized germ-free mice with a sphingolipid-deficient Bacteroides thetaiotaomicron strain. A lack of Bacteroides-derived sphingolipids increased intestinal inflammation, dysregulated innate immunity and altered the host ceramide pool. Using metabolomic analysis, we described the Bacteroides sphingolipid biosynthesis pathway and revealed a greater variety of Bacteroides-derived sphingolipids than previously recognized, including ceramide phosphoinositol and deoxy-sphingolipids. We annotated Bacteroides sphingolipids in an IBD metabolomic dataset, discovering lower abundances in IBD and negative correlations with gut inflammation and host sphingolipid production. These data highlight the role of sphingolipids in maintaining host-bacterial symbiosis and intestinal homeostasis. |
| Institute: | Broad Institute of MIT and Harvard |
| Last Name: | Avila-Pacheco |
| First Name: | Julian |
| Address: | 415 Main Street, Cambridge MA |
| Email: | jravilap@broadinstitute.org |
| Phone: | 617-714-8264 |
| Funding Source: | National Institutes of Health (P30 DK043351 and R01 AT009708) |
| Contributors: | Eric M. Brown, Xiaobo Ke, Daniel Hitchcock, Timothy D. Arthur, Toru Nakata, Nadine Fornelos, Cortney Heim, Eric A. Franzosa1,4, Curtis Huttenhower1,4, Henry J. Haiser3, Glen 6 Dillow3, Daniel B. Graham1, B. Brett Finlay, Aleksandar D. Kostic, Jeffrey A. Porter, Hera Vlamakis, Sarah Jeanfavre, Julian Avila-Pacheco, Clary B. Clish, and Ramnik J. Xavier |
Subject:
| Subject ID: | SU001186 |
| Subject Type: | Bacteria |
| Subject Species: | Bacteroides thetaiotaomicron VPI-5482;Bacteroides ovatus ATCC 8483 |
| Taxonomy ID: | 226186;411476 |
| Genotype Strain: | WT (VPI-5482), SPT K.O. (BT_0870), (ATCC_8483), SPT K.O. (BACOVA_02588) |
| Age Or Age Range: | 411476 |
| Cell Biosource Or Supplier: | ATCC |
| Cell Strain Details: | Wild type Bacteroides strains and serine palmitoyltransferase (SPT) knockouts |
| Subject Comments: | Grown in BHI Media (Bacteroides tethaiotamicron, Bateroides ovatus : VPI-5482/ATCC_8483) |
Factors:
Subject type: Bacteria; Subject species: Bacteroides thetaiotaomicron VPI-5482;Bacteroides ovatus ATCC 8483 (Factor headings shown in green)
| mb_sample_id | local_sample_id | Species | Strain |
|---|---|---|---|
| SA078040 | BOSPT_1 | Bacteroides ovatus | SPT K.O. (BACOVA_02588) |
| SA078041 | BOSPT_2 | Bacteroides ovatus | SPT K.O. (BACOVA_02588) |
| SA078042 | BOWT_2 | Bacteroides ovatus | WT (ATCC_8483) |
| SA078043 | BOWT_1 | Bacteroides ovatus | WT (ATCC_8483) |
| SA078044 | BTSPT_1 | Bacteroides thetaiotaomicron | SPT K.O. (BT_0870) |
| SA078045 | BTSPT_2 | Bacteroides thetaiotaomicron | SPT K.O. (BT_0870) |
| SA078046 | BTWT_2 | Bacteroides thetaiotaomicron | WT (VPI-5482) |
| SA078047 | BTWT_1 | Bacteroides thetaiotaomicron | WT (VPI-5482) |
| Showing results 1 to 8 of 8 |
Collection:
| Collection ID: | CO001180 |
| Collection Summary: | WT and ΔSPT cultures of B. thetaiotaomicron and B. ovatus were grown in 5 mL of BHI liquid media supplemented with vitamin K and hemin. After 24 hrs, cultures were normalized by OD600 and centrifuged at 8,000 rpm for 10 min to pellet cells. Resulting pellets were washed and further centrifuged twice to remove residual media. Resulting pellets were resuspended in 5 mL isopropanol + 0.1 ng/μL C24:0 PC, incubated at RT for 1 hr, and centrifuged for 10 min at 10,000g. From each sample, 2 μL was injected. |
| Sample Type: | Bacterial cells |
| Storage Conditions: | -80℃ |
Treatment:
| Treatment ID: | TR001201 |
