Summary of Study ST002473

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 PR001596. The data can be accessed directly via it's Project DOI: 10.21228/M8D701 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	ST002473
Study Title	Linking bacterial metabolites to disease-associated microbes to uncover mechanisms of host-microbial interactions in intestinal inflammation. Veillonella parvula media profiling of IBD drug metabolites
Study Summary	Understanding the role of the gut microbiome in inflammatory and autoimmune diseases requires the identification of microbial molecular effectors and their link to host pathophysiology. Here, we present a framework to identify and characterize novel microbial metabolites in patient samples and to directly link their production to disease-associated microbes. We applied this approach to investigate the spectrum of disease severity and treatment response in ulcerative colitis (UC) using longitudinal metabolite and strain profiles combined with paired plasma profiles.
Institute	Broad Institute of MIT and Harvard
Last Name	Xavier
First Name	Ramnik
Address	415 Main Street
Email	rxavier@broadinstitute.org
Phone	617717084
Submit Date	2023-02-10
Raw Data Available	Yes
Raw Data File Type(s)	raw(Thermo)
Analysis Type Detail	LC-MS
Release Date	2024-02-12
Release Version	1

Select appropriate tab below to view additional metadata details:

Project:

Project ID:	PR001596
Project DOI:	doi: 10.21228/M8D701
Project Title:	Linking bacterial metabolites to disease-associated microbes to uncover mechanisms of host-microbial interactions in intestinal inflammation
Project Type:	Metabolomic profiling of human fecal and plasma samples and bacterial strains
Project Summary:	Understanding the role of the gut microbiome in inflammatory and autoimmune diseases requires the identification of microbial molecular effectors and their link to host pathophysiology. Here, we present a framework to identify and characterize novel microbial metabolites in patient samples and to directly link their production to disease-associated microbes. We applied this approach to investigate the spectrum of disease severity and treatment response in ulcerative colitis (UC) using longitudinal metabolite and strain profiles combined with paired plasma profiles.
Institute:	Broad Institute of MIT and Harvard
Last Name:	Xavier
First Name:	Ramnik
Address:	415 Main Street
Email:	rxavier@broadinstitute.org
Phone:	6177147080
Publications:	Schirmer, M., Stražar, M., Avila-Pacheco, J., Rojas-Tapias, D. F., Brown, E. M., Temple, E., Deik, A., Bullock, K., Jeanfavre, S., Pierce, K., Jin, S., Invernizzi, R., Pust, M.-M., Costliow, Z., Mack, D. R., Griffiths, A. M., Walters, T., Boyle, B. M., Kugathasan, S., … Xavier, R. J. (2024). Linking microbial genes to plasma and stool metabolites uncovers host-microbial interactions underlying ulcerative colitis disease course. Cell Host & Microbe. https://doi.org/10.1016/j.chom.2023.12.013
Contributors:	Melanie Schirmer, Martin Strazar, Julian Avila-Pacheco, Daniel F. Rojas-Tapias, Eric Brown, Emily Temple, Subra Kugathasan, Zach Costliow, Hera Vlamakis, Jeff Hyams, Lee Denson, Clary B. Clish, Ramnik J. Xavier

Subject:

Subject ID:	SU002563
Subject Type:	Bacteria
Subject Species:	Veillonella parvula
Taxonomy ID:	29466
Species Group:	Bacteria

Factors:

Subject type: Bacteria; Subject species: Veillonella parvula (Factor headings shown in green)

