#METABOLOMICS WORKBENCH common_aslab_20250818_044210 DATATRACK_ID:6302 STUDY_ID:ST004134 ANALYSIS_ID:AN006854 PROJECT_ID:PR002596
VERSION             	1
CREATED_ON             	August 20, 2025, 5:31 pm
#PROJECT
PR:PROJECT_TITLE                 	Bioenergetic reprogramming of macrophages reduces drug tolerance in
PR:PROJECT_TITLE                 	Mycobacterium tuberculosis
PR:PROJECT_TYPE                  	Research
PR:PROJECT_SUMMARY               	Effective clearance of Mycobacterium tuberculosis (Mtb) requires targeting
PR:PROJECT_SUMMARY               	drug-tolerant populations within host macrophages. Here, we show that macrophage
PR:PROJECT_SUMMARY               	metabolic states govern redox heterogeneity and drug response in intracellular
PR:PROJECT_SUMMARY               	Mtb. Using a redox-sensitive fluorescent reporter (Mrx1-roGFP2), flow cytometry,
PR:PROJECT_SUMMARY               	and transcriptomics, we found that macrophages with high oxidative
PR:PROJECT_SUMMARY               	phosphorylation (OXPHOS) and low glycolysis harbor reductive, drug-tolerant Mtb,
PR:PROJECT_SUMMARY               	whereas glycolytically active macrophages generate mitochondrial ROS via reverse
PR:PROJECT_SUMMARY               	electron transport, imposing oxidative stress on Mtb and enhancing drug
PR:PROJECT_SUMMARY               	efficacy. Computational and genetic analyses identified Nrf2 as a key regulator
PR:PROJECT_SUMMARY               	linking host metabolism to bacterial redox state and drug tolerance.
PR:PROJECT_SUMMARY               	Pharmacological reprogramming of macrophages with the FDA-approved drug
PR:PROJECT_SUMMARY               	meclizine (MEC) shifted metabolism toward glycolysis, suppressed redox
PR:PROJECT_SUMMARY               	heterogeneity, and reduced Mtb drug tolerance in macrophages and mice. MEC
PR:PROJECT_SUMMARY               	exhibited no adverse interactions with frontline anti-TB drugs. These findings
PR:PROJECT_SUMMARY               	demonstrate the therapeutic potential of host metabolic reprogramming to
PR:PROJECT_SUMMARY               	overcome Mtb drug tolerance.
PR:INSTITUTE                     	Indian Institute of Science
PR:DEPARTMENT                    	Microbiology and Cell Biology
PR:LAST_NAME                     	Singh
PR:FIRST_NAME                    	Amit
PR:ADDRESS                       	Centre for Infectious Disease Research, Bangalore, Karnataka, 560012, India
PR:EMAIL                         	common.aslab@gmail.com
PR:PHONE                         	+918022933273
#STUDY
ST:STUDY_TITLE                   	Metabolomic analysis of glycolytic intermediates in Mycobacterium
ST:STUDY_TITLE                   	tuberculosis-infected BMDMs (Bone marrow-derived macrophages)
ST:STUDY_SUMMARY                 	Glycolytic intermediates were analyzed for Mycobacterium tuberculosis (Mtb)
ST:STUDY_SUMMARY                 	(Mtb)-infected BMDMs (Bone marrow-derived macrophages) either treated with
ST:STUDY_SUMMARY                 	vehicle control (0.2% DMSO) or 20 μM meclizine. In another set, the same
ST:STUDY_SUMMARY                 	metabolites were measured for Mtb-infected BMDMs grown in 10 mM glucose or
ST:STUDY_SUMMARY                 	galactose as the sole sugar sources in DMEM medium.
ST:INSTITUTE                     	Indian Institute of Science
ST:LAST_NAME                     	Singh
ST:FIRST_NAME                    	Amit
ST:ADDRESS                       	Centre for Infectious Disease Research, Bangalore, Karnataka, 560012, India
ST:EMAIL                         	common.aslab@gmail.com
ST:PHONE                         	+918022933273
#SUBJECT
SU:SUBJECT_TYPE                  	Cultured cells
SU:SUBJECT_SPECIES               	Mus musculus
SU:TAXONOMY_ID                   	10090
SU:GENOTYPE_STRAIN               	C57BL/6
SU:AGE_OR_AGE_RANGE              	8-10 weeks
SU:WEIGHT_OR_WEIGHT_RANGE        	20-25
SU:GENDER                        	Female
SU:CELL_BIOSOURCE_OR_SUPPLIER    	Isolated from the bone marrow.
