#METABOLOMICS WORKBENCH marian3101_20210622_142630 DATATRACK_ID:2715 STUDY_ID:ST001904 ANALYSIS_ID:AN003101 PROJECT_ID:PR001199 VERSION 1 CREATED_ON August 16, 2021, 3:19 pm #PROJECT PR:PROJECT_TITLE Lipidomic study PR:PROJECT_TYPE LC-MS analysis PR:PROJECT_SUMMARY Lipidomic analysis of total membranes and outer membrane vesicles from the human PR:PROJECT_SUMMARY gut comensal Bacteroides thetaiotaomicron. Strains used to perform the analysis: PR:PROJECT_SUMMARY wild-tipe and mutants lacking the genes BT1522, BT1523, BT1524 and BT1526, PR:PROJECT_SUMMARY involved in the synthesis of phosphoinosytol and Ceramide phosphinosytol PR:INSTITUTE Washington University, St. Louis PR:DEPARTMENT Molecular Microbiology PR:LABORATORY Feldman lab PR:LAST_NAME Sartorio PR:FIRST_NAME Mariana PR:ADDRESS 660 S Euclid avenue, campus box 8230, 63110 PR:EMAIL mgsartorio@wustl.edu PR:PHONE 3147474477 PR:FUNDING_SOURCE NIH PR:PUBLICATIONS Lipidomics analysis of outer membrane vesicles and elucidation of the ceramide PR:PUBLICATIONS phosphoinositol biosynthetic pathway in Bacteroides thetaiotaomicron PR:CONTRIBUTORS Ezequiel Valguarnera, Mariana G. Sartorio, Fong-Fu Hsu and Mario F. Feldman #STUDY ST:STUDY_TITLE Lipidomics analysis of outer membrane vesicles and elucidation of the ceramide ST:STUDY_TITLE phosphoinositol biosynthetic pathway in Bacteroides thetaiotaomicron ST:STUDY_SUMMARY In this work, we characterized the lipid composition of membranes and OMV from ST:STUDY_SUMMARY Bacteroides thetaiotaomicron VPI-5482. LC-MS analysis indicate that OMV carry ST:STUDY_SUMMARY sphingolipids, glycerophospholipids and serine-dipeptide lipids. Sphingolipid ST:STUDY_SUMMARY species represent more than 50% of the total lipid content of OMV. The most ST:STUDY_SUMMARY abundant sphingolipids in OMV are ceramide phosphoethanolamine (CerPE) and ST:STUDY_SUMMARY ceramide phosphoinositol (CerPI). Bioinformatic analysis allowed the ST:STUDY_SUMMARY identification of the BT1522-1526 operon putatively involved in CerPI synthesis. ST:STUDY_SUMMARY Mutagenesis studies revealed BT1522-1526 are essential for synthesis of PI and ST:STUDY_SUMMARY CerPI, confirming the role of this operon in biosynthesis of CerPI. BT1522-1526 ST:STUDY_SUMMARY mutant strains lacking CerPI produced OMV that were indistinguishable from the ST:STUDY_SUMMARY wild-type strain, indicating that CerPI sphingolipid species are not involved in ST:STUDY_SUMMARY OMV biogenesis. Bacteroides sphingolipids are thought to modulate host-commensal ST:STUDY_SUMMARY interactions, and based on our data, we propose that OMV could act as long ST:STUDY_SUMMARY distance delivery vehicles for these molecules. ST:INSTITUTE Washington University, St. Louis ST:DEPARTMENT Molecular Microbiology ST:LABORATORY Feldman lab ST:LAST_NAME Sartorio ST:FIRST_NAME Mariana ST:ADDRESS 660 S Euclid avenue, campus box 8230, 63110 ST:EMAIL mgsartorio@wustl.edu ST:STUDY_TYPE Lipidic profile in wild-type and mutant strains ST:PHONE 3147474477 #SUBJECT SU:SUBJECT_TYPE Bacteria SU:SUBJECT_SPECIES Bacteroides thetaiotaomicron SU:TAXONOMY_ID 818 #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 - OMV-WT-1 Genotype:wt SUBJECT_SAMPLE_FACTORS - OMV-WT-2 Genotype:wt SUBJECT_SAMPLE_FACTORS - OMV-1522-1 Genotype:deltaBT1522 SUBJECT_SAMPLE_FACTORS - OMV-1522-2 Genotype:deltaBT1522 SUBJECT_SAMPLE_FACTORS - OMV-1523-1 Genotype:deltaBT1523 SUBJECT_SAMPLE_FACTORS - OMV-1523-2 Genotype:deltaBT1523 SUBJECT_SAMPLE_FACTORS - OMV-1524-1 Genotype:deltaBT1524 SUBJECT_SAMPLE_FACTORS - OMV-1524-2 Genotype:deltaBT1524 SUBJECT_SAMPLE_FACTORS - OMV-1526-1 Genotype:deltaBT1526 SUBJECT_SAMPLE_FACTORS - OMV-1526-2 Genotype:deltaBT1526 SUBJECT_SAMPLE_FACTORS - TM-WT-1 Genotype:wt SUBJECT_SAMPLE_FACTORS - TM-WT-2 Genotype:wt SUBJECT_SAMPLE_FACTORS - TM-1522-1 Genotype:deltaBT1522 SUBJECT_SAMPLE_FACTORS - TM-1522-2 Genotype:deltaBT1522 SUBJECT_SAMPLE_FACTORS - TM-1523-1 Genotype:deltaBT1523 SUBJECT_SAMPLE_FACTORS - TM-1523-2 Genotype:deltaBT1523 SUBJECT_SAMPLE_FACTORS - TM-1524-1 Genotype:deltaBT1524 SUBJECT_SAMPLE_FACTORS - TM-1524-2 Genotype:deltaBT1524 SUBJECT_SAMPLE_FACTORS - TM-1526-1 Genotype:deltaBT1526 SUBJECT_SAMPLE_FACTORS - TM-1526-2 Genotype:deltaBT1526 #COLLECTION CO:COLLECTION_SUMMARY B. thetaiotaomicron strains (wild-type and ΔBT1522-BT1526 mutants) were grown CO:COLLECTION_SUMMARY overnight in an anaerobic chamber (Coy Laboratories) using an atmosphere of 10% CO:COLLECTION_SUMMARY H2, 5% CO2, 85% N2. For liquid growth, Brain heart infusion (BHI) supplemented CO:COLLECTION_SUMMARY with hemin and vitamin K3 was used. Cultures were then subjected to subcellular CO:COLLECTION_SUMMARY fractionation to obtain total membranes and outer membrane vesicles (OMV) CO:COLLECTION_SUMMARY preparation. OMV preparations: OMV were purified by ultracentrifugation of CO:COLLECTION_SUMMARY filtered spent media from 150 ml of liquid culture as described previously (1). CO:COLLECTION_SUMMARY OMV preparations were resuspended in PBS before lipid analyses. Protein content CO:COLLECTION_SUMMARY was quantified using a DC protein assay kit (Bio-Rad). Fractions were aliquoted CO:COLLECTION_SUMMARY and stored at -80°C until analyzed. Membrane preparations: Total membrane CO:COLLECTION_SUMMARY preparations were performed by cell lysis and ultracentrifugation as previously CO:COLLECTION_SUMMARY described (1). Total membranes from 150 ml of liquid culture were resuspended in CO:COLLECTION_SUMMARY PBS using a 2-ml glass tissue grinder with a polytetrafluoroethylene (PTFE) CO:COLLECTION_SUMMARY pestle (VWR). Protein content was quantified using a DC protein assay kit CO:COLLECTION_SUMMARY (Bio-Rad). Fractions were stored aliquoted and stored at -80°C until analyzed. CO:COLLECTION_SUMMARY References: 1. Elhenawy W, Debelyy MO, Feldman MF. Preferential packing of CO:COLLECTION_SUMMARY acidic glycosidases and proteases into Bacteroides outer membrane vesicles. CO:COLLECTION_SUMMARY mBio. 2014;5(2):e00909-14. CO:SAMPLE_TYPE