#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