Summary of Study ST004099

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 PR002575. The data can be accessed directly via it's Project DOI: 10.21228/M8SV6X This work is supported by NIH grant, U2C- DK119886. See: https://www.metabolomicsworkbench.org/about/howtocite.php

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Study ID	ST004099
Study Title	A genome-scale metabolic model for deciphering the host metabolic perturbations during Mycobacterium tuberculosis infection
Study Type	Metabolomics
Study Summary	Conventional tuberculosis (TB) research predominantly relies on forward-designed experiments, such as studying host responses to Mycobacterium tuberculosis (Mtb) gene knockouts. While informative, these approaches offer only a partial view of host-pathogen interactions and often overlook the broader alterations in the host microenvironment during infection. To address this limitation, we adopted a backtracing strategy to retrospectively identify host metabolic modulators and link them to specific Mtb virulence factors. Using RNA-seq data from Mtb-infected mouse lung tissue, we integrated transcriptomic profiles into a genome-scale metabolic model to predict host metabolic genes essential for Mtb survival. This analysis identified 18 host proteins as putative modulators. We then constructed a host-pathogen protein interaction network, which connected these host factors to 9 Mtb proteins. Experimental validation using Mtb knockdown strains confirmed that three genes Rv1970, Rv0243, and Rv2234 play key roles in manipulating host responses rather than supporting intrinsic bacterial viability. Further, we employed targeted proteomics and untargeted metabolomics analyses on THP-1 macrophages infected with these KD strains to dissect the host-pathogen interaction mechanisms in greater detail. These findings in troduce a novel framework for decoding host-pathogen interactions and lay the groundwork for future host-directed therapy (HDT) strategies targeting Mtb-induced host vulnerabilities.
Institute	Translational health science and technology institute
Department	NCD
Laboratory	Biomarker lab
Last Name	Kumar
First Name	Yashwant
Address	NCR Biotech Science Cluster,, Faridabad, Haryana, 121001, India
Email	y.kumar@thsti.res.in
Phone	+911292876496
Submit Date	2025-08-04
Raw Data Available	Yes
Raw Data File Type(s)	mzXML
Analysis Type Detail	LC-MS
Release Date	2025-08-25
Release Version	1

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Project:

Project ID:	PR002575
Project DOI:	doi: 10.21228/M8SV6X
Project Title:	A genome-scale metabolic model for deciphering the host metabolic perturbations during Mycobacterium tuberculosis infection
Project Type:	Metabolomics
Project Summary:	Conventional tuberculosis (TB) research predominantly relies on forward-designed experiments, such as studying host responses to Mycobacterium tuberculosis (Mtb) gene knockouts. While informative, these approaches offer only a partial view of host-pathogen interactions and often overlook the broader alterations in the host microenvironment during infection. To address this limitation, we adopted a backtracing strategy to retrospectively identify host metabolic modulators and link them to specific Mtb virulence factors. Using RNA-seq data from Mtb-infected mouse lung tissue, we integrated transcriptomic profiles into a genome-scale metabolic model to predict host metabolic genes essential for Mtb survival. This analysis identified 18 host proteins as putative modulators. We then constructed a host-pathogen protein interaction network, which connected these host factors to 9 Mtb proteins. Experimental validation using Mtb knockdown strains confirmed that three genes Rv1970, Rv0243, and Rv2234 play key roles in manipulating host responses rather than supporting intrinsic bacterial viability. Further, we employed targeted proteomics and untargeted metabolomics analyses on THP-1 macrophages infected with these KD strains to dissect the host-pathogen interaction mechanisms in greater detail. These findings in troduce a novel framework for decoding host-pathogen interactions and lay the groundwork for future host-directed therapy (HDT) strategies targeting Mtb-induced host vulnerabilities.
Institute:	Translational health science and technology institute
Department:	NCD
Laboratory:	Biomarker lab
Last Name:	Kumar
First Name:	Yashwant
Address:	NCR Biotech Science Cluster,, Faridabad, Haryana, 121001, India
Email:	y.kumar@thsti.res.in
Phone:	01292876496
Funding Source:	THSTI

Subject:

Subject ID:	SU004246
Subject Type:	Cultured cells
Subject Species:	Homo sapiens
Taxonomy ID:	9606

Factors:

