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.

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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.

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Study IDST001125
Study TitleWT and ΔSPT cultures of B. thetaiotaomicron and B. ovatus grown BHI liquid media (part I)
Study SummaryLipid 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
DepartmentGastrointestinal Unit, Center for the Study of Inflammatory Bowel Disease, University Medical Center Groningen
Last NameAvila-Pacheco
First NameJulian
Address415 Main Street
Emailjravilap@broadinstitute.org
Phone617-714-8264
Submit Date2019-01-15
Num Groups4
Total Subjects8
Raw Data AvailableYes
Raw Data File Type(s).raw
Analysis Type DetailLC-MS
Release Date2019-03-06
Release Version1
Julian Avila-Pacheco Julian Avila-Pacheco
https://dx.doi.org/10.21228/M8709G
ftp://www.metabolomicsworkbench.org/Studies/ application/zip

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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 tethaiotamicron, Bateroides ovatus
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 tethaiotamicron, Bateroides ovatus (Factor headings shown in green)

mb_sample_id local_sample_id Species Strain
SA078040BOSPT_1Bacteroides ovatus SPT K.O. (BACOVA_02588)
SA078041BOSPT_2Bacteroides ovatus SPT K.O. (BACOVA_02588)
SA078042BOWT_2Bacteroides ovatus WT (ATCC_8483)
SA078043BOWT_1Bacteroides ovatus WT (ATCC_8483)
SA078044BTSPT_1Bacteroides thetaiotaomicron SPT K.O. (BT_0870)
SA078045BTSPT_2Bacteroides thetaiotaomicron SPT K.O. (BT_0870)
SA078046BTWT_2Bacteroides thetaiotaomicron WT (VPI-5482)
SA078047BTWT_1Bacteroides 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
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