#METABOLOMICS WORKBENCH Codreags00_20240205_121806 DATATRACK_ID:4627 STUDY_ID:ST003068 ANALYSIS_ID:AN005025 PROJECT_ID:PR001913 VERSION 1 CREATED_ON February 7, 2024, 10:06 am #PROJECT PR:PROJECT_TITLE Attenuation of Helicobacter pylori VacA toxin-induced cell death by modulation PR:PROJECT_TITLE of intracellular taurine metabolism - Study #2 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 #2 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:Control RAW_FILE_NAME=SC_20230107_RPLCp_FMS_Cover_A1 SUBJECT_SAMPLE_FACTORS - A2 Treatment:Control RAW_FILE_NAME=SC_20230107_RPLCp_FMS_Cover_A2 SUBJECT_SAMPLE_FACTORS - A3 Treatment:Control RAW_FILE_NAME=SC_20230107_RPLCp_FMS_Cover_A3 SUBJECT_SAMPLE_FACTORS - A4 Treatment:Control RAW_FILE_NAME=SC_20230107_RPLCp_FMS_Cover_A4 SUBJECT_SAMPLE_FACTORS - A5 Treatment:Control RAW_FILE_NAME=SC_20230107_RPLCp_FMS_Cover_A5 SUBJECT_SAMPLE_FACTORS - B1 Treatment:VacA_WT_Toxin RAW_FILE_NAME=SC_20230107_RPLCp_FMS_Cover_B1 SUBJECT_SAMPLE_FACTORS - B2 Treatment:VacA_WT_Toxin RAW_FILE_NAME=SC_20230107_RPLCp_FMS_Cover_B2 SUBJECT_SAMPLE_FACTORS - B3 Treatment:VacA_WT_Toxin RAW_FILE_NAME=SC_20230107_RPLCp_FMS_Cover_B3 SUBJECT_SAMPLE_FACTORS - B4 Treatment:VacA_WT_Toxin RAW_FILE_NAME=SC_20230107_RPLCp_FMS_Cover_B4 SUBJECT_SAMPLE_FACTORS - B5 Treatment:VacA_WT_Toxin RAW_FILE_NAME=SC_20230107_RPLCp_FMS_Cover_B5 SUBJECT_SAMPLE_FACTORS - C1 Treatment:VacA_Δ6-27 Toxin RAW_FILE_NAME=SC_20230107_RPLCp_FMS_Cover_C1 SUBJECT_SAMPLE_FACTORS - C2 Treatment:VacA_Δ6-27 Toxin RAW_FILE_NAME=SC_20230107_RPLCp_FMS_Cover_C2 SUBJECT_SAMPLE_FACTORS - C3 Treatment:VacA_Δ6-27 Toxin RAW_FILE_NAME=SC_20230107_RPLCp_FMS_Cover_C3 SUBJECT_SAMPLE_FACTORS - C4 Treatment:VacA_Δ6-27 Toxin RAW_FILE_NAME=SC_20230107_RPLCp_FMS_Cover_C4 SUBJECT_SAMPLE_FACTORS - C5 Treatment:VacA_Δ6-27 Toxin RAW_FILE_NAME=SC_20230107_RPLCp_FMS_Cover_C5 SUBJECT_SAMPLE_FACTORS - D1 Treatment:VacA_s2m1 Toxin RAW_FILE_NAME=SC_20230107_RPLCp_FMS_Cover_D1 SUBJECT_SAMPLE_FACTORS - D2 Treatment:VacA_s2m1 Toxin RAW_FILE_NAME=SC_20230107_RPLCp_FMS_Cover_D2 SUBJECT_SAMPLE_FACTORS - D3 Treatment:VacA_s2m1 Toxin RAW_FILE_NAME=SC_20230107_RPLCp_FMS_Cover_D3 SUBJECT_SAMPLE_FACTORS - D4 Treatment:VacA_s2m1 Toxin RAW_FILE_NAME=SC_20230107_RPLCp_FMS_Cover_D4 SUBJECT_SAMPLE_FACTORS - D5 Treatment:VacA_s2m1 Toxin RAW_FILE_NAME=SC_20230107_RPLCp_FMS_Cover_D5 #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. AGS cells were seeded at 2x104 cells/well into 96-well plates and CO:COLLECTION_SUMMARY incubated overnight. Cultured cells were then incubated with 20 ug/mL purified CO:COLLECTION_SUMMARY VacA [activated with by addition of HCl to a pH of 3] in medium supplemented CO:COLLECTION_SUMMARY with 5 mM NH4Cl. Following intoxication, the media was removed, and cells were CO:COLLECTION_SUMMARY washed with PBS. Cells were detached by incubation with trypsin for 5 minutes CO:COLLECTION_SUMMARY and collected via centrifugation at 4°C at 1,000 rpm for 4 minutes. Trypsin was CO:COLLECTION_SUMMARY removed, cells were once again washed with PBS, and the cells were then CO:COLLECTION_SUMMARY flash-frozen in liquid nitrogen and stored at -70C. CO:SAMPLE_TYPE Cultured cells CO:STORAGE_CONDITIONS -80℃ #TREATMENT TR:TREATMENT_SUMMARY Cells were incubated in media containing 20 ug/mL of purified VacA toxin and 5 TR:TREATMENT_SUMMARY mM ammonium chloride. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Briefly, cell pellets were normalized by total protein (200 ug) and the SP:SAMPLEPREP_SUMMARY corresponding cell supernatants were normalized by volume (200 uL). Metabolites SP:SAMPLEPREP_SUMMARY were extracted with methanol/water 80:20. Heavy labeled phenylalanine-D8 and SP:SAMPLEPREP_SUMMARY biotin-D2 were added to individual samples prior to protein precipitation. SP:SAMPLEPREP_SUMMARY Following overnight incubation at -80°C, precipitated proteins were pelleted by SP:SAMPLEPREP_SUMMARY centrifugation at 10,000 rpm for 10 min and metabolite extracts were transferred SP:SAMPLEPREP_SUMMARY into two Eppendorf tubes in equal amounts and dried down in vacuo and stored at SP:SAMPLEPREP_SUMMARY -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 ST003068_AN005025_Results.txt UNITS:time_m/z Has m/z:Yes Has RT:Yes RT units:Minutes #END