Bacterial cells #TREATMENT TR:TREATMENT_SUMMARY No treatment displayed. Wild-type and mutant bacteria were grown in BHI TR:TREATMENT_SUMMARY supplemented with vitamin K and Hemin #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Total lipids from OMV and TM were extracted based on the Bligh and Dyer SP:SAMPLEPREP_SUMMARY chloroform:methanol method (1). Briefly, 2 volumes of methanol, 1 volume of SP:SAMPLEPREP_SUMMARY chloroform, and 0.8 volumes of Milli-Q water were added to 1 volume of SP:SAMPLEPREP_SUMMARY PBS-resuspended OMV or TM fractions into solvent-resistant glass tubes. Contents SP:SAMPLEPREP_SUMMARY were mixed for 1 min by vortexing and 1 volume of chloroform was added to the SP:SAMPLEPREP_SUMMARY mixture. Contents were mixed for another minute and tubes were centrifuged for 5 SP:SAMPLEPREP_SUMMARY min at 4000 rpm. After centrifugation, bottom phase (organic) was recovered SP:SAMPLEPREP_SUMMARY using a glass Pasteur pipette and stored in solvent-sealed vials at -80°C until SP:SAMPLEPREP_SUMMARY lipid analysis by LC-MS. References: 1. Bligh EG, Dyer WJ. A rapid method of SP:SAMPLEPREP_SUMMARY total lipid extraction and purification. Can J Biochem Physiol. SP:SAMPLEPREP_SUMMARY 1959;37(8):911-7. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY LC/MS analyses were conducted on an Agilent 6550 A QTOF instrument with an CH:CHROMATOGRAPHY_SUMMARY Agilent 1290 HPLC with autosampler, operated by Agilent Masshunter software CH:CHROMATOGRAPHY_SUMMARY (Santa Clara, CA USA). Separation of the total lipid extracts was achieved by a CH:CHROMATOGRAPHY_SUMMARY Thermo (Waltham MA, USA) 100 × 2.1 mm BETASIL 5 μm C18 column at a flow rate CH:CHROMATOGRAPHY_SUMMARY of 300 μl/min at room temperature. The mobile phase contained 5 mM ammonium CH:CHROMATOGRAPHY_SUMMARY formate (pH 5.0) both in solvent A, acetonitrile:water (60:40, v/v), and solvent CH:CHROMATOGRAPHY_SUMMARY B, isopropanol:acetonitrile (90:10, v/v). A gradient elution in the following CH:CHROMATOGRAPHY_SUMMARY manner was applied: 68% A, 0–1.5 min; 68–55% A, 1.5–4 min; 55–48% A, CH:CHROMATOGRAPHY_SUMMARY 4–5 min; 48–42% A, 5–8 min; 42–34% A, 8–11 min; 34–30% A, 11–14 CH:CHROMATOGRAPHY_SUMMARY min; 30–25% A, 14–18 min; 25–3% A, 18–23 min; 3–0% A, 25–30 min; 0% CH:CHROMATOGRAPHY_SUMMARY A, 30–35 min; 68% A, 35–40 min. Both the positive-ion and negative-ion ESI CH:CHROMATOGRAPHY_SUMMARY MS scans were acquired in the mass range of 200-2000 Da at a rate of 2 CH:CHROMATOGRAPHY_SUMMARY scans/min. CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Agilent 6550 CH:COLUMN_NAME Thermo Betasil C18 (100 x 2.1mm, 5um) #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Agilent 6550 QTOF MS:INSTRUMENT_TYPE QTOF MS:MS_TYPE ESI MS:ION_MODE POSITIVE/NEGATIVE MS:MS_COMMENTS ESI MS scans were acquired in the mass range of 200-2000 Da at a rate of 2 scans MS:MS_COMMENTS /min. MS:MS_RESULTS_FILE ST001904_AN003101_Results.txt UNITS:peak area Has m/z:Yes Has RT:No RT units:No RT data #END