#SUBJECT_SAMPLE_FACTORS:         	SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Raw file names and additional sample data
SUBJECT_SAMPLE_FACTORS           	-	DMSO_a1	Sample source:Bone marrow-derived macrophages | Treatment:DMSO	RAW_FILE_NAME(Raw file name 1)=DMSO_1.wiff; RAW_FILE_NAME(Raw file name 2)=DMSO_1.wiff.scan
SUBJECT_SAMPLE_FACTORS           	-	DMSO_a2	Sample source:Bone marrow-derived macrophages | Treatment:DMSO	RAW_FILE_NAME(Raw file name 1)=DMSO_2.wiff; RAW_FILE_NAME(Raw file name 2)=DMSO_2.wiff.scan
SUBJECT_SAMPLE_FACTORS           	-	DMSO_a3	Sample source:Bone marrow-derived macrophages | Treatment:DMSO	RAW_FILE_NAME(Raw file name 1)=DMSO_3.wiff; RAW_FILE_NAME(Raw file name 2)=DMSO_3.wiff.scan
SUBJECT_SAMPLE_FACTORS           	-	Mec_a1	Sample source:Bone marrow-derived macrophages | Treatment:Mec 20uM	RAW_FILE_NAME(Raw file name 1)=Mec_1.wiff; RAW_FILE_NAME(Raw file name 2)=Mec_1.wiff.scan
SUBJECT_SAMPLE_FACTORS           	-	Mec_a2	Sample source:Bone marrow-derived macrophages | Treatment:Mec 20uM	RAW_FILE_NAME(Raw file name 1)=Mec_2.wiff; RAW_FILE_NAME(Raw file name 2)=Mec_2.wiff.scan
SUBJECT_SAMPLE_FACTORS           	-	Uk5_a2	Sample source:Bone marrow-derived macrophages | Treatment:UK5099 10uM	RAW_FILE_NAME(Raw file name 1)=Uk5_2.wiff; RAW_FILE_NAME(Raw file name 2)=Uk5_2.wiff.scan
SUBJECT_SAMPLE_FACTORS           	-	Uk5_a3	Sample source:Bone marrow-derived macrophages | Treatment:UK5099 10uM	RAW_FILE_NAME(Raw file name 1)=Uk5_3.wiff; RAW_FILE_NAME(Raw file name 2)=Uk5_3.wiff.scan
SUBJECT_SAMPLE_FACTORS           	-	Glu_a1	Sample source:Bone marrow-derived macrophages | Treatment:Glucose 10mM	RAW_FILE_NAME(Raw file name 1)=Glu_1.wiff; RAW_FILE_NAME(Raw file name 2)=Glu_1.wiff.scan
SUBJECT_SAMPLE_FACTORS           	-	Glu_b1	Sample source:Bone marrow-derived macrophages | Treatment:Glucose 10mM	RAW_FILE_NAME(Raw file name 1)=Glu_2.wiff; RAW_FILE_NAME(Raw file name 2)=Glu_2.wiff.scan
SUBJECT_SAMPLE_FACTORS           	-	Glu_c1	Sample source:Bone marrow-derived macrophages | Treatment:Glucose 10mM	RAW_FILE_NAME(Raw file name 1)=Glu_3.wiff; RAW_FILE_NAME(Raw file name 2)=Glu_3.wiff.scan
SUBJECT_SAMPLE_FACTORS           	-	Gal_a1	Sample source:Bone marrow-derived macrophages | Treatment:Galactose 10mM	RAW_FILE_NAME(Raw file name 1)=Gal_1.wiff; RAW_FILE_NAME(Raw file name 2)=Gal_1.wiff.scan
SUBJECT_SAMPLE_FACTORS           	-	Gal_b1	Sample source:Bone marrow-derived macrophages | Treatment:Galactose 10mM	RAW_FILE_NAME(Raw file name 1)=Gal_2.wiff; RAW_FILE_NAME(Raw file name 2)=Gal_2.wiff.scan
SUBJECT_SAMPLE_FACTORS           	-	Gal_c1	Sample source:Bone marrow-derived macrophages | Treatment:Galactose 10mM	RAW_FILE_NAME(Raw file name 1)=Gal_3.wiff; RAW_FILE_NAME(Raw file name 2)=Gal_3.wiff.scan
#COLLECTION
CO:COLLECTION_SUMMARY            	Bone marrow of female C57BL/6 mice were isolated from the long bones of the
CO:COLLECTION_SUMMARY            	legs, femur and tibia. The entire marrow was incubated for 6 days in culture
CO:COLLECTION_SUMMARY            	medium- DMEM+10%FBS+2 mM glutamine+10 mM HEPES+1 mM sodium pyruvate+30 ng/ml
