#METABOLOMICS WORKBENCH Codreags00_20240202_133633 DATATRACK_ID:4626 STUDY_ID:ST003067 ANALYSIS_ID:AN005024 PROJECT_ID:PR001913 VERSION 1 CREATED_ON February 7, 2024, 9:56 am #PROJECT PR:PROJECT_TITLE Attenuation of Helicobacter pylori VacA toxin-induced cell death by modulation PR:PROJECT_TITLE of intracellular taurine metabolism - Study #1 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 #1 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:CELL_BIOSOURCE_OR_SUPPLIER ATCC 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 - rT2_1 Treatment:Control | Treatment:2h RAW_FILE_NAME=SC_20220124_RPLCp_FMS_Cover_T2_1 SUBJECT_SAMPLE_FACTORS - rT2_2 Treatment:Control | Treatment:2h RAW_FILE_NAME=SC_20220124_RPLCp_FMS_Cover_T2_2 SUBJECT_SAMPLE_FACTORS - rT2_3 Treatment:Control | Treatment:2h RAW_FILE_NAME=SC_20220124_RPLCp_FMS_Cover_T2_3 SUBJECT_SAMPLE_FACTORS - rT2_4 Treatment:Control | Treatment:2h RAW_FILE_NAME=SC_20220124_RPLCp_FMS_Cover_T2_4 SUBJECT_SAMPLE_FACTORS - rT2_5 Treatment:VacA | Treatment:2h RAW_FILE_NAME=SC_20220124_RPLCp_FMS_Cover_T2_5 SUBJECT_SAMPLE_FACTORS - rT2_6 Treatment:VacA | Treatment:2h RAW_FILE_NAME=SC_20220124_RPLCp_FMS_Cover_T2_6 SUBJECT_SAMPLE_FACTORS - rT2_7 Treatment:VacA | Treatment:2h RAW_FILE_NAME=SC_20220124_RPLCp_FMS_Cover_T2_7 SUBJECT_SAMPLE_FACTORS - rT2_8 Treatment:VacA | Treatment:2h RAW_FILE_NAME=SC_20220124_RPLCp_FMS_Cover_T2_8 SUBJECT_SAMPLE_FACTORS - rT8_1 Treatment:Control | Treatment:8h RAW_FILE_NAME=SC_20220124_RPLCp_FMS_Cover_T8_1 SUBJECT_SAMPLE_FACTORS - rT8_2 Treatment:Control | Treatment:8h RAW_FILE_NAME=SC_20220124_RPLCp_FMS_Cover_T8_2 SUBJECT_SAMPLE_FACTORS - rT8_3 Treatment:Control | Treatment:8h RAW_FILE_NAME=SC_20220124_RPLCp_FMS_Cover_T8_3 SUBJECT_SAMPLE_FACTORS - rT8_4 Treatment:Control | Treatment:8h RAW_FILE_NAME=SC_20220124_RPLCp_FMS_Cover_T8_4 SUBJECT_SAMPLE_FACTORS - rT8_5 Treatment:VacA | Treatment:8h RAW_FILE_NAME=SC_20220124_RPLCp_FMS_Cover_T8_5 SUBJECT_SAMPLE_FACTORS - rT8_6 Treatment:VacA | Treatment:8h RAW_FILE_NAME=SC_20220124_RPLCp_FMS_Cover_T8_6 SUBJECT_SAMPLE_FACTORS - rT8_7 Treatment:VacA | Treatment:8h RAW_FILE_NAME=SC_20220124_RPLCp_FMS_Cover_T8_7 SUBJECT_SAMPLE_FACTORS - rT8_8 Treatment:VacA | Treatment:8h RAW_FILE_NAME=SC_20220124_RPLCp_FMS_Cover_T8_8 SUBJECT_SAMPLE_FACTORS - rT12_1 Treatment:Control | Treatment:12h RAW_FILE_NAME=SC_20220124_RPLCp_FMS_Cover_T12_1 SUBJECT_SAMPLE_FACTORS - rT12_2 Treatment:Control | Treatment:12h RAW_FILE_NAME=SC_20220124_RPLCp_FMS_Cover_T12_2 SUBJECT_SAMPLE_FACTORS - rT12_3 Treatment:Control | Treatment:12h RAW_FILE_NAME=SC_20220124_RPLCp_FMS_Cover_T12_3 