#METABOLOMICS WORKBENCH Codreags00_20240207_121456 DATATRACK_ID:4634 STUDY_ID:ST003071 ANALYSIS_ID:AN005029 PROJECT_ID:PR001913
VERSION             	1
CREATED_ON             	February 7, 2024, 12:31 pm
#PROJECT
PR:PROJECT_TITLE                 	Attenuation of Helicobacter pylori VacA toxin-induced cell death by modulation
PR:PROJECT_TITLE                 	of intracellular taurine metabolism - Study #4
PR:PROJECT_TYPE                  	Untargeted Metabolomics analysis
PR:PROJECT_SUMMARY               	Colonization of the human stomach with H. pylori strains producing active forms
PR:PROJECT_SUMMARY               	of a secreted toxin (VacA) is associated with an increased risk of peptic ulcer
PR:PROJECT_SUMMARY               	disease and gastric cancer, compared to colonization with strains producing
PR:PROJECT_SUMMARY               	hypoactive forms of VacA. Previous studies have shown that VacA causes cell
PR:PROJECT_SUMMARY               	vacuolation and mitochondrial dysfunction. In this study, we sought to define
PR:PROJECT_SUMMARY               	the cellular metabolic consequences of VacA intoxication. Untargeted metabolomic
PR:PROJECT_SUMMARY               	analyses revealed that several hundred metabolites were significantly altered in
PR:PROJECT_SUMMARY               	VacA-treated gastroduodenal cells (AGS and AZ-521) compared to control cells.
PR:PROJECT_SUMMARY               	Pathway analysis suggested that VacA caused alterations in taurine and
PR:PROJECT_SUMMARY               	hypotaurine metabolism. Treatment of cells with the purified active s1m1 form of
PR:PROJECT_SUMMARY               	VacA, but not hypoactive s2m1 or 6-27 VacA mutant proteins (defective in
PR:PROJECT_SUMMARY               	membrane channel formation), caused reductions in taurine and hypotaurine
PR:PROJECT_SUMMARY               	levels. Supplementation of the tissue culture medium with taurine or hypotaurine
PR:PROJECT_SUMMARY               	protected AZ-521 cells against VacA-induced cell death. Untargeted global
PR:PROJECT_SUMMARY               	metabolomics of AZ-521 cells or AGS cells intoxicated with VacA in the presence
PR:PROJECT_SUMMARY               	or absence of extracellular taurine showed that taurine was the main
PR:PROJECT_SUMMARY               	intracellular metabolite significantly altered by extracellular taurine
PR:PROJECT_SUMMARY               	supplementation. These results indicate that VacA causes alterations in cellular
PR:PROJECT_SUMMARY               	taurine metabolism and indicate that repletion of taurine is sufficient to
PR:PROJECT_SUMMARY               	attenuate VacA-induced cell death.
PR:INSTITUTE                     	Vanderbilt University
PR:DEPARTMENT                    	Chemistry
PR:LABORATORY                    	Center for Innovative Technology
PR:LAST_NAME                     	CODREANU
PR:FIRST_NAME                    	SIMONA
PR:ADDRESS                       	1234 STEVENSON CENTER LANE
PR:EMAIL                         	SIMONA.CODREANU@VANDERBILT.EDU
PR:PHONE                         	6158758422
#STUDY
ST:STUDY_TITLE                   	Attenuation of Helicobacter pylori VacA toxin-induced cell death by modulation
ST:STUDY_TITLE                   	of intracellular taurine metabolism - Study #4
ST:STUDY_TYPE                    	untargeted metabolomics analysis
ST:STUDY_SUMMARY                 	Colonization of the human stomach with H. pylori strains producing active forms
ST:STUDY_SUMMARY                 	of a secreted toxin (VacA) is associated with an increased risk of peptic ulcer
ST:STUDY_SUMMARY                 	disease and gastric cancer, compared to colonization with strains producing
ST:STUDY_SUMMARY                 	hypoactive forms of VacA. Previous studies have shown that VacA causes cell
ST:STUDY_SUMMARY                 	vacuolation and mitochondrial dysfunction. In this study, we sought to define
ST:STUDY_SUMMARY                 	the cellular metabolic consequences of VacA intoxication. Untargeted metabolomic
ST:STUDY_SUMMARY                 	analyses revealed that several hundred metabolites were significantly altered in
ST:STUDY_SUMMARY                 	VacA-treated gastroduodenal cells (AGS and AZ-521) compared to control cells.
