#METABOLOMICS WORKBENCH pavaoa_20230105_133948 DATATRACK_ID:3683 STUDY_ID:ST002433 ANALYSIS_ID:AN003963 VERSION 1 CREATED_ON 02-08-2024 #PROJECT PR:PROJECT_TITLE Elucidating dynamic anaerobe metabolism with HRMAS 13C NMR and genome-scale PR:PROJECT_TITLE modeling PR:PROJECT_SUMMARY Anaerobic microbial metabolism drives critical functions within global PR:PROJECT_SUMMARY ecosystems, host-microbiota interactions, and industrial applications, yet PR:PROJECT_SUMMARY remains ill-defined. Here we advance a versatile approach to elaborate cellular PR:PROJECT_SUMMARY metabolism in obligate anaerobes using the pathogen Clostridioides difficile, an PR:PROJECT_SUMMARY amino acid and carbohydrate-fermenting Clostridia. High-Resolution Magic Angle PR:PROJECT_SUMMARY Spinning (HRMAS) Nuclear Magnetic Resonance (NMR) spectroscopy of C. difficile, PR:PROJECT_SUMMARY grown with fermentable 13C substrates, informed dynamic flux balance analysis PR:PROJECT_SUMMARY (dFBA) of the pathogen’s genome-scale metabolism. Analyses identified dynamic PR:PROJECT_SUMMARY recruitment of oxidative and supporting reductive pathways, with integration of PR:PROJECT_SUMMARY high-flux amino acid and glycolytic metabolism at alanine’s biosynthesis to PR:PROJECT_SUMMARY support efficient energy generation, nitrogen handling, and biomass generation. PR:PROJECT_SUMMARY Model predictions informed an approach leveraging the sensitivity of 13C NMR PR:PROJECT_SUMMARY spectroscopy to simultaneously track cellular carbon and nitrogen flow from PR:PROJECT_SUMMARY [U-13C]glucose and [15N]leucine, confirming the formation of [13C,15N]alanine. PR:PROJECT_SUMMARY Findings identify metabolic strategies used by C. difficile to support its rapid PR:PROJECT_SUMMARY colonization and expansion in gut ecosystems. PR:INSTITUTE Brigham and Women's Hospital PR:DEPARTMENT Pathology PR:LABORATORY Bry Lab (Massachusetts Host-Microbiome Center, BWH) and Cheng Lab (Massachusetts PR:LABORATORY General Hospital) PR:LAST_NAME Pavao PR:FIRST_NAME Aidan PR:ADDRESS 221 Longwood Ave, EBRC-411, Boston, MA, 02115, USA PR:EMAIL apavao2@bwh.harvard.edu PR:PHONE 617-525-7184 PR:FUNDING_SOURCE NIH R01AI153653, R03AI174158, P30DK056338, S10OD023406, R21CA243255, and PR:FUNDING_SOURCE R01AG070257; BWH Precision Medicine Institute; MGH A. A. Martinos Center for PR:FUNDING_SOURCE Biomedical Imaging PR:PUBLICATIONS https://doi.org/10.1038/s41589-023-01275-9 PR:DOI http://dx.doi.org/10.21228/M88M5G PR:CONTRIBUTORS Aidan Pavao, Brintha Girinathan, Johann Peltier, Pamela Altamirano Silva, Bruno PR:CONTRIBUTORS Dupuy, Isabella H. Muti, Craig Malloy, Leo L. Cheng, Lynn Bry #STUDY ST:STUDY_TITLE Elucidating dynamic anaerobe metabolism with HRMAS 13C NMR and genome-scale ST:STUDY_TITLE modeling ST:STUDY_SUMMARY Anaerobic microbial metabolism drives critical functions within global