Summary of Study ST003068
This data is available at the NIH Common Fund's National Metabolomics Data Repository (NMDR) website, the Metabolomics Workbench, https://www.metabolomicsworkbench.org, where it has been assigned Project ID PR001913. The data can be accessed directly via it's Project DOI: 10.21228/M8FH9H This work is supported by NIH grant, U2C- DK119886.
See: https://www.metabolomicsworkbench.org/about/howtocite.php
This study contains a large results data set and is not available in the mwTab file. It is only available for download via FTP as data file(s) here.
| Study ID | ST003068 |
| Study Title | Attenuation of Helicobacter pylori VacA toxin-induced cell death by modulation of intracellular taurine metabolism _ Study #2 |
| Study Type | untargeted metabolomics analysis |
| Study Summary | AGS cells treated with wild-type VacA have metabolic profiles different from those of AGS cells treated with mutant forms of VacA. Most VacA-induced cellular alterations are attributed to VacA’s ability to form channels within host cell membranes. To determine if the observed VacA-induced metabolic alterations were dependent on VacA’s ability to form membrane channels, we treated AGS cells with the wild-type s1m1 wild-type (WT) toxin (which has robust channel-forming activity), as well as two mutant forms of the toxin, Delta_6-27 or s2m1. VacA Delta_6-27 has a deletion in the VacA amino-terminal hydrophobic region and is defective in channel formation. In the s2m1 mutant toxin, the active s1 isotype sequence of the WT toxin is replaced with the less active s2 sequence, resulting in markedly diminished channel-forming activity. AGS cells treated with the Delta_6-27 or s2m1 mutant VacA did not undergo vacuolation, while AGS cells treated with WT VacA experienced robust vacuolation, consistent with previously published results. |
| Institute | Vanderbilt University |
| Department | Chemistry |
| Laboratory | Center for Innovative Technology |
| Last Name | CODREANU |
| First Name | SIMONA |
| Address | 1234 STEVENSON CENTER LANE |
| SIMONA.CODREANU@VANDERBILT.EDU | |
| Phone | 6158758422 |
| Submit Date | 2024-02-05 |
| Raw Data Available | Yes |
| Raw Data File Type(s) | raw(Thermo) |
| Analysis Type Detail | LC-MS |
| Release Date | 2024-06-12 |
| Release Version | 1 |
Select appropriate tab below to view additional metadata details:
Project:
| Project ID: | PR001913 |
| Project DOI: | doi: 10.21228/M8FH9H |
| Project Title: | Attenuation of Helicobacter pylori VacA toxin-induced cell death by modulation of intracellular taurine metabolism |
| Project Type: | Untargeted Metabolomics analysis |
| Project Summary: | Colonization of the human stomach with H. pylori strains producing active forms of a secreted toxin (VacA) is associated with an increased risk of peptic ulcer disease and gastric cancer, compared to colonization with strains producing hypoactive forms of VacA. Previous studies have shown that VacA causes cell vacuolation and mitochondrial dysfunction. In this study, we sought to define the cellular metabolic consequences of VacA intoxication. Untargeted metabolomic analyses revealed that several hundred metabolites were significantly altered in VacA-treated gastroduodenal cells (AGS and AZ-521) compared to control cells. Pathway analysis suggested that VacA caused alterations in taurine and hypotaurine metabolism. Treatment of cells with the purified active s1m1 form of VacA, but not hypoactive s2m1 or Delta_6-27 VacA mutant proteins (defective in membrane channel formation), caused reductions in taurine and hypotaurine levels. Supplementation of the tissue culture medium with taurine or hypotaurine protected AZ-521 cells against VacA-induced cell death. Untargeted global metabolomics of AZ-521 cells or AGS cells intoxicated with VacA in the presence or absence of extracellular taurine showed that taurine was the main intracellular metabolite significantly altered by extracellular taurine supplementation. These results indicate that VacA causes alterations in cellular taurine metabolism and indicate that repletion of taurine is sufficient to attenuate VacA-induced cell death. |
| Institute: | Vanderbilt University |
| Department: | Chemistry |
| Laboratory: | Center for Innovative Technology |
| Last Name: | CODREANU |
| First Name: | SIMONA |
| Address: | 1234 STEVENSON CENTER LANE |