ST:STUDY_SUMMARY                 	Pathway analysis suggested that VacA caused alterations in taurine and
ST:STUDY_SUMMARY                 	hypotaurine metabolism. Treatment of cells with the purified active s1m1 form of
ST:STUDY_SUMMARY                 	VacA, but not hypoactive s2m1 or 6-27 VacA mutant proteins (defective in
ST:STUDY_SUMMARY                 	membrane channel formation), caused reductions in taurine and hypotaurine
ST:STUDY_SUMMARY                 	levels. Supplementation of the tissue culture medium with taurine or hypotaurine
ST:STUDY_SUMMARY                 	protected AZ-521 cells against VacA-induced cell death. Untargeted global
ST:STUDY_SUMMARY                 	metabolomics of AZ-521 cells or AGS cells intoxicated with VacA in the presence
ST:STUDY_SUMMARY                 	or absence of extracellular taurine showed that taurine was the main
ST:STUDY_SUMMARY                 	intracellular metabolite significantly altered by extracellular taurine
ST:STUDY_SUMMARY                 	supplementation. These results indicate that VacA causes alterations in cellular
ST:STUDY_SUMMARY                 	taurine metabolism and indicate that repletion of taurine is sufficient to
ST:STUDY_SUMMARY                 	attenuate VacA-induced cell death.
ST:INSTITUTE                     	Vanderbilt University
ST:DEPARTMENT                    	Chemistry
ST:LABORATORY                    	Center for Innovative Technology
ST:LAST_NAME                     	CODREANU
ST:FIRST_NAME                    	SIMONA
ST:ADDRESS                       	1234 STEVENSON CENTER LANE
ST:EMAIL                         	SIMONA.CODREANU@VANDERBILT.EDU
ST:PHONE                         	6158758422
#SUBJECT
SU:SUBJECT_TYPE                  	Cultured cells
SU:SUBJECT_SPECIES               	Homo sapiens
SU:TAXONOMY_ID                   	9606
SU:GENOTYPE_STRAIN               	AGS and AZ-521
SU:SPECIES_GROUP                 	AGS and AZ-521
#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           	-	A1	Treatment:12h_PBS-treated | Genotype:AGS cells | Treatment:0mM extra taurine	RAW_FILE_NAME=SC_20231002_RPLCp_FMS_Cover_Taurine_A1
SUBJECT_SAMPLE_FACTORS           	-	A2	Treatment:12h_PBS-treated | Genotype:AGS cells | Treatment:0mM extra taurine	RAW_FILE_NAME=SC_20231002_RPLCp_FMS_Cover_Taurine_A2
SUBJECT_SAMPLE_FACTORS           	-	A3	Treatment:12h_PBS-treated | Genotype:AGS cells | Treatment:0mM extra taurine	RAW_FILE_NAME=SC_20231002_RPLCp_FMS_Cover_Taurine_A3
SUBJECT_SAMPLE_FACTORS           	-	A4	Treatment:12h_PBS-treated | Genotype:AGS cells | Treatment:0mM extra taurine	RAW_FILE_NAME=SC_20231002_RPLCp_FMS_Cover_Taurine_A4
SUBJECT_SAMPLE_FACTORS           	-	A5	Treatment:12h_PBS-treated | Genotype:AGS cells | Treatment:0mM extra taurine	RAW_FILE_NAME=SC_20231002_RPLCp_FMS_Cover_Taurine_A5
SUBJECT_SAMPLE_FACTORS           	-	B1	Treatment:12h_VacA-treated | Genotype:AGS cells | Treatment:0mM extra taurine	RAW_FILE_NAME=SC_20231002_RPLCp_FMS_Cover_Taurine_B1