| Treatment Summary: | In-frame deletion of the serine palmitoyl transferase gene (spt) in B. thetaiotaomicron Δtdk was generated using a counter-selectable allelic exchange procedure (Koropatkin et al., 2008). Briefly, a 2 kb fragment concatenating the 700 bp upstream sequence, the first 291bp of the SPT gene (BT0870) followed by a stop, and the 1 kb downstream sequences of SPT was synthesized by IDT. The in-frame deletion in spt encodes only the first 97 amino acids of the B. thetaiotaomicron Spt protein. This 2 kb fragment was amplified using primer pairs BT0870_Up700b_BamHI _F and BT0870_Down1kb_NotI_R and ligated into the suicide vector pExchange_tdk (obtained from Dr. Harry Brumer, UBC). The resulting pExchange_tdk_BT0870_DEL vector was electroporated into E. coli S17-1 λ pir and then conjugated into B. thetaiotaomicron VPI-5482. Single recombinants were selected on BHI agar plates containing gentamicin and erythromycin, cultured in liquid BHI medium overnight without antibiotics and then plated onto BHI agar plates containing 200 μg/mL 5-fluoro-2-deoxyuridine (FUdR). Single colonies of SPT deletion candidates were confirmed by PCR using the diagnostic primers BT0870_Up1kb_F and BT0870_Down1150b_R. The B. ovatus spt deletion mutant was generated in a similar fashion. Briefly, ~900 bp fragments upstream and downstream of the B. ovatus SPT (BACOVA_02588) were cloned and fused using primer pairs ΔSPT Xba1-UPF (5’AGTCACGACGTTGTAAAACGACGGCCAGT-3’), BamH1 UPR-(5’- GGCGTAATCATGGTCATAGCTGTTTCCTG-3’), EcoR1-DNF (5’- GTTGTAAAACGACGGCCAGT-3’) and HindIII-DNR (5’-GGCGTAATCATGGTCATAGC-3’), respectively, and ligated into pExchange_tdk. The resulting pExchange_tdk_BACOVA_02588_DEL vector was electroporated into E. coli S17-1 λ pir and then conjugated into B. ovatus ATCC 8483. Single recombinants were selected on BHI agar plates containing gentamicin and erythromycin, cultured in liquid BHI plus medium [BHI lemented with 5% heat inactivated fetal bovine serum, 1% vitamin 500 K1-hemin solution (Becton Dickinson), 1% trace mineral supplement (ATCC), 1% vitamin supplement (ATCC), 2.9 mM (+)-cellobiose (Becton Dickinson), 2.9 mM maltose (Hardy Diagnostics), 5.8 mM D-(−)- Fructose (Sigma) and 2.8 mM L-Cysteine hydrochloride monohydrate (Sigma)] overnight without antibiotics, and then plated onto BHI plus agar plates containing 200 μg/mL FUdR. Single colonies of SPT deletion candidates were screened and confirmed by PCR using the diagnostic primers BACOVA_02588 _Up1kb_F and BACOVA_02588 _Down1kb_R. |
| Cell Storage: | -80C |
| Cell Growth Container: | anaerobic chamber (Coy Laboratory Products) with an atmosphere of 20% CO2, 5% H2, and 75% N2 at 37°C. |
| Cell Media: | Brain Heart Infusion (BHI, Becton Dickinson) medium supplemented with 1% Vitamin K1-Hemin Solution (VitK/Hemin, Becton Dickinson), or on BHI agar (Becton Dickinson) supplemented with 1% VitK/Hemin. |
Sample Preparation:
| Sampleprep ID: | SP001194 |
| Sampleprep Summary: | Bacterial cell pellets from 5ml overnight BHI cultures were resuspended in 5 mL isopropanol + 0.1 ng/μL C24:0 PC, incubated at RT for 1 hr, and centrifuged for 10 min at 10,000g. |
Combined analysis:
| Analysis ID | AN001850 |
|---|---|
| Analysis type | MS |
| Chromatography type | Reversed phase |
| Chromatography system | Shimadzu Nexera X2 |
| Column | Waters Acquity BEH C8 (100 x 2.1mm,1.7um) |
| MS Type | ESI |
| MS instrument type | Orbitrap |
| MS instrument name | Thermo Q Exactive Focus |
| Ion Mode | POSITIVE |
| Units | abundance |
Chromatography:
| Chromatography ID: | CH001338 |
| Instrument Name: | Shimadzu Nexera X2 |
| Column Name: | Waters Acquity BEH C8 (100 x 2.1mm,1.7um) |
| Chromatography Type: | Reversed phase |
MS:
| MS ID: | MS001711 |
| Analysis ID: | AN001850 |
| Instrument Name: | Thermo Q Exactive Focus |
| Instrument Type: | Orbitrap |
| MS Type: | ESI |
| MS Comments: | Raw data were processed using TraceFinder 3.1 (Thermo Fisher) and Progenesis QI (Nonlinear dynamics). Retention time, m/z, MS/MS fragmentation were used to confirm the identity of metabolite using authentic reference standards when available (level 1 identifications), in addition to monitoring the product ions produced and the retention time and mass of species belonging to the each lipid class (level 2-3 annotation). |
| Ion Mode: | POSITIVE |
