#METABOLOMICS WORKBENCH common_aslab_20250818_030126 DATATRACK_ID:6300 STUDY_ID:ST004129 ANALYSIS_ID:AN006843 PROJECT_ID:PR002596
VERSION             	1
CREATED_ON             	August 18, 2025, 11:13 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 TCA cycle intermediates in Mycobacterium
ST:STUDY_TITLE                   	tuberculosis-infected BMDMs.
ST:STUDY_SUMMARY                 	TCA cycle intermediates were analysed for Mycobacterium tuberculosis (Mtb)
ST:STUDY_SUMMARY                 	(Mtb)-infected BMDMs either treated with vehicle control (0.2% DMSO) or 20uM
ST:STUDY_SUMMARY                 	meclizine. In another set, the same metabolites were measured for Mtb-infected
ST:STUDY_SUMMARY                 	BMDMs grown in 10mM glucose or galactose as the sole sugar sources in DMEM
ST:STUDY_SUMMARY                 	medium.
ST:INSTITUTE                     	Indian Institute of Science
ST:LAST_NAME                     	Singh
ST:FIRST_NAME                    	Amit
ST:ADDRESS                       	CIDR, IISc, Bangalore, Karnataka, 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:BMDMs | 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:BMDMs | 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:BMDMs | 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:BMDMs | 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:BMDMs | 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           	-	Mec_a3	Sample source:BMDMs | Treatment:Mec 20uM	RAW_FILE_NAME(Raw file name 1)=Mec_3.wiff; RAW_FILE_NAME(Raw file name 2)=Mec_3.wiff.scan
SUBJECT_SAMPLE_FACTORS           	-	Uk5_a1	Sample source:BMDMs | Treatment:UK5099 10uM	RAW_FILE_NAME(Raw file name 1)=Uk5_1.wiff; RAW_FILE_NAME(Raw file name 2)=Uk5_1.wiff.scan
SUBJECT_SAMPLE_FACTORS           	-	Uk5_a2	Sample source:BMDMs | 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:BMDMs | 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:BMDMs | 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:BMDMs | 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:BMDMs | 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:BMDMs | 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:BMDMs | 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:BMDMs | 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.
CO:COLLECTION_SUMMARY            	Post heating, they were immediately transferred to an ice bath for 5 minutes.
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 (50% methanol for
SP:SAMPLEPREP_SUMMARY            	TCA cycle intermediates) and separated on a Synergi 4-µm Fusion-RP 80 Å LC
SP:SAMPLEPREP_SUMMARY            	column (150 × 4.6 mm, Phenomenex) using a Shimadzu Nexera UHPLC system. TCA
SP:SAMPLEPREP_SUMMARY            	cycle intermediates were derivatized prior to separation. Solvent system
SP:SAMPLEPREP_SUMMARY            	employed for TCA intermediates—Solvent A was 0.1% formic acid in water, and
SP:SAMPLEPREP_SUMMARY            	Solvent B was 0.1% formic acid in methanol. Chromatographic flow parameters
SP:SAMPLEPREP_SUMMARY            	followed previously established settings. Metabolite detection was performed
SP:SAMPLEPREP_SUMMARY            	using an AB Sciex Qtrap 5500 mass spectrometer with data acquired via Analyst
SP:SAMPLEPREP_SUMMARY            	1.6.2 software (Sciex). TCA intermediates were analyzed in positive 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                 	Gradient 1: T = 0 min, 50% B; T = 2 min, 75% B; T = 6 min, 100% B; T = 15 min,
CH:FLOW_GRADIENT                 	100% B; T = 17 min, 50% B; T = 21 min, stop; Gradient 2: T = 0 min, 50% B; T = 2
CH:FLOW_GRADIENT                 	min, 65% B; T = 12 min, 90% B; T = 12.01 min, 100% B; T = 15 min, 100% B; T = 20
CH:FLOW_GRADIENT                 	min, 50% B; T = 26 min, stop
CH:FLOW_RATE                     	0.4 ml/min
CH:COLUMN_TEMPERATURE            	40
#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                      	POSITIVE
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).
MS:MS_COMMENTS                   	Quantification was carried out by calculating peak areas using MultiQuant
MS:MS_COMMENTS                   	software (version 3.0.1) as explained in detail in Niphadkar et al., STAR
MS:MS_COMMENTS                   	protocols (2025) doi:https://doi.org/10.1016/j.xpro.2025.103786. The MS analysis
MS:MS_COMMENTS                   	protocol is as follows: 1. Make a quantitation method using data from the
MS:MS_COMMENTS                   	standard. a. Set retention times for the metabolites. b. Set integration
MS:MS_COMMENTS                   	parameters as follows: Gaussian smooth width-2, RT half window-30 s, Minimum
MS:MS_COMMENTS                   	peak width-3 points, minimum peak height-0, noise percentage-75%, baseline sub.
