#METABOLOMICS WORKBENCH juliehaines_20241126_072642 DATATRACK_ID:5404 STUDY_ID:ST003609 ANALYSIS_ID:AN005932 PROJECT_ID:PR002231 VERSION 1 CREATED_ON December 2, 2024, 3:03 pm #PROJECT PR:PROJECT_TITLE Macrophages recycle phagocytosed bacteria to fuel immunometabolic responses PR:PROJECT_SUMMARY Macrophages specialize in phagocytosis, a cellular process that eliminates PR:PROJECT_SUMMARY extracellular matter, including microbes, through internalization and PR:PROJECT_SUMMARY degradation. Despite the critical role of phagocytosis during bacterial PR:PROJECT_SUMMARY infection, the fate of phagocytosed microbial cargo and its impact on host cell PR:PROJECT_SUMMARY is poorly understood. Here, we reveal that ingested bacteria constitute an PR:PROJECT_SUMMARY alternative nutrient source that skews immunometabolic host responses. Tracing PR:PROJECT_SUMMARY stable isotope-labelled bacteria, we found that phagolysosomal degradation of PR:PROJECT_SUMMARY bacteria provides carbon atoms and amino acids that are recycled into various PR:PROJECT_SUMMARY metabolic pathways, including glutathione and itaconate biosynthesis, and PR:PROJECT_SUMMARY satisfy macrophage bioenergetic needs. Metabolic recycling of PR:PROJECT_SUMMARY microbially-derived nutrients is regulated by the nutrient sensing mTORC1 and PR:PROJECT_SUMMARY intricately tied to microbial viability. Dead bacteria, as opposed to live ones, PR:PROJECT_SUMMARY are enriched in cyclic- adenosine monophosphate (AMP), sustain the cellular AMP PR:PROJECT_SUMMARY pool and subsequently activate AMP protein kinase (AMPK) to inhibit mTORC1. PR:PROJECT_SUMMARY Consequently, killed bacteria strongly fuel metabolic recycling and support PR:PROJECT_SUMMARY macrophage survival, but elicit decreased reactive oxygen species (ROS) PR:PROJECT_SUMMARY production and a reduced IL-1β secretion compared to viable bacteria. These PR:PROJECT_SUMMARY results reveal a novel insight into the fate of engulfed microbes and highlights PR:PROJECT_SUMMARY a microbial viability-associated metabolite that triggers host metabolic and PR:PROJECT_SUMMARY immune responses. Our findings hold promise for shaping immunometabolic PR:PROJECT_SUMMARY intervention in various immune-related pathologies. PR:INSTITUTE University of Colorado Anschutz Medical Campus PR:LABORATORY Lab of Angelo D'Alessandro in collaboration with lab of Johan Garaude (INSERM, PR:LABORATORY Fr) PR:LAST_NAME Haines PR:FIRST_NAME Julie PR:ADDRESS 12801 E 17th Ave, Room 1303, Aurora, Colorado, 80045, USA PR:EMAIL julie.haines@cuanschutz.edu PR:PHONE 3037243339 #STUDY ST:STUDY_TITLE 13C-tracing of central energy metabolism in thymidine-auxotroph E. Coli ST:STUDY_SUMMARY 13C-tracing metabolomics of live thymidine-auxotroph Escherichia Coli K12 ST:STUDY_SUMMARY cultured (1h) or not (0h) in RPMI medium. ST:INSTITUTE University of Colorado Anschutz Medical Campus ST:LAST_NAME Haines ST:FIRST_NAME Julie ST:ADDRESS 12801 E 17th Ave, Room 1303, Aurora, Colorado, 80045, USA ST:EMAIL julie.haines@cuanschutz.edu ST:PHONE 3037243339 #SUBJECT SU:SUBJECT_TYPE Cultured cells SU:SUBJECT_SPECIES Escherichia coli SU:GENDER Not applicable #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 - E. coli T0_1 time (h):0 | Sample source:cells RAW_FILE_NAME(raw file name)=AA83eC-7_r3- SUBJECT_SAMPLE_FACTORS - E. coli T0_2 time (h):0 | Sample source:cells RAW_FILE_NAME(raw file name)=AA83eC-8_r10- SUBJECT_SAMPLE_FACTORS - E. coli T0_3 time (h):0 | Sample source:cells RAW_FILE_NAME(raw file name)=AA83eC-9_r9- SUBJECT_SAMPLE_FACTORS - E. coli T1_1 time (h):1 | Sample source:cells RAW_FILE_NAME(raw file name)=AA83eC-10_r4- SUBJECT_SAMPLE_FACTORS - E. coli T1_2 time (h):1 | Sample source:cells RAW_FILE_NAME(raw file name)=AA83eC-11_r7- SUBJECT_SAMPLE_FACTORS - E. coli T1_3 time (h):1 | Sample source:cells RAW_FILE_NAME(raw file name)=AA83eC-12_r2- #COLLECTION CO:COLLECTION_SUMMARY 1E9 Bacteria were washed with cold PBS, frozen as dry cell pellet, and stored at CO:COLLECTION_SUMMARY -80˚C until processing. CO:SAMPLE_TYPE Bacterial cells #TREATMENT TR:TREATMENT_SUMMARY ThyA- E. coli were grown overnight with shaking in LB supplemented with TR:TREATMENT_SUMMARY thymidine (500 ug/ml) and trimethoprim (50 ug/ml), diluted 1/40, and grown until TR:TREATMENT_SUMMARY log-phase [optical density at 600 nm (OD600) of 0.8-1.2]. For labeling of TR:TREATMENT_SUMMARY bacteria, 10 ul of an overnight cultured of thyA- E. coli was added to 20 ml of TR:TREATMENT_SUMMARY a filtered M9 minimal medium salts (Life Technologies) supplemented with 1 mM TR:TREATMENT_SUMMARY thiamine, 1 mM MgSO4, 0.1 M CaCl2, 500 ug/ml thymidine, 50 ug/ml trimethoprim, TR:TREATMENT_SUMMARY and 0.5% U-13C glucose (Campro Scientific). Bacteria were harvested at time zero TR:TREATMENT_SUMMARY or 1h. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Metabolites from frozen pellets were extracted at 2e9 cells per mL using ice SP:SAMPLEPREP_SUMMARY cold 5:3:2 methanol:acetonitrile:water (v/v/v) with vigorous vortexing at 4 SP:SAMPLEPREP_SUMMARY degrees C followed by centrifugation as described for 10 min at 18,000 g. 50 uL SP:SAMPLEPREP_SUMMARY aliquots of supernatants were diluted 1:1 with cold 5:3:2 SP:SAMPLEPREP_SUMMARY methanol:acetonitrile:water. Resulting samples were maintained at 4°C until SP:SAMPLEPREP_SUMMARY analysis that same day. SP:PROCESSING_STORAGE_CONDITIONS 4℃ SP:EXTRACT_STORAGE -80℃ #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY Positive C18 CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Thermo Vanquish CH:COLUMN_NAME Phenomenex Kinetex C18 (150 x 2.1mm,1.7um) CH:SOLVENT_A 100% water; 0.1% formic acid CH:SOLVENT_B 100% acetonitrile; 0.1% formic acid CH:FLOW_GRADIENT 0-0.5 min 5% B, 0.5-1.1 min 5-95% B, 1.1-2.75 min hold at 95% B, 2.75-3 min CH:FLOW_GRADIENT 95-5% B, 3-5 min hold at 5% B CH:FLOW_RATE 450 uL/min CH:COLUMN_TEMPERATURE 45 CH:SAMPLE_INJECTION 10 uL #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Thermo Orbitrap Exploris 120 MS:INSTRUMENT_TYPE Orbitrap MS:MS_TYPE ESI MS:ION_MODE POSITIVE MS:MS_COMMENTS We use a Thermo Orbitrap Exploris 120. Resolution 120,000, scan range 65-975 MS:MS_COMMENTS m/z, maximum injection time 100 ms, microscans 1, automatic gain control (AGC) MS:MS_COMMENTS detection duration 20 msec, source voltage 3.5 kV, capillary temperature 320 C, MS:MS_COMMENTS vaporizer temp 200 C, and sheath gas 50, auxiliary gas 10, and sweep gas 1 (all MS:MS_COMMENTS nitrogen). Data converted to mzXML using RawConverter. Metabolites were MS:MS_COMMENTS annotated and integrated using Maven in conjunction with the KEGG database. #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS peak area MS_METABOLITE_DATA_START Samples E. coli T0_1 E. coli T0_2 E. coli T0_3 E. coli T1_1 E. coli T1_2 E. coli T1_3 Factors time (h):0 | Sample source:cells time (h):0 | Sample source:cells time (h):0 | Sample source:cells time (h):1 | Sample source:cells time (h):1 | Sample source:cells time (h):1 | Sample source:cells Arginine 23726.05 56042.08 3774431.75 1848732.62 1658745.62 1693482 Arginine 13C3 5337.05 