#METABOLOMICS WORKBENCH juliehaines_20241126_071750 DATATRACK_ID:5403 STUDY_ID:ST003608 ANALYSIS_ID:AN005929 PROJECT_ID:PR002231 VERSION 1 CREATED_ON December 2, 2024, 2:48 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 Global metabolomics to compare metabolic profiles of live vs heat-killed ST:STUDY_TITLE thymidine-auxotrophe E. Coli ST:STUDY_SUMMARY Global metabolomics of live and heat-killed thymidine-auxotrophe Escherichia ST:STUDY_SUMMARY Coli K12 cultured or not in RPMI medium for 1h. 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 - AA83eC-1_r5- factor:live | Sample source:cells RAW_FILE_NAME(raw file name)=AA83eC-1_r5- SUBJECT_SAMPLE_FACTORS - AA83eC-2_r8- factor:live | Sample source:cells RAW_FILE_NAME(raw file name)=AA83eC-2_r8- SUBJECT_SAMPLE_FACTORS - AA83eC-3_r1- factor:live | Sample source:cells RAW_FILE_NAME(raw file name)=AA83eC-3_r1- SUBJECT_SAMPLE_FACTORS - AA83eC-4_r11- factor:dead | Sample source:cells RAW_FILE_NAME(raw file name)=AA83eC-4_r11- SUBJECT_SAMPLE_FACTORS - AA83eC-5_r12- factor:dead | Sample source:cells RAW_FILE_NAME(raw file name)=AA83eC-5_r12- SUBJECT_SAMPLE_FACTORS - AA83eC-6_r6- factor:dead | Sample source:cells RAW_FILE_NAME(raw file name)=AA83eC-6_r6- #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 Preparation of viable and killed bacteria: ThyA- E. coli were grown overnight TR:TREATMENT_SUMMARY with shaking in LB supplemented with thymidine (500 ug/ml) and trimethoprim (50 TR:TREATMENT_SUMMARY ug/ml), diluted 1/40, and grown until log-phase [optical density at 600 nm TR:TREATMENT_SUMMARY (OD600) of 0.8-1.2]. Bacteria were washed with PBS and subjected to heat-killing TR:TREATMENT_SUMMARY by re-suspension in PBS and subsequently incubation at 60˚C for 60-90 min. TR:TREATMENT_SUMMARY Efficient killing was confirmed by overnight plating on LB-agar plates. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Metabolites from frozen pellets were extracted at 1e9 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. SP:SAMPLEPREP_SUMMARY Supernatants were maintained at 4°C until 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 AA83eC-1_r5- AA83eC-2_r8- AA83eC-3_r1- AA83eC-4_r11- AA83eC-5_r12- AA83eC-6_r6- Factors factor:live | Sample source:cells factor:live | Sample source:cells factor:live | Sample source:cells factor:dead | Sample source:cells factor:dead | Sample source:cells factor:dead | Sample source:cells Aspartate 1409291.62 1422479.12 1482790.62 50124.42 66228.27 2594779.75 Glutamate 9370041 9965182 9830204 478054.53 531809.81 2062914.12 Glutamine 79350.1 107543.12 106924.78 0 2614.04 8220.31 Proline 26164.38 40644.21 32125.55 4670.14 1577.74 80248.98 ATP 104299.53 109805.81 80054.21 0 0 0 ADP 1134279.38 1111581.62 1643609.88 6785.48 5481.42 5092.03 AMP 2283457.25 2387506 3142198.75 21313.98 21126.64 19943.98 dAMP 421190.59 483367.38 547162.56 0 0 0 GDP 274251.03 250313.62 301401.34 1041.13 4184.64 2459.57 GMP 1361403.12 1513631.12 2018037.88 20090.47 17887.51 21270.26 CTP 69679.74 71014.25 69346.3 0 0 0 CDP 684191 667164.19 882066.25 211860.83 195865.2 