#METABOLOMICS WORKBENCH juliehaines_20241126_072642 DATATRACK_ID:5404 STUDY_ID:ST003609 ANALYSIS_ID:AN005931 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 Negative 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 95% water/5% acetonitrile; 1 mM ammonium acetate CH:SOLVENT_B 95% acetonitrile/5% water; 1 mM ammonium acetate CH:FLOW_GRADIENT 0-0.5 min 0% B, 0.5-1.1 min 0-100% B, 1.1-2.75 min hold at 100% B, 2.75-3 min CH:FLOW_GRADIENT 100-0% B, 3-5 min hold at 0% 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 NEGATIVE 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 2.0 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 Glutamate 86055.39 45499.02 1465204.5 27764006 21346200 20835272 Glutamate 13C1 1314.14355 136.2339 0 162311.17 155855.62 112759.04 Glutamate 13C2 88925.06245 82459.51546 71537.31639 805300.4419 676840.245 675060.8022 Glutamate 13C3 811192.44 802720.94 830597.19 468915.25 379806.28 332913.16 Glutamate 13C4 2227030.5 2108102 1988053 350793.59 311264.88 324521.38 Glutamate 13C5 13744087 13504339 13221563 2199177 1794646 1830142 ADP 3522.01 2405.3 1748.52 374740.16 312110.28 327258.09 ADP 13C1 0 0 0 15482.0824 1774.0098 28287.9539 ADP 13C2 7050.243679 20209.39587 14482.77291 10613.11406 7198.045612 8942.957111 ADP 13C3 83891.63 66639.98 86548.32 47789.79 16330.31 41763.83 ADP 13C4 282777.38 282128.41 302268.66 120525.96 119845.21 116961.26 ADP 13C5 0 0 0 50146.53 67631.07 65493.22 ADP 13C9 48753.57 51584.2 57633.91 46001.14 9513.73 52636.06 ADP 13C10 225544.69 225525.14 312269.03 52640.16 63221.31 54874.13 AMP 5156.9 1913.75 0 433710.25 322538.53 303382 AMP 13C1 0 0 0 34031.7525 37182.4217 4437.7 AMP 13C8 14464.58 9313.19 14299.66 0 3816.02 0 AMP 13C9 47110.44 79829.09 122388.6 12609 26892.74 5341.84 AMP 13C10 297494.47 404031.12 499158.25 42682.21 28796.22 40869.88 Adenine 104735.24 188097 186293.33 89376.32 156321.89 73836 Citrate 404765.34 275164.59 319542.62 3139604.75 2714684.75 3173371 Citrate 13C1 3209.24756 3226.05706 0 34403.0265 45372.3065 44699.874 Citrate 13C2 0 6433.943046 633.5723473 226619.8017 166284.7532 242630.3159 Citrate 13C3 23257.44 28000.99 7617.08 146935.94 90861.3 106532.3 Citrate 13C4 156929.11 279093.25 127529.01 77032.22 58916.41 66283.13 Citrate 13C5 1197100.62 1638734.62 811528.44 43194.44 12586.52 39627.07 Citrate 13C6 889729.5 1279160.88 1195040.12 51155.61 25528.73 51377.57 2-Oxoglutarate 0 0 0 435498.41 183244.73 367046.5 2-Oxoglutarate 13C5 202146.77 217618.92 101136.16 31466.06 11806.48 14562.1 Succinate 41354.92 54478.46 137866.23 6767553.5 6617559.5 6404632 Succinate 13C1 39166.51352 38624.25776 35519.51588 61447.236 26201.632 38203.282 Succinate 13C2 305036.9069 293561.6497 310219.871 162758.0164 131183.2948 150117.4624 Succinate 13C3 749347.5 702044.19 719349.31 52877.84 41859.78 84773.27 Succinate 13C4 8395311 8171753.5 8645002 273143.66 244827.56 268112.34 Fumarate 10753.49 23651.16 39230.16 179998.88 155879.08 209283.2 Fumarate 13C1 0 0 0 5689.00928 0 1410.3792 Fumarate 13C3 169020.25 152330.67 28035.3 18208.04 17357.98 23838.98 Fumarate 13C4 174744.03 180114.81 138139.44 5830.46 4277.24 4532.09 Malate 135868.3 109592.7 279113.75 2665018.25 2140907.25 3045582.75 Malate 13C1 10478.8348 8476.7712 7115.245 77706.427 35056.791 90720.299 Malate 13C2 155385.959 168737.7485 103043.8958 57075.22467 47248.86356 68379.8818 Malate 13C3 2332082 2234053.5 1340370.88 254104.58 76008.85 295915.78 Malate 13C4 1953955 1784654.38 1547985.12 57822.44 71925.42 