Subject type: Cultured cells; Subject species: Homo sapiens (Factor headings shown in green)

mb_sample_id	local_sample_id	Treatment	Sample source
SA474528	37	Rv0243+Atc1	Acute monocytic leukemia cell line
SA474534	31	Rv0243-Atc1	Acute monocytic leukemia cell line
SA474529	38	Rv0243+Atc2	Acute monocytic leukemia cell line
SA474535	32	Rv0243-Atc2	Acute monocytic leukemia cell line
SA474530	39	Rv0243+Atc3	Acute monocytic leukemia cell line
SA474536	33	Rv0243-Atc3	Acute monocytic leukemia cell line
SA474531	40	Rv0243+Atc4	Acute monocytic leukemia cell line
SA474537	34	Rv0243-Atc4	Acute monocytic leukemia cell line
SA474532	41	Rv0243+Atc5	Acute monocytic leukemia cell line
SA474538	35	Rv0243-Atc5	Acute monocytic leukemia cell line
SA474533	42	Rv0243+Atc6	Acute monocytic leukemia cell line
SA474539	36	Rv0243-Atc6	Acute monocytic leukemia cell line
SA474540	13	Rv0410+Atc1	Acute monocytic leukemia cell line
SA474546	7	Rv0410-Atc1	Acute monocytic leukemia cell line
SA474541	14	Rv0410+Atc2	Acute monocytic leukemia cell line
SA474547	8	Rv0410-Atc2	Acute monocytic leukemia cell line
SA474542	15	Rv0410+Atc3	Acute monocytic leukemia cell line
SA474548	9	Rv0410-Atc3	Acute monocytic leukemia cell line
SA474543	16	Rv0410+Atc4	Acute monocytic leukemia cell line
SA474549	10	Rv0410-Atc4	Acute monocytic leukemia cell line
SA474544	17	Rv0410+Atc5	Acute monocytic leukemia cell line
SA474550	11	Rv0410-Atc5	Acute monocytic leukemia cell line
SA474545	18	Rv0410+Atc6	Acute monocytic leukemia cell line
SA474551	12	Rv0410-Atc6	Acute monocytic leukemia cell line
SA474552	61	Rv1970+Atc1	Acute monocytic leukemia cell line
SA474558	55	Rv1970-Atc1	Acute monocytic leukemia cell line
SA474553	62	Rv1970+Atc2	Acute monocytic leukemia cell line
SA474559	56	Rv1970-Atc2	Acute monocytic leukemia cell line
SA474554	63	Rv1970+Atc3	Acute monocytic leukemia cell line
SA474560	57	Rv1970-Atc3	Acute monocytic leukemia cell line
SA474555	64	Rv1970+Atc4	Acute monocytic leukemia cell line
SA474561	58	Rv1970-Atc4	Acute monocytic leukemia cell line
SA474556	65	Rv1970+Atc5	Acute monocytic leukemia cell line
SA474562	59	Rv1970-Atc5	Acute monocytic leukemia cell line
SA474557	66	Rv1970+Atc6	Acute monocytic leukemia cell line
SA474563	60	Rv1970-Atc6	Acute monocytic leukemia cell line
SA474564	25	Rv2234+Atc1	Acute monocytic leukemia cell line
SA474570	19	Rv2234-Atc1	Acute monocytic leukemia cell line
SA474565	26	Rv2234+Atc2	Acute monocytic leukemia cell line
SA474571	20	Rv2234-Atc2	Acute monocytic leukemia cell line
SA474566	27	Rv2234+Atc3	Acute monocytic leukemia cell line
SA474572	21	Rv2234-Atc3	Acute monocytic leukemia cell line
SA474567	28	Rv2234+Atc4	Acute monocytic leukemia cell line
SA474573	22	Rv2234-Atc4	Acute monocytic leukemia cell line
SA474568	29	Rv2234+Atc5	Acute monocytic leukemia cell line
SA474574	23	Rv2234-Atc5	Acute monocytic leukemia cell line
SA474569	30	Rv2234+Atc6	Acute monocytic leukemia cell line
SA474575	24	Rv2234-Atc6	Acute monocytic leukemia cell line
SA474576	49	Rv3774+Atc1	Acute monocytic leukemia cell line
SA474582	43	Rv3774-Atc1	Acute monocytic leukemia cell line
SA474577	50	Rv3774+Atc2	Acute monocytic leukemia cell line
SA474583	44	Rv3774-Atc2	Acute monocytic leukemia cell line
SA474578	51	Rv3774+Atc3	Acute monocytic leukemia cell line
SA474584	45	Rv3774-Atc3	Acute monocytic leukemia cell line
SA474579	52	Rv3774+Atc4	Acute monocytic leukemia cell line
SA474585	46	Rv3774-Atc4	Acute monocytic leukemia cell line
SA474580	53	Rv3774+Atc5	Acute monocytic leukemia cell line
SA474586	47	Rv3774-Atc5	Acute monocytic leukemia cell line
SA474581	54	Rv3774+Atc6	Acute monocytic leukemia cell line
SA474587	48	Rv3774-Atc6	Acute monocytic leukemia cell line
SA474588	1	Uninfected1	Acute monocytic leukemia cell line
SA474589	2	Uninfected2	Acute monocytic leukemia cell line
SA474590	3	Uninfected3	Acute monocytic leukemia cell line
SA474591	4	Uninfected4	Acute monocytic leukemia cell line
SA474592	5	Uninfected5	Acute monocytic leukemia cell line
SA474593	6	Uninfected6	Acute monocytic leukemia cell line