CO:COLLECTION_SUMMARY            	macrophage colony-stimulating factor (MCSF) to differentiate monocytes into
CO:COLLECTION_SUMMARY            	macrophages. Post 6 days, the attached cells were kept, and the supernatant was
CO:COLLECTION_SUMMARY            	washed off. These cells were infected with Mycobacterium tuberculosis at an moi
CO:COLLECTION_SUMMARY            	of 2 for 3 hours and 24 hours post infection kept under different treatment
CO:COLLECTION_SUMMARY            	conditions described in the next section. During the entirety of the experiment,
CO:COLLECTION_SUMMARY            	the cells were incubated at 37 °C and 5% CO2. Post-treatment, the cells were
CO:COLLECTION_SUMMARY            	scraped off in 80% ethanol, heated at 80°C for 90 seconds, vortexed and heated
CO:COLLECTION_SUMMARY            	again for 90 seconds at 80°C. Post heating, they were immediately transferred
CO:COLLECTION_SUMMARY            	to an ice bath for 5 minutes.Post that,the cells were centrifuged at 10000 rpm
CO:COLLECTION_SUMMARY            	for 5 minutes with the temperature maintained at 4°C. The supernatant was
CO:COLLECTION_SUMMARY            	collected, lyophilised and stored at -80°C until further analysis described in
CO:COLLECTION_SUMMARY            	"Sample prep".Post-treatment, the cells were scraped off in 80% ethanol, heated
CO:COLLECTION_SUMMARY            	at 80°C for 90 seconds, vortexed and heated again for 90 seconds at 80°C. Post
CO:COLLECTION_SUMMARY            	heating, they were immediately transferred to an ice bath for 5 minutes.
CO:COLLECTION_PROTOCOL_FILENAME  	BMDM_isolation_protocol.pdf
CO:SAMPLE_TYPE                   	Macrophages
#TREATMENT
TR:TREATMENT_SUMMARY             	BMDMs were infected with Mtb at a multiplicity of infection (moi) of 2, post
TR:TREATMENT_SUMMARY             	which the cells were divided into different treatment groups. They were either
TR:TREATMENT_SUMMARY             	treated for 24 hours with vehicle control (0.2% DMSO), 10 μM UK5099, 20 μM
TR:TREATMENT_SUMMARY             	Meclizine hydrochloride, 10 mM glucose or 10 mM galactose. During the treatment,
TR:TREATMENT_SUMMARY             	cells were incubated at 37°C and 5% CO2. Post treatment, cells were scraped off
TR:TREATMENT_SUMMARY             	and prepared for analysis as described in the "Sample prep" section.
#SAMPLEPREP
SP:SAMPLEPREP_SUMMARY            	Metabolites were extracted, resuspended in required solvents (water for sugar
SP:SAMPLEPREP_SUMMARY            	phosphates) and separated on a Synergi 4-µm Fusion-RP 80 Å LC column (150 ×
SP:SAMPLEPREP_SUMMARY            	4.6 mm, Phenomenex) using a Shimadzu Nexera UHPLC system. Solvent system
SP:SAMPLEPREP_SUMMARY            	employed for sugar phosphates—Solvent A was 5 mM ammonium acetate in water,
SP:SAMPLEPREP_SUMMARY            	and Solvent B was 100% acetonitrile. Metabolite detection was performed using an
SP:SAMPLEPREP_SUMMARY            	AB Sciex Qtrap 5500 mass spectrometer with data acquired via Analyst 1.6.2
SP:SAMPLEPREP_SUMMARY            	software (Sciex). Sugar phosphates were measured in negative ion mode.