MS:MS_COMMENTS                   	window- 2 min, report largest peak-yes. 2. Use this quantitation method, to
MS:MS_COMMENTS                   	analyze data from the samples. 3. Wherever needed, manual integration of peaks
MS:MS_COMMENTS                   	can be done. 4. 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	Mec_a3	Uk5_a1	Uk5_a2	Uk5_a3
Factors	Sample source:BMDMs | Treatment:DMSO	Sample source:BMDMs | Treatment:DMSO	Sample source:BMDMs | Treatment:DMSO	Sample source:BMDMs | Treatment:Galactose 10mM	Sample source:BMDMs | Treatment:Galactose 10mM	Sample source:BMDMs | Treatment:Galactose 10mM	Sample source:BMDMs | Treatment:Glucose 10mM	Sample source:BMDMs | Treatment:Glucose 10mM	Sample source:BMDMs | Treatment:Glucose 10mM	Sample source:BMDMs | Treatment:Mec 20uM	Sample source:BMDMs | Treatment:Mec 20uM	Sample source:BMDMs | Treatment:Mec 20uM	Sample source:BMDMs | Treatment:UK5099 10uM	Sample source:BMDMs | Treatment:UK5099 10uM	Sample source:BMDMs | Treatment:UK5099 10uM
Methylglyoxal	5.20E+07	7.64E+07	7.22E+07	6.96E+07	6.54E+07	6.70E+07	7.88E+07	7.76E+07	7.16E+07	5.36E+07	5.16E+07	7.82E+07	7.54E+07	7.07E+07	7.18E+07
Lactate	1.90E+08	2.03E+08	2.01E+08	1.41E+08	1.49E+08	1.55E+08	2.13E+08	2.06E+08	2.15E+08	1.91E+08	2.05E+08	2.07E+08	1.88E+08	2.11E+08	1.96E+08
Pyruvate	1.76E+08	1.52E+08	1.36E+08	9.42E+07	8.73E+07	1.01E+08	1.88E+08	1.69E+08	1.71E+08	1.35E+08	1.63E+08	2.10E+08	1.72E+08	2.01E+08	2.48E+08
Oxaloacetate	1.57E+07	8.31E+06	9.80E+06	8.25E+06	8.41E+06	9.05E+06	7.91E+06	8.18E+06	8.80E+06	6.58E+06	8.95E+06	9.84E+06	8.54E+06	1.02E+07	1.02E+07
Citrate	1.01E+08	9.16E+07	8.34E+07	9.58E+07	9.50E+07	9.86E+07	9.78E+07	8.26E+07	9.21E+07	3.37E+07	4.21E+07	4.87E+07	4.56E+07	6.15E+07	5.86E+07
2-ketoglutarate	1.38E+08	1.41E+08	1.15E+08	6.50E+07	6.00E+07	6.33E+07	9.56E+07	1.13E+08	1.09E+08	6.42E+07	8.00E+07	1.14E+08	7.13E+07	9.53E+07	1.01E+08
Succinate	1.37E+08	1.36E+08	1.29E+08	1.29E+08	1.27E+08	1.34E+08	1.28E+08	1.26E+08	1.30E+08	1.19E+08	1.28E+08	1.27E+08	1.21E+08	1.29E+08	1.19E+08
Fumarate	1.51E+08	1.38E+08	1.35E+08	1.40E+08	1.36E+08	1.44E+08	1.42E+08	1.29E+08	1.36E+08	1.07E+08	9.47E+07	1.30E+08	1.35E+08	1.34E+08	1.37E+08
Malate	1.03E+08	1.07E+08	1.08E+08	1.05E+08	9.60E+07	1.01E+08	1.07E+08	1.05E+08	1.03E+08	1.04E+08	1.11E+08	1.14E+08	9.89E+07	1.04E+08	1.05E+08
MS_METABOLITE_DATA_END
#METABOLITES
METABOLITES_START
metabolite_name	Q1/Q3 (parent/product)	Retention time (min)
Methylglyoxal	283.0/91.2	10.89
Lactate	196.0/91.2	6.5
Pyruvate	299.0/181.0	8.95
Oxaloacetate	448.0/325.0	10.09
Citrate	508./91.2	8.22
2-ketoglutarate	462.0/91.2	8.86
Succinate	327.0/204.0	8.29
Fumarate	329.0/206.0	7.46
Malate	345.0/91.2	7.21
METABOLITES_END
#END