SUBJECT_SAMPLE_FACTORS - rT12_4 Treatment:Control | Treatment:12h RAW_FILE_NAME=SC_20220124_RPLCp_FMS_Cover_T12_4 SUBJECT_SAMPLE_FACTORS - rT12_5 Treatment:VacA | Treatment:12h RAW_FILE_NAME=SC_20220124_RPLCp_FMS_Cover_T12_5 SUBJECT_SAMPLE_FACTORS - rT12_6 Treatment:VacA | Treatment:12h RAW_FILE_NAME=SC_20220124_RPLCp_FMS_Cover_T12_6 SUBJECT_SAMPLE_FACTORS - rT12_7 Treatment:VacA | Treatment:12h RAW_FILE_NAME=SC_20220124_RPLCp_FMS_Cover_T12_7 SUBJECT_SAMPLE_FACTORS - rT12_8 Treatment:VacA | Treatment:12h RAW_FILE_NAME=SC_20220124_RPLCp_FMS_Cover_T12_8 SUBJECT_SAMPLE_FACTORS - hT2_1 Treatment:Control | Treatment:2h RAW_FILE_NAME=SC_20220131_HILICn_FMS_Cover_T2_1 SUBJECT_SAMPLE_FACTORS - hT2_2 Treatment:Control | Treatment:2h RAW_FILE_NAME=SC_20220131_HILICn_FMS_Cover_T2_2 SUBJECT_SAMPLE_FACTORS - hT2_3 Treatment:Control | Treatment:2h RAW_FILE_NAME=SC_20220131_HILICn_FMS_Cover_T2_3 SUBJECT_SAMPLE_FACTORS - hT2_4 Treatment:Control | Treatment:2h RAW_FILE_NAME=SC_20220131_HILICn_FMS_Cover_T2_4 SUBJECT_SAMPLE_FACTORS - hT2_5 Treatment:VacA | Treatment:2h RAW_FILE_NAME=SC_20220131_HILICn_FMS_Cover_T2_5 SUBJECT_SAMPLE_FACTORS - hT2_6 Treatment:VacA | Treatment:2h RAW_FILE_NAME=SC_20220131_HILICn_FMS_Cover_T2_6 SUBJECT_SAMPLE_FACTORS - hT2_7 Treatment:VacA | Treatment:2h RAW_FILE_NAME=SC_20220131_HILICn_FMS_Cover_T2_7 SUBJECT_SAMPLE_FACTORS - hT2_8 Treatment:VacA | Treatment:2h RAW_FILE_NAME=SC_20220131_HILICn_FMS_Cover_T2_8 SUBJECT_SAMPLE_FACTORS - hT8_1 Treatment:Control | Treatment:8h RAW_FILE_NAME=SC_20220131_HILICn_FMS_Cover_T8_1 SUBJECT_SAMPLE_FACTORS - hT8_2 Treatment:Control | Treatment:8h RAW_FILE_NAME=SC_20220131_HILICn_FMS_Cover_T8_2 SUBJECT_SAMPLE_FACTORS - hT8_3 Treatment:Control | Treatment:8h RAW_FILE_NAME=SC_20220131_HILICn_FMS_Cover_T8_3 SUBJECT_SAMPLE_FACTORS - hT8_4 Treatment:Control | Treatment:8h RAW_FILE_NAME=SC_20220131_HILICn_FMS_Cover_T8_4 SUBJECT_SAMPLE_FACTORS - hT8_5 Treatment:VacA | Treatment:8h RAW_FILE_NAME=SC_20220131_HILICn_FMS_Cover_T8_5 SUBJECT_SAMPLE_FACTORS - hT8_6 Treatment:VacA | Treatment:8h RAW_FILE_NAME=SC_20220131_HILICn_FMS_Cover_T8_6 SUBJECT_SAMPLE_FACTORS - hT8_7 Treatment:VacA | Treatment:8h RAW_FILE_NAME=SC_20220131_HILICn_FMS_Cover_T8_7 SUBJECT_SAMPLE_FACTORS - hT8_8 Treatment:VacA | Treatment:8h RAW_FILE_NAME=SC_20220131_HILICn_FMS_Cover_T8_8 SUBJECT_SAMPLE_FACTORS - hT12_1 Treatment:Control | Treatment:12h RAW_FILE_NAME=SC_20220131_HILICn_FMS_Cover_T12_1 SUBJECT_SAMPLE_FACTORS - hT12_2 Treatment:Control | Treatment:12h RAW_FILE_NAME=SC_20220131_HILICn_FMS_Cover_T12_2 SUBJECT_SAMPLE_FACTORS - hT12_3 Treatment:Control | Treatment:12h RAW_FILE_NAME=SC_20220131_HILICn_FMS_Cover_T12_3 SUBJECT_SAMPLE_FACTORS - hT12_4 Treatment:Control | Treatment:12h RAW_FILE_NAME=SC_20220131_HILICn_FMS_Cover_T12_4 SUBJECT_SAMPLE_FACTORS - hT12_5 Treatment:VacA | Treatment:12h RAW_FILE_NAME=SC_20220131_HILICn_FMS_Cover_T12_5 SUBJECT_SAMPLE_FACTORS - hT12_6 Treatment:VacA | Treatment:12h RAW_FILE_NAME=SC_20220131_HILICn_FMS_Cover_T12_6 SUBJECT_SAMPLE_FACTORS - hT12_7 Treatment:VacA | Treatment:12h RAW_FILE_NAME=SC_20220131_HILICn_FMS_Cover_T12_7 SUBJECT_SAMPLE_FACTORS - hT12_8 Treatment:VacA | Treatment:12h RAW_FILE_NAME=SC_20220131_HILICn_FMS_Cover_T12_8 #FACTORS #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. Following TR:TREATMENT_SUMMARY intoxication, the media was removed, and cells were washed with PBS. Cells were TR:TREATMENT_SUMMARY detached by incubation with trypsin for 5 minutes and collected via TR:TREATMENT_SUMMARY centrifugation at 4°C at 1,000 rpm for 4 minutes. Trypsin was removed, cells TR:TREATMENT_SUMMARY were once again washed with PBS, and the cells were then flash-frozen in liquid TR:TREATMENT_SUMMARY nitrogen and stored at -70°C. TR:TREATMENT_PROTOCOL_FILENAME Cell_Culture_Methods.pdf #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 methods. Briefly, cell pellets were normalized by total protein (200 SP:SAMPLEPREP_SUMMARY ug) and the corresponding cell supernatants were normalized by volume (200 uL). SP:SAMPLEPREP_SUMMARY Metabolites were extracted with methanol/water 80:20. Heavy labeled SP:SAMPLEPREP_SUMMARY phenylalanine-D8 and biotin-D2 were added to individual samples prior to protein SP:SAMPLEPREP_SUMMARY precipitation. Following overnight incubation at -80°C, precipitated proteins SP:SAMPLEPREP_SUMMARY were pelleted by centrifugation at 10,000 rpm for 10 min and metabolite extracts SP:SAMPLEPREP_SUMMARY were transferred into two Eppendorf tubes in equal amounts and dried down in SP:SAMPLEPREP_SUMMARY vacuo and stored at -80°C. SP:PROCESSING_STORAGE_CONDITIONS -80℃ SP:EXTRACT_STORAGE -80℃ #CHROMATOGRAPHY CH:CHROMATOGRAPHY_TYPE HILIC CH:INSTRUMENT_NAME Thermo Vanquish CH:COLUMN_NAME Waters ACQUITY UPLC BEH Amide (100 x 2.1mm,1.7um) CH:SOLVENT_A 90% water, 10% acetonitrile, 5mM Ammonium Formate, 0.1%FA CH:SOLVENT_B 10% water, 90% acetonitrile, 5mM Ammonium Formate, 0.1%FA CH:FLOW_GRADIENT 30 min; 5%A, 95%B CH:FLOW_RATE 0.20 mL/min CH:COLUMN_TEMPERATURE 30 #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 NEGATIVE 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 ST003067_AN005024_Results.txt UNITS:time_m/z Has m/z:Yes Has RT:Yes RT units:Minutes #END