SUBJECT_SAMPLE_FACTORS           	-	B2	Treatment:12h_VacA-treated | Genotype:AGS cells | Treatment:0mM extra taurine	RAW_FILE_NAME=SC_20231002_RPLCp_FMS_Cover_Taurine_B2
SUBJECT_SAMPLE_FACTORS           	-	B3	Treatment:12h_VacA-treated | Genotype:AGS cells | Treatment:0mM extra taurine	RAW_FILE_NAME=SC_20231002_RPLCp_FMS_Cover_Taurine_B3
SUBJECT_SAMPLE_FACTORS           	-	B4	Treatment:12h_VacA-treated | Genotype:AGS cells | Treatment:0mM extra taurine	RAW_FILE_NAME=SC_20231002_RPLCp_FMS_Cover_Taurine_B4
SUBJECT_SAMPLE_FACTORS           	-	B5	Treatment:12h_VacA-treated | Genotype:AGS cells | Treatment:0mM extra taurine	RAW_FILE_NAME=SC_20231002_RPLCp_FMS_Cover_Taurine_B5
SUBJECT_SAMPLE_FACTORS           	-	C1	Treatment:12h_PBS-treated | Genotype:AGS cells | Treatment:10 mM extra taurine	RAW_FILE_NAME=SC_20231002_RPLCp_FMS_Cover_Taurine_C1
SUBJECT_SAMPLE_FACTORS           	-	C2	Treatment:12h_PBS-treated | Genotype:AGS cells | Treatment:10 mM extra taurine	RAW_FILE_NAME=SC_20231002_RPLCp_FMS_Cover_Taurine_C2
SUBJECT_SAMPLE_FACTORS           	-	C3	Treatment:12h_PBS-treated | Genotype:AGS cells | Treatment:10 mM extra taurine	RAW_FILE_NAME=SC_20231002_RPLCp_FMS_Cover_Taurine_C3
SUBJECT_SAMPLE_FACTORS           	-	C4	Treatment:12h_PBS-treated | Genotype:AGS cells | Treatment:10 mM extra taurine	RAW_FILE_NAME=SC_20231002_RPLCp_FMS_Cover_Taurine_C4
SUBJECT_SAMPLE_FACTORS           	-	C5	Treatment:12h_PBS-treated | Genotype:AGS cells | Treatment:10 mM extra taurine	RAW_FILE_NAME=SC_20231002_RPLCp_FMS_Cover_Taurine_C5
SUBJECT_SAMPLE_FACTORS           	-	D1	Treatment:12h_VacA-treated | Genotype:AGS cells | Treatment:10 mM extra taurine	RAW_FILE_NAME=SC_20231002_RPLCp_FMS_Cover_Taurine_D1
SUBJECT_SAMPLE_FACTORS           	-	D2	Treatment:12h_VacA-treated | Genotype:AGS cells | Treatment:10 mM extra taurine	RAW_FILE_NAME=SC_20231002_RPLCp_FMS_Cover_Taurine_D2
SUBJECT_SAMPLE_FACTORS           	-	D3	Treatment:12h_VacA-treated | Genotype:AGS cells | Treatment:10 mM extra taurine	RAW_FILE_NAME=SC_20231002_RPLCp_FMS_Cover_Taurine_D3
SUBJECT_SAMPLE_FACTORS           	-	D4	Treatment:12h_VacA-treated | Genotype:AGS cells | Treatment:10 mM extra taurine	RAW_FILE_NAME=SC_20231002_RPLCp_FMS_Cover_Taurine_D4
SUBJECT_SAMPLE_FACTORS           	-	D5	Treatment:12h_VacA-treated | Genotype:AGS cells | Treatment:10 mM extra taurine	RAW_FILE_NAME=SC_20231002_RPLCp_FMS_Cover_Taurine_D5
SUBJECT_SAMPLE_FACTORS           	-	E1	Treatment:3h_PBS-treated | Genotype:AZ521 cells | Treatment:0mM extra taurine	RAW_FILE_NAME=SC_20231002_RPLCp_FMS_Cover_Taurine_E1
SUBJECT_SAMPLE_FACTORS           	-	E2	Treatment:3h_PBS-treated | Genotype:AZ521 cells | Treatment:0mM extra taurine	RAW_FILE_NAME=SC_20231002_RPLCp_FMS_Cover_Taurine_E2
SUBJECT_SAMPLE_FACTORS           	-	E3	Treatment:3h_PBS-treated | Genotype:AZ521 cells | Treatment:0mM extra taurine	RAW_FILE_NAME=SC_20231002_RPLCp_FMS_Cover_Taurine_E3
SUBJECT_SAMPLE_FACTORS           	-	E4	Treatment:3h_PBS-treated | Genotype:AZ521 cells | Treatment:0mM extra taurine	RAW_FILE_NAME=SC_20231002_RPLCp_FMS_Cover_Taurine_E4