Showing results 1 to 66 of 66

Showing results 1 to 66 of 66

Collection:

Collection ID:	CO004239
Collection Summary:	THP-1 cells (2x10^6) were seeded in 6-well plates and treated with 50 ng/mL PMA for 36 hours to induce differentiation. After PMA treatment, the medium was replaced with fresh RPMI, and the cells were allowed to mature for an additional 24 hours. Mtb infection was performed at a MOI of 5 and un-infected cells were maintained in parallel culture as control. After 4 hours of infection, extracellular bacteria were removed by three washes with pre-warmed PBS. A complete RPMI medium supplemented with 400 ng/mL ATc was added to the wells to induce the knockdown effect. The infected cells were incubated for 48 hours before being washed again with pre-warmed PBS.
Sample Type:	Human acute monocytic leukemia cell line

Treatment:

Treatment ID:	TR004255
Treatment Summary:	THP-1 cells (2x10^6) were seeded in 6-well plates and treated with 50 ng/mL PMA for 36 hours to induce differentiation. After PMA treatment, the medium was replaced with fresh RPMI, and the cells were allowed to mature for an additional 24 hours. Mtb infection was performed at a MOI of 5 and un-infected cells were maintained in parallel culture as control. After 4 hours of infection, extracellular bacteria were removed by three washes with pre-warmed PBS. A complete RPMI medium supplemented with 400 ng/mL ATc was added to the wells to induce the knockdown effect. The infected cells were incubated for 48 hours before being washed again with pre-warmed PBS.

Treatment ID:

TR004255

Treatment Summary:

THP-1 cells (2x10^6) were seeded in 6-well plates and treated with 50 ng/mL PMA for 36 hours to induce differentiation. After PMA treatment, the medium was replaced with fresh RPMI, and the cells were allowed to mature for an additional 24 hours. Mtb infection was performed at a MOI of 5 and un-infected cells were maintained in parallel culture as control. After 4 hours of infection, extracellular bacteria were removed by three washes with pre-warmed PBS. A complete RPMI medium supplemented with 400 ng/mL ATc was added to the wells to induce the knockdown effect. The infected cells were incubated for 48 hours before being washed again with pre-warmed PBS.

Sample Preparation:

Sampleprep ID:	SP004252
Sampleprep Summary:	To quench metabolism and extract metabolites, 1 mL of chilled methanol (HPLC grade; Sigma) was added to the cells. The cells were harvested on ice using a scraper, and the resulting extracts were transferred to pre-cooled microcentrifuge tubes (MCTs) maintained at 4°C. The extracts were vortexed for 15 minutes and centrifuged at 12000 rpm for 10 minutes at 4°C. After centrifugation, the supernatants were transferred to fresh pre-cooled MCTs (4°C), dried under vacuum, and reconstituted. The reconstituted samples were stored at -80°C until further analysis by liquid chromatography-mass spectrometry (LC-MS).