| Email: | SIMONA.CODREANU@VANDERBILT.EDU |
| Phone: | 6158758422 |
Subject:
| Subject ID: | SU003183 |
| Subject Type: | Cultured cells |
| Subject Species: | Homo sapiens |
| Taxonomy ID: | 9606 |
| Genotype Strain: | AGS and AZ-521 |
| Species Group: | AGS and AZ-521 |
Factors:
Subject type: Cultured cells; Subject species: Homo sapiens (Factor headings shown in green)
| mb_sample_id | local_sample_id | Treatment |
|---|---|---|
| SA332076 | A1 | Control |
| SA332077 | A5 | Control |
| SA332078 | A4 | Control |
| SA332079 | A2 | Control |
| SA332080 | A3 | Control |
| SA332086 | D1 | VacA_s2m1 Toxin |
| SA332087 | D3 | VacA_s2m1 Toxin |
| SA332088 | D5 | VacA_s2m1 Toxin |
| SA332089 | D2 | VacA_s2m1 Toxin |
| SA332090 | D4 | VacA_s2m1 Toxin |
| SA332081 | B5 | VacA_WT_Toxin |
| SA332082 | B4 | VacA_WT_Toxin |
| SA332083 | B3 | VacA_WT_Toxin |
| SA332084 | B1 | VacA_WT_Toxin |
| SA332085 | B2 | VacA_WT_Toxin |
| SA332091 | C1 | VacA_Δ6-27 Toxin |
| SA332092 | C2 | VacA_Δ6-27 Toxin |
| SA332093 | C3 | VacA_Δ6-27 Toxin |
| SA332094 | C4 | VacA_Δ6-27 Toxin |
| SA332095 | C5 | VacA_Δ6-27 Toxin |
| Showing results 1 to 20 of 20 |
Collection:
| Collection ID: | CO003176 |
| Collection Summary: | AGS cells or AZ-521 cells were cultured in Roswell Park Memorial Institute (RPMI) 1640 medium containing 10% fetal bovine serum, or minimal essential medium (MEM) containing 10% fetal bovine serum and 5% nonessential amino acids, respectively. AGS cells were seeded at 2x104 cells/well into 96-well plates and incubated overnight. Cultured cells were then incubated with 20 ug/mL purified VacA [activated with by addition of HCl to a pH of 3] in medium supplemented with 5 mM NH4Cl. Following intoxication, the media was removed, and cells were washed with PBS. Cells were detached by incubation with trypsin for 5 minutes and collected via centrifugation at 4°C at 1,000 rpm for 4 minutes. Trypsin was removed, cells were once again washed with PBS, and the cells were then flash-frozen in liquid nitrogen and stored at -70°C. |
| Collection Protocol Filename: | Cell_Culture_Methods.pdf |
| Sample Type: | Cultured cells |
| Storage Conditions: | -80℃ |
Treatment:
| Treatment ID: | TR003192 |
| Treatment Summary: | Cells were incubated in media containing 20 ug/mL of purified VacA toxin and 5 mM ammonium chloride. |
| Treatment Protocol Filename: | Cell_Culture_Methods.pdf |
Sample Preparation:
| Sampleprep ID: | SP003189 |
| Sampleprep Summary: | Briefly, cell pellets were normalized by total protein (200 ug) and the corresponding cell supernatants were normalized by volume (200 uL). Metabolites were extracted with methanol/water 80:20. Heavy labeled phenylalanine-D8 and biotin-D2 were added to individual samples prior to protein precipitation. Following overnight incubation at -80°C, precipitated proteins were pelleted by centrifugation at 10,000 rpm for 10 min and metabolite extracts were transferred into two Eppendorf tubes in equal amounts and dried down in vacuo and stored at -80°C. |
| Sampleprep Protocol Filename: | MS_Methods_Cover_VacA_Mutants.pdf |
| Processing Storage Conditions: | -80℃ |
| Extract Storage: | -80℃ |
Combined analysis:
| Analysis ID | AN005025 |
|---|---|
| Analysis type | MS |
| Chromatography type | Reversed phase |
| Chromatography system | Thermo Vanquish |
| Column | Thermo Hypersil GOLD aQ (100 x 2.1mm,1.9um) |
| MS Type | ESI |
| MS instrument type | Orbitrap |
| MS instrument name | Thermo Q Exactive HF hybrid Orbitrap |
| Ion Mode | POSITIVE |
| Units | time_m/z |
Chromatography:
| Chromatography ID: | CH003797 |
| Methods Filename: | MS_Methods_Cover_VacA_Mutants.pdf |
| Instrument Name: | Thermo Vanquish |
| Column Name: | Thermo Hypersil GOLD aQ (100 x 2.1mm,1.9um) |
| Column Temperature: | 40 |
| Flow Gradient: | 30 min; 95%A, 5%B |
| Flow Rate: | 0.25 mL/min |
| Solvent A: | 100% water, 0.1% Formic Acid |
| Solvent B: | 80:20 acetonitrile:water, 0.1% Formic Acid |
| Chromatography Type: | Reversed phase |
MS:
| MS ID: | MS004764 |
| Analysis ID: | AN005025 |
| Instrument Name: | Thermo Q Exactive HF hybrid Orbitrap |
| Instrument Type: | Orbitrap |
| MS Type: | ESI |
| MS Comments: | Mass spectrometry raw data was imported, processed, normalized, and reviewed using Progenesis QI v.3.0 (Non-linear Dynamics, Newcastle, UK). |
| Ion Mode: | POSITIVE |