3450.83 5668.25 0 1210.94 0 Arginine 13C4 26723.67 21823.04 25993.33 2516.98 4135.46 4981.48 Arginine 13C5 56537.44 49299.48 56871.64 16922.73 14125.05 17994.48 Arginine 13C6 201133.38 200913.42 200885.05 46152.03 51099.66 62485.5 Cysteine 7808.6 9614.07 5991.72 133452.47 185523.83 99944.46 Glutamine 9751.47 7676.16 23375.4 723469.69 350817.59 281533.62 Glutamine 13C3 6115.69 8553.26 4562.34 0 0 0 Glutamine 13C4 38237.34 28129.72 26145.14 0 0 0 Glutamine 13C5 176634.05 167560.33 176874.48 21951.28 18084.55 19121.11 Methionine 0 0 87822.9 503454.16 449060.41 332445.66 Methionine 13C4 35122.91 31136.25 31231.97 1888.79 2014.83 2738.77 Methionine 13C5 70876.4 63823.52 46631.07 13766.49 25106.05 17420.12 Adenosine 58526.23 23960.96 89009.38 111352.13 37839.71 66155.06 Adenosine 13C1 3816.5747 5214.6044 1270.1482 4091.5357 0 2316.9634 Adenosine 13C5 0 0 0 31259.47 28100.57 17937.38 Adenosine 13C8 5104.23 2781.48 5115.85 20697.3 10543.25 9065.58 Adenosine 13C9 20139.72 24367.93 6328.42 7663.92 2540.68 13019.32 Adenosine 13C10 91116.59 99862.09 48504.23 25464.4 16583.23 22571.12 S-Adenosyl-L-methionine 0 0 0 56678.24 48605.47 37504.82 S-Adenosyl-L-methionine 13C15 22385.48 23370.69 19619.58 4778.83 4725.5 3590.53 Putrescine 6435.63 7446.06 4077.81 49839.81 70048.96 67363.71 Putrescine 13C2 13418.33062 15501.85443 18012.37536 25168.21013 26978.26521 26520.15386 Putrescine 13C3 7316.74 10122.29 9191.54 10761.08 16304.43 18983.27 Putrescine 13C4 111623.35 98901.81 91351.52 132982.02 141460.02 156625.81 Spermidine 1460.64 2594.42 8154.97 30789.71 26486.46 22119.33 Spermidine 13C1 0 0 0 7708.64233 4218.26258 1343.73159 Spermidine 13C2 2124.799865 1622.357684 1477.529183 25367.89781 18829.19178 13912.96449 Spermidine 13C3 8259.12 4322.56 3061.87 24841.63 18636.65 19226.75 Spermidine 13C4 12142.67 3580.57 7582.4 98307.5 92091.24 57851.83 Spermidine 13C5 24093.58 16644.21 12462.31 57830.17 56094.91 33903.07 Spermidine 13C6 17686.36 8237.23 11754.41 40382.08 40101.36 17826.43 Spermidine 13C7 64297.97 31319.3 37561.28 167287.67 139354.67 97835.3 Ornithine 6241.41 11253.94 52351.95 27024.23 24209.91 12645.82 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name KEGG ID m/z r.t. Arginine C00062 175.118896 0.588 Arginine 13C3 C00062 178.129517 0.615 Arginine 13C4 C00062 179.132324 0.591 Arginine 13C5 C00062 180.136337 0.602 Arginine 13C6 C00062 181.139053 0.585 Cysteine C00097 122.027222 0.686 Glutamine C00064 147.076477 0.61 Glutamine 13C3 C00064 150.086533 0.605 Glutamine 13C4 C00064 151.089859 0.609 Glutamine 13C5 C00064 152.093201 0.605 Methionine C00073 150.058365 0.659 Methionine 13C4 C00073 154.073059 0.641 Methionine 13C5 C00073 155.075104 0.659 Adenosine C00212 268.103912 0.664 Adenosine 13C1 C00212 269.105682 0.662 Adenosine 13C5 C00212 273.121521 0.641 Adenosine 13C8 C00212 276.131531 0.658 Adenosine 13C9 C00212 277.136414 0.657 Adenosine 13C10 C00212 278.137848 0.659 S-Adenosyl-L-methionine C00019 399.144379 0.577 S-Adenosyl-L-methionine 13C15 C00019 414.194824 0.589 Putrescine C00134 89.107468 0.573 Putrescine 13C2 C00134 91.11422 0.56 Putrescine 13C3 C00134 92.117615 0.566 Putrescine 13C4 C00134 93.120934 0.561 Spermidine C00750 146.165222 0.559 Spermidine 13C1 C00750 147.168579 0.55 Spermidine 13C2 C00750 148.171921 0.543 Spermidine 13C3 C00750 149.175293 0.542 Spermidine 13C4 C00750 150.178635 0.538 Spermidine 13C5 C00750 151.182007 0.541 Spermidine 13C6 C00750 152.185333 0.544 Spermidine 13C7 C00750 153.188675 0.536 Ornithine C00062 133.097168 0.587 METABOLITES_END #END