mb_sample_id	local_sample_id	Strain	Drug
SA247736	None_6_mercaptopurine_R1	None	6-mercaptopurine
SA247737	None_6_mercaptopurine_R4	None	6-mercaptopurine
SA247738	None_6_mercaptopurine_R3	None	6-mercaptopurine
SA247739	None_6_mercaptopurine_R2	None	6-mercaptopurine
SA247740	None_Azathioprine_R3	None	Azathioprine
SA247741	None_Azathioprine_R4	None	Azathioprine
SA247742	None_Azathioprine_R2	None	Azathioprine
SA247743	None_Azathioprine_R1	None	Azathioprine
SA247744	None_None_R3	None	None
SA247745	None_None_R4	None	None
SA247746	None_None_R2	None	None
SA247747	None_None_R1	None	None
SA247760	pucD_6_mercaptopurine_R4	pucD	6-mercaptopurine
SA247761	pucD_6_mercaptopurine_R1	pucD	6-mercaptopurine
SA247762	pucD_6_mercaptopurine_R2	pucD	6-mercaptopurine
SA247763	pucD_6_mercaptopurine_R3	pucD	6-mercaptopurine
SA247764	pucD_Azathioprine_R3	pucD	Azathioprine
SA247765	pucD_Azathioprine_R2	pucD	Azathioprine
SA247766	pucD_Azathioprine_R1	pucD	Azathioprine
SA247767	pucD_Azathioprine_R4	pucD	Azathioprine
SA247768	pucD_None_R2	pucD	None
SA247769	pucD_None_R3	pucD	None
SA247770	pucD_None_R1	pucD	None
SA247771	pucD_None_R4	pucD	None
SA247748	WT_6_mercaptopurine_R2	WT	6-mercaptopurine
SA247749	WT_6_mercaptopurine_R4	WT	6-mercaptopurine
SA247750	WT_6_mercaptopurine_R3	WT	6-mercaptopurine
SA247751	WT_6_mercaptopurine_R1	WT	6-mercaptopurine
SA247752	WT_Azathioprine_R1	WT	Azathioprine
SA247753	WT_Azathioprine_R3	WT	Azathioprine
SA247754	WT_Azathioprine_R2	WT	Azathioprine
SA247755	WT_Azathioprine_R4	WT	Azathioprine
SA247756	WT_None_R2	WT	None
SA247757	WT_None_R4	WT	None
SA247758	WT_None_R3	WT	None
SA247759	WT_None_R1	WT	None
SA247772	xdhA_6_mercaptopurine_R4	xdhA	6-mercaptopurine
SA247773	xdhA_6_mercaptopurine_R1	xdhA	6-mercaptopurine
SA247774	xdhA_6_mercaptopurine_R3	xdhA	6-mercaptopurine
SA247775	xdhA_6_mercaptopurine_R2	xdhA	6-mercaptopurine
SA247776	xdhA_Azathioprine_R3	xdhA	Azathioprine
SA247777	xdhA_Azathioprine_R4	xdhA	Azathioprine
SA247778	xdhA_Azathioprine_R2	xdhA	Azathioprine
SA247779	xdhA_Azathioprine_R1	xdhA	Azathioprine
SA247780	xdhA_None_R4	xdhA	None
SA247781	xdhA_None_R2	xdhA	None
SA247782	xdhA_None_R1	xdhA	None
SA247783	xdhA_None_R3	xdhA	None

Showing results 1 to 48 of 48

Showing results 1 to 48 of 48

Collection:

Collection ID:	CO002556
Collection Summary:	All strain experiments were done in the Xavier lab. Strains and growth conditions: we utilized the following strains of Veillonella for growth and metabolic analysis; Veillonella parvula SKV38 [DR071], Veillonella parvula SKV38 xdh::cat* [DR214], Veillonella parvula SKV38 pucD::cat* [DR213]. All strains were first streaked on an agar plate with SK media (composition: yeast extract 10 gL-1, casitone 10 gL-1, NaCl 2 gL-1, K2HPO4 0.4 gL-1) supplemented with 50 mM lactate and 40 mM KNO3 (SKLN medium) and antibiotics if required. From this agar plate, a single colony was selected and inoculated in 5 mL SKLN media and grown for 24 hours. Next, overnight cells from this inoculum were grown using a 1/50 inoculum of the overnight culture, on either SK, SK + 50 mM lactate (SKL), SK + 40 mM nitrate (SKN), and SKLN. Supernatants were collected at the mid-exponential phase (OD600~0.4) and collected for metabolomic analyses (HILIC-pos metabolite profiling method described above). For analysis of the metabolism of the IBD drugs, cells were prepared as described above, and mid-exponential phase cells were collected, washed twice with sterile SK, and resuspended in 100 µl of SK. A volume of 6 ml of SKN was then inoculated using 90 µl of the bacterial suspension. Those 6 ml were split in three parts: a) 1.5 ml SK, b) 1.5 ml SK + 20 µM 6-mercaptopurine, and c) 1.5 ml SK + 20 µM 6-azathioprine. Additionally, sterile SK was prepared in an identical fashion to serve as a control for the experiment. Cells were incubated in a plate reader (4 wells/treatment) with low shaking and 37ºC for about ~5 h, and when the OD600 reached 0.4 cells were collected. For collection, 700 µl of the suspensions were centrifuged at 21,000 g for 2 min, and 400 µl of the supernatant harvested and stored at -20 ºC for analysis. Preparation of thiopurine drugs: 20 mM solutions were prepared using 4.25 mg of 6-mercaptopurine (Sigma 852678) or 6.9 mg of azathioprine (Sigma A4638) dissolved in 1 ml of DMSO. The drugs were prepared and used the same day.
Sample Type:	Culture Media

Treatment:

Treatment ID:	TR002575
Treatment Summary:	A volume of 6 ml of SKN was then inoculated using 90 µl of the bacterial suspension. Those 6 ml were split in three parts: a) 1.5 ml SK, b) 1.5 ml SK + 20 µM 6-mercaptopurine, and c) 1.5 ml SK + 20 µM 6-azathioprine. Preparation of thiopurine drugs: 20 mM solutions were prepared using 4.25 mg of 6-mercaptopurine (Sigma 852678) or 6.9 mg of azathioprine (Sigma A4638) dissolved in 1 ml of DMSO. The drugs were prepared and used the same day.

Treatment ID:

TR002575

Treatment Summary:

A volume of 6 ml of SKN was then inoculated using 90 µl of the bacterial suspension. Those 6 ml were split in three parts: a) 1.5 ml SK, b) 1.5 ml SK + 20 µM 6-mercaptopurine, and c) 1.5 ml SK + 20 µM 6-azathioprine. Preparation of thiopurine drugs: 20 mM solutions were prepared using 4.25 mg of 6-mercaptopurine (Sigma 852678) or 6.9 mg of azathioprine (Sigma A4638) dissolved in 1 ml of DMSO. The drugs were prepared and used the same day.

Sample Preparation:

Sampleprep ID:	SP002569
Sampleprep Summary:	Bacterial supernatant (media) metabolites were profiled using the HILIC-pos and HILIC-neg methods in order to estimate purines and thiopurines metabolism. Media samples were prepared as follows: mid-exponential Veillonella cultures (OD600 = 0.3-0.4) were harvested by centrifugation at 20,000g at 4°C for 1 minute, supernatants (spent media) were aliquoted and stored at -80°C until metabolite profiling was conducted. For the HILIC-pos method, media samples (10 µL) were extracted using 90 µL HILIC extraction solution (74.9:24.9:0.2 v/v/v acetonitrile/methanol/formic acid) with internal standards (valine-d8, Sigma-Aldrich; St. Louis, MO; and phenylalanine-d8) and extracts were cleared by centrifugation (10 min, 9,000 x g, Room Temperature). For media profiled in the HILIC-neg mode, 30 µL of media and metabolites extracted using 80% methanol containing inosine-15N4, thymine-d4 and glycocholate-d4 internal standards (Cambridge Isotope Laboratories; Andover, MA). Extracts were cleared by centrifugation (10 min, 9,000 x g, 4C) prior to analysis.