SP:SAMPLEPREP_SUMMARY            	Quantification was carried out by calculating peak areas using MultiQuant
SP:SAMPLEPREP_SUMMARY            	software (version 3.0.1).
SP:PROCESSING_STORAGE_CONDITIONS 	-20℃
#CHROMATOGRAPHY
CH:CHROMATOGRAPHY_SUMMARY        	Liquid Chromatography
CH:CHROMATOGRAPHY_TYPE           	Reversed phase
CH:INSTRUMENT_NAME               	Shimadzu Nexera UHPLC system
CH:COLUMN_NAME                   	Phenomenex Synergi Fusion-RP (100 x 4.6mm,4um)
CH:SOLVENT_A                     	0.1% formic acid in water
CH:SOLVENT_B                     	0.1% formic acid in methanol
CH:FLOW_GRADIENT                 	T = 0 min, 0% B; T = 3 min, 5% B; T = 10 min, 60% B; T = 11 min, 95% B; T = 14
CH:FLOW_GRADIENT                 	min, 95% B; T = 15 min, 5% B; T = 16 min, 0% B; T = 21 min, stop
CH:FLOW_RATE                     	0.4 ml/min
CH:COLUMN_TEMPERATURE            	25
#ANALYSIS
AN:ANALYSIS_TYPE                 	MS
#MS
MS:INSTRUMENT_NAME               	ABI Sciex 5500 QTrap
MS:INSTRUMENT_TYPE               	Triple quadrupole
MS:MS_TYPE                       	ESI
MS:ION_MODE                      	NEGATIVE
MS:MS_COMMENTS                   	Metabolite detection was performed using an AB Sciex Qtrap 5500 mass
MS:MS_COMMENTS                   	spectrometer with data acquired via Analyst 1.6.2 software (Sciex). TCA
MS:MS_COMMENTS                   	intermediates were analyzed in positive ion mode, while sugar phosphates were
MS:MS_COMMENTS                   	measured in negative ion mode. Quantification was carried out by calculating
MS:MS_COMMENTS                   	peak areas using MultiQuant software (version 3.0.1) as explained in detail in
MS:MS_COMMENTS                   	Niphadkar and Sreedharan et al., STAR protocols (2025)
MS:MS_COMMENTS                   	doi:https://doi.org/10.1016/j.xpro.2025.103786. The MS analysis protocol is as
MS:MS_COMMENTS                   	follows: 1. Make a quantitation method using data from the standard. a. Set
MS:MS_COMMENTS                   	retention times for the metabolites. b. Set integration parameters as follows:
MS:MS_COMMENTS                   	Gaussian smooth width-2, RT half window-30 s, Minimum peak width-3 points,
MS:MS_COMMENTS                   	minimum peak height-0, noise percentage-75%, baseline sub. window- 2 min, report
MS:MS_COMMENTS                   	largest peak-yes. 2. Use this quantitation method, to analyze data from the
MS:MS_COMMENTS                   	samples. 3. Wherever needed, manual integration of peaks can be done. 4.
MS:MS_COMMENTS                   	Calculate the area under the curve for the metabolites.