106225.84 Itaconate 19581.13 49635.74 51814.4 103576.41 85702.6 113018.94 Itaconate 13C2 2804.177082 1897.381887 4196.32144 1913.16136 5105.179635 1780.967707 Itaconate 13C4 15749.59 25722.2 50172.14 66774.24 40392.41 43590.46 Itaconate 13C5 108773.04 130363.96 100238.02 16357.26 27504.62 9654.32 Glutathione 0 0 0 22032836 20614408 20971330 Glutathione 13C2 0 0 0 2161756.934 1989678.413 2146875.157 Glutathione 13C3 0 0 0 713900.5 715573 764534.81 Glutathione 13C4 6067.14 5142.39 6696.53 802659.69 684717.31 856702.19 Glutathione 13C5 32762.92 39076.42 30558.37 3532272.75 3502789.25 3731260.75 Glutathione 13C6 103729.02 89479.69 102389.62 495423.34 430422.03 445342.34 Glutathione 13C7 449115.53 524860.81 427962.25 929296 977055.25 997726 Glutathione 13C8 2406546.25 2468683.75 1989015.38 2714616.75 2653365 2779758 Glutathione 13C9 3115470 3294388 2763278.75 2720577.75 2787280.25 2824504 Glutathione 13C10 17955306 18813222 15336215 15945488 15337195 16401588 Glutathione disulfide 0 0 0 19736.65 17239.39 9562.42 Glutathione disulfide 13C10 0 0 0 36726.39 27575.33 17277.4 Glutathione disulfide 13C20 6165.81 4619.45 45732.52 11108.92 10671.74 2592.93 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name KEGG ID m/z r.t. Glutamate C00025 146.045883 0.582 Glutamate 13C1 C00025 147.04924 0.57 Glutamate 13C2 C00025 148.051804 0.585 Glutamate 13C3 C00025 149.055939 0.571 Glutamate 13C4 C00025 150.059326 0.572 Glutamate 13C5 C00025 151.062515 0.573 ADP C00008 426.021973 0.554 ADP 13C1 C00008 427.0255 0.5508 ADP 13C2 C00008 428.026672 0.599 ADP 13C3 C00008 429.032074 0.571 ADP 13C4 C00008 430.035736 0.572 ADP 13C5 C00008 431.039001 0.561 ADP 13C9 C00008 435.052002 0.563 ADP 13C10 C00008 436.055847 0.557 AMP C00020 346.055878 0.63 AMP 13C1 C00020 347.058197 0.616 AMP 13C8 C00020 354.082245 0.634 AMP 13C9 C00020 355.084778 0.621 AMP 13C10 C00020 356.089325 0.616 Adenine C00047 134.047256 0.692 Citrate C00158 191.01976 0.565 Citrate 13C1 C00158 192.023102 0.572 Citrate 13C2 C00158 193.026596 0.552 Citrate 13C3 C00158 194.029816 0.571 Citrate 13C4 C00158 195.032578 0.559 Citrate 13C5 C00158 196.036942 0.568 Citrate 13C6 C00158 197.039688 0.562 2-Oxoglutarate C00026 145.014236 0.569 2-Oxoglutarate 13C5 C00026 150.030975 0.567 Succinate C00042 117.019325 0.556 Succinate 13C1 C00042 118.022896 0.547 Succinate 13C2 C00042 119.024361 0.559 Succinate 13C3 C00042 120.031448 0.601 Succinate 13C4 C00042 121.033852 0.557 Fumarate C00122 115.003677 0.555 Fumarate 13C1 C00122 116.00705 0.543 Fumarate 13C3 C00122 118.015648 0.572 Fumarate 13C4 C00122 119.017738 0.549 Malate C00149 133.014267 0.559 Malate 13C1 C00149 134.0177 0.568 Malate 13C2 C00149 135.019165 0.561 Malate 13C3 C00149 136.025772 0.578 Malate 13C4 C00149 137.02829 0.564 Itaconate C00490 129.019318 0.563 Itaconate 13C2 C00490 131.023773 0.522 Itaconate 13C4 C00490 133.03331 0.571 Itaconate 13C5 C00490 134.034927 0.573 Glutathione C00051 306.07663 0.589 Glutathione 13C2 C00051 308.083557 0.591 Glutathione 13C3 C00051 309.086884 0.588 Glutathione 13C4 C00051 310.089722 0.589 Glutathione 13C5 C00051 311.093536 0.588 Glutathione 13C6 C00051 312.096497 0.591 Glutathione 13C7 C00051 313.100098 0.587 Glutathione 13C8 C00051 314.103485 0.586 Glutathione 13C9 C00051 315.106873 0.588 Glutathione 13C10 C00051 316.110138 0.587 Glutathione disulfide C00127 611.144836 0.529 Glutathione disulfide 13C10 C00127 621.178406 0.532 Glutathione disulfide 13C20 C00127 631.211731 0.533 METABOLITES_END #END