SUBJECT_SAMPLE_FACTORS           	-	E5	Treatment:3h_PBS-treated | Genotype:AZ521 cells | Treatment:0mM extra taurine	RAW_FILE_NAME=SC_20231002_RPLCp_FMS_Cover_Taurine_E5
SUBJECT_SAMPLE_FACTORS           	-	F1	Treatment:3h_VacA-treated | Genotype:AZ521 cells | Treatment:0mM extra taurine	RAW_FILE_NAME=SC_20231002_RPLCp_FMS_Cover_Taurine_F1
SUBJECT_SAMPLE_FACTORS           	-	F2	Treatment:3h_VacA-treated | Genotype:AZ521 cells | Treatment:0mM extra taurine	RAW_FILE_NAME=SC_20231002_RPLCp_FMS_Cover_Taurine_F2
SUBJECT_SAMPLE_FACTORS           	-	F3	Treatment:3h_VacA-treated | Genotype:AZ521 cells | Treatment:0mM extra taurine	RAW_FILE_NAME=SC_20231002_RPLCp_FMS_Cover_Taurine_F3
SUBJECT_SAMPLE_FACTORS           	-	F4	Treatment:3h_VacA-treated | Genotype:AZ521 cells | Treatment:0mM extra taurine	RAW_FILE_NAME=SC_20231002_RPLCp_FMS_Cover_Taurine_F4
SUBJECT_SAMPLE_FACTORS           	-	F5	Treatment:3h_VacA-treated | Genotype:AZ521 cells | Treatment:0mM extra taurine	RAW_FILE_NAME=SC_20231002_RPLCp_FMS_Cover_Taurine_F5
SUBJECT_SAMPLE_FACTORS           	-	G1	Treatment:3h_PBS-treated | Genotype:AZ521 cells | Treatment:10 mM extra taurine	RAW_FILE_NAME=SC_20231002_RPLCp_FMS_Cover_Taurine_G1
SUBJECT_SAMPLE_FACTORS           	-	G2	Treatment:3h_PBS-treated | Genotype:AZ521 cells | Treatment:10 mM extra taurine	RAW_FILE_NAME=SC_20231002_RPLCp_FMS_Cover_Taurine_G2
SUBJECT_SAMPLE_FACTORS           	-	G3	Treatment:3h_PBS-treated | Genotype:AZ521 cells | Treatment:10 mM extra taurine	RAW_FILE_NAME=SC_20231002_RPLCp_FMS_Cover_Taurine_G3
SUBJECT_SAMPLE_FACTORS           	-	G4	Treatment:3h_PBS-treated | Genotype:AZ521 cells | Treatment:10 mM extra taurine	RAW_FILE_NAME=SC_20231002_RPLCp_FMS_Cover_Taurine_G4
SUBJECT_SAMPLE_FACTORS           	-	G5	Treatment:3h_PBS-treated | Genotype:AZ521 cells | Treatment:10 mM extra taurine	RAW_FILE_NAME=SC_20231002_RPLCp_FMS_Cover_Taurine_G5
SUBJECT_SAMPLE_FACTORS           	-	H1	Treatment:3h_VacA-treated | Genotype:AZ521 cells | Treatment:10 mM extra taurine	RAW_FILE_NAME=SC_20231002_RPLCp_FMS_Cover_Taurine_H1
SUBJECT_SAMPLE_FACTORS           	-	H2	Treatment:3h_VacA-treated | Genotype:AZ521 cells | Treatment:10 mM extra taurine	RAW_FILE_NAME=SC_20231002_RPLCp_FMS_Cover_Taurine_H2
SUBJECT_SAMPLE_FACTORS           	-	H3	Treatment:3h_VacA-treated | Genotype:AZ521 cells | Treatment:10 mM extra taurine	RAW_FILE_NAME=SC_20231002_RPLCp_FMS_Cover_Taurine_H3
SUBJECT_SAMPLE_FACTORS           	-	H4	Treatment:3h_VacA-treated | Genotype:AZ521 cells | Treatment:10 mM extra taurine	RAW_FILE_NAME=SC_20231002_RPLCp_FMS_Cover_Taurine_H4
SUBJECT_SAMPLE_FACTORS           	-	H5	Treatment:3h_VacA-treated | Genotype:AZ521 cells | Treatment:10 mM extra taurine	RAW_FILE_NAME=SC_20231002_RPLCp_FMS_Cover_Taurine_H5
#COLLECTION
CO:COLLECTION_SUMMARY            	AGS cells or AZ-521 cells were cultured in Roswell Park Memorial Institute
CO:COLLECTION_SUMMARY            	(RPMI) 1640 medium containing 10% fetal bovine serum, or minimal essential
CO:COLLECTION_SUMMARY            	medium (MEM) containing 10% fetal bovine serum and 5% nonessential amino acids,
CO:COLLECTION_SUMMARY            	respectively.