#MS_METABOLITE_DATA
MS_METABOLITE_DATA:UNITS	peak area
MS_METABOLITE_DATA_START
Samples	DMSO_a1	DMSO_a2	DMSO_a3	Gal_a1	Gal_b1	Gal_c1	Glu_a1	Glu_b1	Glu_c1	Mec_a1	Mec_a2	Uk5_a2	Uk5_a3
Factors	Sample source:Bone marrow-derived macrophages | Treatment:DMSO	Sample source:Bone marrow-derived macrophages | Treatment:DMSO	Sample source:Bone marrow-derived macrophages | Treatment:DMSO	Sample source:Bone marrow-derived macrophages | Treatment:Galactose 10mM	Sample source:Bone marrow-derived macrophages | Treatment:Galactose 10mM	Sample source:Bone marrow-derived macrophages | Treatment:Galactose 10mM	Sample source:Bone marrow-derived macrophages | Treatment:Glucose 10mM	Sample source:Bone marrow-derived macrophages | Treatment:Glucose 10mM	Sample source:Bone marrow-derived macrophages | Treatment:Glucose 10mM	Sample source:Bone marrow-derived macrophages | Treatment:Mec 20uM	Sample source:Bone marrow-derived macrophages | Treatment:Mec 20uM	Sample source:Bone marrow-derived macrophages | Treatment:UK5099 10uM	Sample source:Bone marrow-derived macrophages | Treatment:UK5099 10uM
Uridine diphosphate N-acetylglucosamine	2.03E+04	6.31E+03	5.83E+03	8.13E+03	7.81E+03	8.17E+03	6.47E+03	1.23E+04	1.29E+04	1.21E+04	1.23E+04	9.83E+03	1.19E+04
Glyceraldehyde 3-phosphate	7.41E+05	1.48E+06	1.25E+06	1.32E+05	2.29E+05	1.39E+05	5.45E+05	7.36E+05	6.74E+05	7.65E+05	1.37E+06	1.07E+06	6.14E+05
3-Phosphoglyceric acid	5.96E+05	1.14E+06	8.80E+05	1.66E+05	2.68E+05	1.92E+05	6.86E+05	9.52E+05	8.38E+05	3.91E+05	8.45E+05	1.65E+06	8.48E+05
Glucose 6-phosphate	1.32E+06	1.62E+06	1.35E+06	8.14E+06	9.27E+06	8.40E+06	9.04E+05	1.21E+06	1.12E+06	6.70E+05	1.06E+06	1.63E+06	1.00E+06
Ribose 5-phosphate	1.17E+05	2.43E+05	1.74E+05	8.78E+04	1.14E+05	1.05E+05	1.30E+05	2.04E+05	1.78E+05	1.37E+05	2.28E+05	2.63E+05	1.48E+05
Sedoheptulose 7-phosphate	3.45E+05	5.20E+05	3.77E+05	2.24E+05	2.40E+05	2.09E+05	3.70E+05	5.41E+05	4.46E+05	1.85E+05	3.29E+05	3.67E+05	2.29E+05
Trehalose	4.65E+04	4.07E+04	4.14E+04	4.30E+04	5.32E+04	4.16E+04	5.99E+04	5.55E+04	5.25E+04	1.17E+04	1.32E+04	3.11E+04	2.77E+04
Fructose 1,6-bisphosphate	6.35E+05	1.06E+06	7.27E+05	2.34E+05	3.23E+05	2.71E+05	7.03E+05	9.60E+05	7.80E+05	3.10E+05	7.36E+05	1.49E+06	6.67E+05
Phosphoenolpyruvic acid	2.06E+05	5.99E+05	3.63E+05	9.06E+04	1.62E+05	1.18E+05	2.49E+05	4.17E+05	3.68E+05	1.54E+05	4.57E+05	6.67E+05	2.82E+05
UDP-Glucose	1.71E+05	8.32E+04	8.67E+04	3.16E+04	4.54E+04	4.32E+04	7.29E+04	8.21E+04	7.89E+04	4.93E+04	7.58E+04	5.14E+04	5.00E+04
6-phosphogluconate	4.29E+04	5.37E+04	4.21E+04	6.57E+05	6.93E+05	5.40E+05	3.47E+04	5.26E+04	3.66E+04	3.49E+04	6.03E+04	6.39E+04	4.04E+04
MS_METABOLITE_DATA_END
#METABOLITES
METABOLITES_START
metabolite_name		Q1/Q3 (parent/product)	Retention time (min)
Uridine diphosphate N-acetylglucosamine		605.8/384.8	3.8
Glyceraldehyde 3-phosphate		169/97	2.79
3-Phosphoglyceric acid		185/97	2.91
Glucose 6-phosphate		259.02/97.0	3.01
Ribose 5-phosphate		229.01/97.0	3.07
Sedoheptulose 7-phosphate		289.03/97.0	3.05
Trehalose		341.3/179.3	3.97
Fructose 1,6-bisphosphate		339.0/97.0	2.79
Phosphoenolpyruvic acid		167.0/79.0	2.89
UDP-Glucose		565.0/323.0	3.57
6-phosphogluconate		275.02/97.0	2.83
METABOLITES_END
#END