ST:STUDY_SUMMARY ecosystems, host-microbiota interactions, and industrial applications, yet ST:STUDY_SUMMARY remains ill-defined. Here we advance a versatile approach to elaborate cellular ST:STUDY_SUMMARY metabolism in obligate anaerobes using the pathogen Clostridioides difficile, an ST:STUDY_SUMMARY amino acid and carbohydrate-fermenting Clostridia. High-Resolution Magic Angle ST:STUDY_SUMMARY Spinning (HRMAS) Nuclear Magnetic Resonance (NMR) spectroscopy of C. difficile, ST:STUDY_SUMMARY grown with fermentable 13C substrates, informed dynamic flux balance analysis ST:STUDY_SUMMARY (dFBA) of the pathogen’s genome-scale metabolism. Analyses identified dynamic ST:STUDY_SUMMARY recruitment of oxidative and supporting reductive pathways, with integration of ST:STUDY_SUMMARY high-flux amino acid and glycolytic metabolism at alanine’s biosynthesis to ST:STUDY_SUMMARY support efficient energy generation, nitrogen handling, and biomass generation. ST:STUDY_SUMMARY Model predictions informed an approach leveraging the sensitivity of 13C NMR ST:STUDY_SUMMARY spectroscopy to simultaneously track cellular carbon and nitrogen flow from ST:STUDY_SUMMARY [U-13C]glucose and [15N]leucine, confirming the formation of [13C,15N]alanine. ST:STUDY_SUMMARY Findings identify metabolic strategies used by C. difficile to support its rapid ST:STUDY_SUMMARY colonization and expansion in gut ecosystems. ST:INSTITUTE Brigham and Women's Hospital ST:DEPARTMENT Pathology ST:LABORATORY Bry Lab, Massachusetts Host-Microbiome Center; Cheng Lab, Massachusetts General ST:LABORATORY Hospital ST:LAST_NAME Pavao ST:FIRST_NAME Aidan ST:ADDRESS 221 Longwood Ave, EBRC-411, Boston, MA, 02115, USA ST:EMAIL apavao2@bwh.harvard.edu ST:PHONE 617-525-7184 ST:SUBMIT_DATE 2023-01-05 #SUBJECT SU:SUBJECT_TYPE Bacteria SU:SUBJECT_SPECIES Clostridioides difficile SU:TAXONOMY_ID NCBI:txid1496 SU:GENOTYPE_STRAIN ATCC 43255 delPaLoc #SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Additional sample data SUBJECT_SAMPLE_FACTORS - Data10_13CGlc_endpt1 Condition:13C-Glucose RAW_FILE_NAME=Data10_13CGlc_endpt1; Type=Culture supernatant; Analysis=Alanine 14N:15N SUBJECT_SAMPLE_FACTORS - Data11_13CGlc_endpt2 Condition:13C-Glucose RAW_FILE_NAME=Data11_13CGlc_endpt2; Type=Culture supernatant; Analysis=Alanine 14N:15N SUBJECT_SAMPLE_FACTORS - Data12_13CGlc_endpt3 Condition:13C-Glucose RAW_FILE_NAME=Data12_13CGlc_endpt3; Type=Culture supernatant; Analysis=Alanine 14N:15N SUBJECT_SAMPLE_FACTORS - Data7_13CGlc1 Condition:13C-Glucose RAW_FILE_NAME=Data7_13CGlc1; Type=Time series; Analysis=dFBA, Alanine 14N:15N SUBJECT_SAMPLE_FACTORS - Data8_13CGlc2 Condition:13C-Glucose RAW_FILE_NAME=Data8_13CGlc2; Type=Time series; Analysis=dFBA SUBJECT_SAMPLE_FACTORS - Data9_13CGlc3 Condition:13C-Glucose RAW_FILE_NAME=Data9_13CGlc3; Type=Time series; Analysis=dFBA SUBJECT_SAMPLE_FACTORS - Data19_13CGlc_standards Condition:13C-Glucose, 13C-Acetate, 13C-Alanine, 13C-Ethanol, 13C-Butyrate RAW_FILE_NAME=Data19_13CGlc_standards; Type=Chemical solutions; Analysis=dFBA SUBJECT_SAMPLE_FACTORS - Data13_13CGlc_15NLeu_endpt1 Condition:13C-Glucose, 15N-Leucine RAW_FILE_NAME=Data13_13CGlc_15NLeu_endpt1; Type=Culture supernatant; Analysis=Alanine 14N:15N SUBJECT_SAMPLE_FACTORS - Data14_13CGlc_15NLeu_endpt2 Condition:13C-Glucose, 15N-Leucine RAW_FILE_NAME=Data14_13CGlc_15NLeu_endpt2; Type=Culture supernatant; Analysis=Alanine 14N:15N SUBJECT_SAMPLE_FACTORS - Data15_13CGlc_15NLeu_endpt3 Condition:13C-Glucose, 15N-Leucine RAW_FILE_NAME=Data15_13CGlc_15NLeu_endpt3; Type=Culture supernatant; Analysis=Alanine 14N:15N SUBJECT_SAMPLE_FACTORS - Data16_13CGlc_15NLeu_stack Condition:13C-Glucose, 15N-Leucine RAW_FILE_NAME=Data16_13CGlc_15NLeu_stack; Type=Time series; Analysis=Alanine 14N:15N SUBJECT_SAMPLE_FACTORS - Data4_13CLeu1 Condition:13C-Leucine RAW_FILE_NAME=Data4_13CLeu1; Type=Time series; Analysis=dFBA SUBJECT_SAMPLE_FACTORS - Data5_13CLeu2 Condition:13C-Leucine RAW_FILE_NAME=Data5_13CLeu2; Type=Time series; Analysis=dFBA SUBJECT_SAMPLE_FACTORS - Data6_13CLeu3 Condition:13C-Leucine RAW_FILE_NAME=Data6_13CLeu3; Type=Time series; Analysis=dFBA SUBJECT_SAMPLE_FACTORS - Data1_13CPro1 Condition:13C-Proline RAW_FILE_NAME=Data1_13CPro1; Type=Time series; Analysis=dFBA SUBJECT_SAMPLE_FACTORS - Data2_13CPro2 Condition:13C-Proline RAW_FILE_NAME=Data2_13CPro2; Type=Time series; Analysis=dFBA SUBJECT_SAMPLE_FACTORS - Data3_13CPro3 Condition:13C-Proline RAW_FILE_NAME=Data3_13CPro3; Type=Time series; Analysis=dFBA SUBJECT_SAMPLE_FACTORS - Data20_2D Condition:13C-Proline, 13C-Leucine, 13C-Glucose RAW_FILE_NAME=Data20_2D; Type=2D time series snapshots; Analysis=Metabolite identification SUBJECT_SAMPLE_FACTORS - Data18_13CPro-Se Condition:13C-Proline, no Selenium source RAW_FILE_NAME=Data18_13CPro-Se; Type=Time series; Analysis=Selenium perturbation SUBJECT_SAMPLE_FACTORS - Data17_13CPro+Se Condition:13C-Proline, Sodium Selenite RAW_FILE_NAME=Data17_13CPro+Se; Type=Time series; Analysis=Selenium perturbation #COLLECTION CO:COLLECTION_SUMMARY For live cell time series, labeled MMM with 10% D2O was inoculated with 100,000 CO:COLLECTION_SUMMARY vegetative Clostridioides difficile ATCC43255 delPaLoc cells in a Kel-F HRMAS CO:COLLECTION_SUMMARY rotor insert under anaerobic atmosphere. The sample was spun at 3600 Hz at 37°C CO:COLLECTION_SUMMARY in a 4mm zirconia rotor and successive spectra were acquired over 36+ hours. For CO:COLLECTION_SUMMARY static culture supernatants, labeled MMM was inoculated with 100,000 vegetative CO:COLLECTION_SUMMARY Clostridioides difficile ATCC43255 delPaLoc cells in an anaerobic chamber and CO:COLLECTION_SUMMARY incubated at 37°C for 48 hours. Cultures were centrifuged and supernatants