Sampleprep ID:

SP002569

Sampleprep Summary:

Bacterial supernatant (media) metabolites were profiled using the HILIC-pos and HILIC-neg methods in order to estimate purines and thiopurines metabolism. Media samples were prepared as follows: mid-exponential Veillonella cultures (OD600 = 0.3-0.4) were harvested by centrifugation at 20,000g at 4°C for 1 minute, supernatants (spent media) were aliquoted and stored at -80°C until metabolite profiling was conducted. For the HILIC-pos method, media samples (10 µL) were extracted using 90 µL HILIC extraction solution (74.9:24.9:0.2 v/v/v acetonitrile/methanol/formic acid) with internal standards (valine-d8, Sigma-Aldrich; St. Louis, MO; and phenylalanine-d8) and extracts were cleared by centrifugation (10 min, 9,000 x g, Room Temperature). For media profiled in the HILIC-neg mode, 30 µL of media and metabolites extracted using 80% methanol containing inosine-15N4, thymine-d4 and glycocholate-d4 internal standards (Cambridge Isotope Laboratories; Andover, MA). Extracts were cleared by centrifugation (10 min, 9,000 x g, 4C) prior to analysis.

Combined analysis:

Analysis ID	AN004039	AN004040
Analysis type	MS	MS
Chromatography type	HILIC	HILIC
Chromatography system	Shimadzu Nexera X2	Shimadzu Nexera X2
Column	Waters Atlantis HILIC (150 x 2 mm, 3 μm)	Phenomenex Luna NH2 (150 x 2.1mm,3um)
MS Type	ESI	ESI
MS instrument type	Orbitrap	Orbitrap
MS instrument name	Thermo Q Exactive Plus Orbitrap	Thermo Q Exactive Plus Orbitrap
Ion Mode	POSITIVE	NEGATIVE
Units	Abundance	Abundance

Chromatography:

Chromatography ID:	CH002987
Instrument Name:	Shimadzu Nexera X2
Column Name:	Waters Atlantis HILIC (150 x 2 mm, 3 μm)
Column Temperature:	30C
Flow Gradient:	Isocratically with 5% mobile phase A for 1 minute followed by a linear gradient to 40% mobile phase B over 10 minutes
Flow Rate:	250 µL/min
Solvent A:	100% water; 10 mM ammonium formate; 0.1% formic acid
Solvent B:	100% acetonitrile; 0.1% formic acid
Chromatography Type:	HILIC

Chromatography ID:	CH002988
Instrument Name:	Shimadzu Nexera X2
Column Name:	Phenomenex Luna NH2 (150 x 2.1mm,3um)
Column Temperature:	30C
Flow Gradient:	The column was eluted with initial conditions of 10% mobile phase A and 90% mobile phase B followed by a 10 min linear gradient to 100% mobile phase A.
Flow Rate:	400 µL/min
Solvent A:	100% water; 20 mM ammonium acetate; 20 mM ammonium hydroxide
Solvent B:	75% acetonitrile/25% methanol; 10 mM ammonium hydroxide
Chromatography Type:	HILIC

MS:

MS ID:	MS003786
Analysis ID:	AN004039
Instrument Name:	Thermo Q Exactive Plus Orbitrap
Instrument Type:	Orbitrap
MS Type:	ESI
MS Comments:	Raw data were processed using TraceFinder 3.3 software (Thermo Fisher Scientific; Waltham, MA) and Progenesis QI (Nonlinear Dynamics; Newcastle upon Tyne, UK). Metabolite identities were confirmed using authentic reference standards or reference samples.
Ion Mode:	POSITIVE

MS ID:	MS003787
Analysis ID:	AN004040
Instrument Name:	Thermo Q Exactive Plus Orbitrap
Instrument Type:	Orbitrap
MS Type:	ESI
MS Comments:	Raw data were processed using TraceFinder 3.3 software (Thermo Fisher Scientific; Waltham, MA) and Progenesis QI (Nonlinear Dynamics; Newcastle upon Tyne, UK). Metabolite identities were confirmed using authentic reference standards or reference samples.
Ion Mode:	NEGATIVE