Sampleprep ID:

SP004252

Sampleprep Summary:

To quench metabolism and extract metabolites, 1 mL of chilled methanol (HPLC grade; Sigma) was added to the cells. The cells were harvested on ice using a scraper, and the resulting extracts were transferred to pre-cooled microcentrifuge tubes (MCTs) maintained at 4°C. The extracts were vortexed for 15 minutes and centrifuged at 12000 rpm for 10 minutes at 4°C. After centrifugation, the supernatants were transferred to fresh pre-cooled MCTs (4°C), dried under vacuum, and reconstituted. The reconstituted samples were stored at -80°C until further analysis by liquid chromatography-mass spectrometry (LC-MS).

Combined analysis:

Analysis ID	AN006795	AN006796	AN006797	AN006798
Chromatography ID	CH005163	CH005163	CH005164	CH005164
MS ID	MS006494	MS006495	MS006496	MS006497
Analysis type	MS	MS	MS	MS
Chromatography type	Reversed phase	Reversed phase	HILIC	HILIC
Chromatography system	Thermo Dionex Ultimate 3000 RS	Thermo Dionex Ultimate 3000 RS	Thermo Dionex Ultimate 3000 RS	Thermo Dionex Ultimate 3000 RS
Column	Waters ACQUITY UPLC HSS T3 (100 x 2.1 mm, 1.8 µm)	Waters ACQUITY UPLC HSS T3 (100 x 2.1 mm, 1.8 µm)	Waters XBridge BEH Amide (100 x 2.1 mm, 2.5 µm)	Waters XBridge BEH Amide (100 x 2.1 mm, 2.5 µm)
MS Type	ESI	ESI	ESI	ESI
MS instrument type	Orbitrap	Orbitrap	Orbitrap	Orbitrap
MS instrument name	Thermo Fusion Tribrid Orbitrap	Thermo Fusion Tribrid Orbitrap	Thermo Fusion Tribrid Orbitrap	Thermo Fusion Tribrid Orbitrap
Ion Mode	POSITIVE	NEGATIVE	POSITIVE	NEGATIVE
Units	relative intensity	relative intensity	relative intensity	relative intensity

Chromatography:

Chromatography ID:	CH005163
Chromatography Summary:	Extracted metabolites were separated on UPLC ultimate 3,000. Data were acquired on the reverse phase and HILIC column with positive and negative ionization modes. The reverse phase column was HSS T3, and the HILIC column was XBridge BEH Amide (Waters Corporation).
Instrument Name:	Thermo Dionex Ultimate 3000 RS
Column Name:	Waters ACQUITY UPLC HSS T3 (100 x 2.1 mm, 1.8 µm)
Column Temperature:	40°C
Flow Gradient:	1% B to 95% B in 10 minutes
Flow Rate:	300 µL/min
Solvent A:	100% Water; 0.1% Formic acid
Solvent B:	100% Methanol; 0.1% Formic acid
Chromatography Type:	Reversed phase

Chromatography ID:	CH005164
Chromatography Summary:	Extracted metabolites were separated on UPLC ultimate 3,000. Data were acquired on the reverse phase and HILIC column with positive and negative ionization modes. The reverse phase column was HSS T3, and the HILIC column was XBridge BEH Amide (Waters Corporation).
Instrument Name:	Thermo Dionex Ultimate 3000 RS
Column Name:	Waters XBridge BEH Amide (100 x 2.1 mm, 2.5 µm)
Column Temperature:	40°C
Flow Gradient:	85% B and proceeds to 10% B over 14 minutes
Flow Rate:	200 µL/min
Solvent A:	100% Water; 20 mM ammonium acetate (pH-9.0)
Solvent B:	100% Acetonitrile
Chromatography Type:	HILIC

MS:

MS ID:	MS006494
Analysis ID:	AN006795
Instrument Name:	Thermo Fusion Tribrid Orbitrap
Instrument Type:	Orbitrap
MS Type:	ESI
MS Comments:	An Orbitrap Fusion mass spectrometer (Thermo Scientific), coupled with a heated electrospray ion source, was used for data acquisition. For MS1 mode, a mass resolution was kept at 120,000, and for MS2 acquisition, a mass resolution was 30,000. The mass range of data acquisition was 60–900 Da. Extracted metabolites were separated on UPLC ultimate 3,000. Data were acquired on the reverse phase and HILIC column with positive and negative ionization modes. The reverse phase column was HSS T3, and the HILIC column was XBridge BEH Amide (Waters Corporation). For polar compound separation, solvent A was 20 mM ammonium acetate in water of PH 9.0, and mobile phase B was 100% acetonitrile. The elution gradient starts from 85% B to 10% B over 14 min with a flow rate of 0.35 ml/min. For the reverse phase, Solvent A was water, and B was methanol with 0.1% formic acid added in both. The elution gradient starts with 1% B to 95% B over 10 min with a flow rate of 0.3 ml/min. sample injection volume was 5 µl.
Ion Mode:	POSITIVE