CO:SAMPLE_TYPE                   	Stomach
CO:STORAGE_CONDITIONS            	-80℃
#TREATMENT
TR:TREATMENT_SUMMARY             	AGS and AZ-521 cells were cultured in T-75 cell culture flasks overnight to a
TR:TREATMENT_SUMMARY             	density of approximately 4x106 cells. Cells were incubated in media containing
TR:TREATMENT_SUMMARY             	20 ug/mL of purified s1m1 VacA toxin and 5 mM ammonium chloride, and
TR:TREATMENT_SUMMARY             	supplemented with 10mM Taurine. Following intoxication, the media was removed,
TR:TREATMENT_SUMMARY             	and cells were washed with PBS. Cells were detached by incubation with trypsin
TR:TREATMENT_SUMMARY             	for 5 minutes and collected via centrifugation at 4°C at 1,000 rpm for 4
TR:TREATMENT_SUMMARY             	minutes. Trypsin was removed, cells were once again washed with PBS, and the
TR:TREATMENT_SUMMARY             	cells were then flash-frozen in liquid nitrogen and stored at -70C.
#SAMPLEPREP
SP:SAMPLEPREP_SUMMARY            	Samples were analyzed via Liquid Chromatography-High Resolution Mass
SP:SAMPLEPREP_SUMMARY            	Spectrometry (LC-HRMS and LC-HRMS/MS)-based metabolomics using previously
SP:SAMPLEPREP_SUMMARY            	described methods26,27,28. Briefly, cell pellets were normalized by total
SP:SAMPLEPREP_SUMMARY            	protein (200 ug) and the corresponding cell supernatants were normalized by
SP:SAMPLEPREP_SUMMARY            	volume (200 uL). Metabolites were extracted with methanol/water 80:20. Heavy
SP:SAMPLEPREP_SUMMARY            	labeled phenylalanine-D8 and biotin-D2 were added to individual samples prior to
SP:SAMPLEPREP_SUMMARY            	protein precipitation. Following overnight incubation at -80°C, precipitated
SP:SAMPLEPREP_SUMMARY            	proteins were pelleted by centrifugation at 10,000 rpm for 10 min and metabolite
SP:SAMPLEPREP_SUMMARY            	extracts were transferred into two Eppendorf tubes in equal amounts and dried
SP:SAMPLEPREP_SUMMARY            	down in vacuo and stored at -80°C.
SP:PROCESSING_STORAGE_CONDITIONS 	-80℃
SP:EXTRACT_STORAGE               	-80℃
#CHROMATOGRAPHY
CH:CHROMATOGRAPHY_TYPE           	Reversed phase
CH:INSTRUMENT_NAME               	Thermo Vanquish
CH:COLUMN_NAME                   	Thermo Hypersil GOLD aQ (100 x 2.1mm,1.9um)
CH:SOLVENT_A                     	100% water, 0.1% Formic Acid
CH:SOLVENT_B                     	80:20 acetonitrile:water, 0.1% Formic Acid
CH:FLOW_GRADIENT                 	30 min; 95%A, 5%B
CH:FLOW_RATE                     	0.25 mL/min
CH:COLUMN_TEMPERATURE            	40
#ANALYSIS
AN:ANALYSIS_TYPE                 	MS
#MS
MS:INSTRUMENT_NAME               	Thermo Q Exactive HF hybrid Orbitrap
MS:INSTRUMENT_TYPE               	Orbitrap
MS:MS_TYPE                       	ESI
MS:ION_MODE                      	POSITIVE
MS:MS_COMMENTS                   	Mass spectrometry raw data was imported, processed, normalized, and reviewed
MS:MS_COMMENTS                   	using Progenesis QI v.3.0 (Non-linear Dynamics, Newcastle, UK).
MS:MS_RESULTS_FILE               	ST003071_AN005029_Results.txt	UNITS:time_m/z	Has m/z:Yes	Has RT:Yes	RT units:Minutes
#END