CO:COLLECTION_SUMMARY collected and lyophilized, then resuspended in D2O prior to NMR spectra CO:COLLECTION_SUMMARY acquisition. CO:SAMPLE_TYPE Bacterial cells CO:VOLUMEORAMOUNT_COLLECTED 30 µL CO:COLLECTION_VIALS Kel-F inserts for 4mm MAS rotor CO:COLLECTION_TUBE_TEMP 37°C #TREATMENT TR:TREATMENT_SUMMARY For live cell time series, cells were grown in MMM with 10% D2O and one or more TR:TREATMENT_SUMMARY labeled substrates (L-[U-13C]Proline, L-[U-13C]Leucine, [U-13C]Glucose, or both TR:TREATMENT_SUMMARY [U-13C]Glucose and L-[15N]Leucine). For culture supernatants, cells were grown TR:TREATMENT_SUMMARY in MMM with [U-13C]Glucose, with or without L-[15N]Leucine. For the selenium TR:TREATMENT_SUMMARY perturbation time series, cells were grown in MMM with 10% L-[U-13C]Proline, TR:TREATMENT_SUMMARY with or without 100µM sodium selenite. TR:TREATMENT_COMPOUND L-[U-13C]Proline, L-[U-13C]Leucine, [U-13C]Glucose, L-[15N]Leucine, sodium TR:TREATMENT_COMPOUND selenite TR:CELL_MEDIA C. difficile Modified Minimal Medium (MMM) with 100µM sodium selenite and 10% TR:CELL_MEDIA D2O TR:CELL_ENVIR_COND anaerobic, 37°C #SAMPLEPREP SP:SAMPLEPREP_SUMMARY For live cell NMR time series, samples were added to the HRMAS rotor neat. For SP:SAMPLEPREP_SUMMARY culture supernatants, lyophilized samples were resuspended in D2O added to the SP:SAMPLEPREP_SUMMARY NMR rotor neat. #CHROMATOGRAPHY #ANALYSIS AN:LABORATORY_NAME Cheng Lab AN:ANALYSIS_TYPE NMR AN:ACQUISITION_PARAMETERS_FILE acqus AN:SOFTWARE_VERSION TopSpin 3.6.2 AN:OPERATOR_NAME Leo L Cheng AN:PROCESSING_PARAMETERS_FILE pdata/1/procs AN:DATA_FORMAT Bruker #NMR NM:INSTRUMENT_NAME Bruker Avance III HD NM:INSTRUMENT_TYPE FT-NMR NM:NMR_EXPERIMENT_TYPE Other NM:NMR_COMMENTS Experiments collected 1D 1H, 1D 13C, 2D 1H, 2D 13C, and 2D 1H-13C data. NM:SPECTROMETER_FREQUENCY 600 MHz NM:NMR_SOLVENT C. difficile Modified Minimal Medium (MMM) with 100µM sodium selenite and 10% NM:NMR_SOLVENT D2O NM:TEMPERATURE 37 #NMR_METABOLITE_DATA NMR_METABOLITE_DATA:UNITS proportion of Alanine NMR_METABOLITE_DATA_START Samples Data10_13CGlc_endpt1 Data11_13CGlc_endpt2 Data12_13CGlc_endpt3 Data13_13CGlc_15NLeu_endpt1 Data14_13CGlc_15NLeu_endpt2 Data15_13CGlc_15NLeu_endpt3 Factors Condition:13C-Glucose Condition:13C-Glucose Condition:13C-Glucose Condition:13C-Glucose, 15N-Leucine Condition:13C-Glucose, 15N-Leucine Condition:13C-Glucose, 15N-Leucine [2_3-13C_ 14N]Alanine 0.4700 0.5300 0.5300 0.1700 0.2200 0.2400 [2_3-13C_ 15N]Alanine 0.2800 0.2600 0.2600 [U-13C_ 14N]Alanine 0.5300 0.4700 0.4700 0.1900 0.2400 0.2300 [U-13C_ 15N]Alanine 0.3600 0.2700 0.2700 NMR_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name pubchem_id inchi_key kegg_id other_id other_id_type ri ri_type moverz_quant [2,3-13C, 14N]Alanine [2,3-13C, 15N]Alanine [U-13C, 14N]Alanine [U-13C, 15N]Alanine METABOLITES_END #END