MS ID:	MS006495
Analysis ID:	AN006796
Instrument Name:	Thermo Fusion Tribrid Orbitrap
Instrument Type:	Orbitrap
MS Type:	ESI
MS Comments:	An Orbitrap Fusion mass spectrometer (Thermo Scientific), coupled with a heated electrospray ion source, was used for data acquisition. For MS1 mode, a mass resolution was kept at 120,000, and for MS2 acquisition, a mass resolution was 30,000. The mass range of data acquisition was 60–900 Da. Extracted metabolites were separated on UPLC ultimate 3,000. Data were acquired on the reverse phase and HILIC column with positive and negative ionization modes. The reverse phase column was HSS T3, and the HILIC column was XBridge BEH Amide (Waters Corporation). For polar compound separation, solvent A was 20 mM ammonium acetate in water of PH 9.0, and mobile phase B was 100% acetonitrile. The elution gradient starts from 85% B to 10% B over 14 min with a flow rate of 0.35 ml/min. For the reverse phase, Solvent A was water, and B was methanol with 0.1% formic acid added in both. The elution gradient starts with 1% B to 95% B over 10 min with a flow rate of 0.3 ml/min. sample injection volume was 5 µl.
Ion Mode:	NEGATIVE

MS ID:	MS006496
Analysis ID:	AN006797
Instrument Name:	Thermo Fusion Tribrid Orbitrap
Instrument Type:	Orbitrap
MS Type:	ESI
MS Comments:	An Orbitrap Fusion mass spectrometer (Thermo Scientific), coupled with a heated electrospray ion source, was used for data acquisition. For MS1 mode, a mass resolution was kept at 120,000, and for MS2 acquisition, a mass resolution was 30,000. The mass range of data acquisition was 60–900 Da. Extracted metabolites were separated on UPLC ultimate 3,000. Data were acquired on the reverse phase and HILIC column with positive and negative ionization modes. The reverse phase column was HSS T3, and the HILIC column was XBridge BEH Amide (Waters Corporation). For polar compound separation, solvent A was 20 mM ammonium acetate in water of PH 9.0, and mobile phase B was 100% acetonitrile. The elution gradient starts from 85% B to 10% B over 14 min with a flow rate of 0.35 ml/min. For the reverse phase, Solvent A was water, and B was methanol with 0.1% formic acid added in both. The elution gradient starts with 1% B to 95% B over 10 min with a flow rate of 0.3 ml/min. sample injection volume was 5 µl.
Ion Mode:	POSITIVE

MS ID:	MS006497
Analysis ID:	AN006798
Instrument Name:	Thermo Fusion Tribrid Orbitrap
Instrument Type:	Orbitrap
MS Type:	ESI
MS Comments:	An Orbitrap Fusion mass spectrometer (Thermo Scientific), coupled with a heated electrospray ion source, was used for data acquisition. For MS1 mode, a mass resolution was kept at 120,000, and for MS2 acquisition, a mass resolution was 30,000. The mass range of data acquisition was 60–900 Da. Extracted metabolites were separated on UPLC ultimate 3,000. Data were acquired on the reverse phase and HILIC column with positive and negative ionization modes. The reverse phase column was HSS T3, and the HILIC column was XBridge BEH Amide (Waters Corporation). For polar compound separation, solvent A was 20 mM ammonium acetate in water of PH 9.0, and mobile phase B was 100% acetonitrile. The elution gradient starts from 85% B to 10% B over 14 min with a flow rate of 0.35 ml/min. For the reverse phase, Solvent A was water, and B was methanol with 0.1% formic acid added in both. The elution gradient starts with 1% B to 95% B over 10 min with a flow rate of 0.3 ml/min. sample injection volume was 5 µl.
Ion Mode:	NEGATIVE