144027.77 CMP 1550957 1818933.38 2263162.75 44465.5 42487.43 29847.42 dCMP 453357 462122.5 478290.38 0 1591.11 1036.78 UTP 55270.92 56518.43 55600.52 0 0 0 UDP 823822.31 778392.75 984218 15999.67 9629.69 0 UMP 4518794.5 4773795 5532267.5 60336.47 53739.35 38298.76 Uracil 1973220.62 1870286 1138379.62 54792.41 60860.81 56914.73 IMP 27241.12 34557.49 15924.6 0 0 0 Urate 99581.56 150954.48 70794.38 0 2124.43 33465.32 3',5'-Cyclic AMP 389790.59 423507.03 355266.78 2682663.25 2801679.75 2069432.5 2',3'-Cyclic CMP 81733.29 132688.23 35106.54 2353026.25 1957499.62 1797292 Adenylosuccinic acid 13068.04 13808.68 11727.88 0 0 0 UDP-glucose 2226508.75 2316108.25 2484964.75 32699.59 34220.48 20917.62 NADP+ 791505.5 868844.81 1227660.88 15618.1 16721.18 8592.33 Phosphate 60902876 51002048 47522836 28500488 24877456 17078946 Diphosphate 10587317 8589571 9385907 2396321.25 1849780.12 846277.25 D-Glucose 15810735 15542803 15776333 78057.06 73570.85 188190.44 Hexose phosphate 416775.91 382438.75 433245.41 18217.86 12283.5 31760.89 D-Fructose 1-6-bisphosphate 39626.25 52382.31 30318.1 0 0 0 D-Glyceraldehyde 3-phosphate/Glycerone phosphate 13865.98 14080.31 12952.79 0 0 0 1-3-Bisphosphoglycerate 309683.66 233154.45 465533.03 0 0 0 2/3-Phospho-D-glycerate 9573651 8693355 10461751 19440.82 13956.45 20383.89 Phosphoenolpyruvate 2398920.25 2485159.75 3985164.75 2119.54 2728.45 0 Pyruvate 917665.75 950558.19 374911.41 45321.02 62041.07 57112.29 Lactate 229884.23 497343.59 495806.66 222648.98 254688.83 249366.05 Mannitol/Sorbitol/Glucitol/Iditol 57623.78 255800.42 139930.42 14607812 12167967 10583830 Ribose/Ribulose/Arabinose/Xylose/Xylulose 210443.55 162360.3 162201.33 23012.67 10008.46 81803.71 Citrate 1342475.38 1208336.5 1652525.12 138124.41 131907.45 175872.25 2-Oxoglutarate 224165.89 263402.16 211173.8 12497.38 10741.91 5184.56 Succinate 5499697 5024886.5 6765640.5 54912.55 33849.5 58404.05 Fumarate 1543282.38 1573177.38 633145.56 8360.42 7388.55 30206.73 Malate 6594121.5 6219900 7221604.5 83687.6 74104.95 104778.23 2-Hydroxyglutarate/Citramalate 384919.25 375412.12 352045.88 17082.89 7451 19850.51 Itaconate 258134.42 256757.7 259467.86 20556.9 14140.59 28060.91 D-Erythrose 4-phosphate 37278.35 25472.23 15825.05 0 0 0 Sedoheptulose 1-phosphate 291492.81 334299.5 295387.84 6452.54 5041.48 2977.86 alpha-D-Ribose 1-phosphate 217259.05 203017.55 150336.83 27965.42 20587.99 24042.02 Glutathione (GSH) 69075608 70345216 63953964 128708.41 87904.84 64516.64 Glutathione disulfide 95954.16 114658.8 92583.25 1153.33 1328.52 1311.27 gamma-L-Glutamyl-L-cysteine 2233927.25 2290614.75 2119980.25 8246.33 16704.91 12497.97 gamma-L-Glutamyl-D-alanine 80847.85 61313.61 82494.96 8447.97 3359.57 11991.14 gamma-Glutamyl-gamma-aminobutyrate 1007451.5 964451.56 881231.5 7669.57 12601.25 5297.74 (5-L-Glutamyl)-L-glutamine 672259.31 622426.62 634276.62 0 0 7693.05 Dimethylglycine 299695.16 291482.78 264529.16 8497.35 13278.61 24819.4 3-Phosphonooxypyruvate 35216.92 32276.15 22376.86 3226.09 5455.31 2599.84 N-Acetylneuraminate 107083.45 101782.15 81598.95 6604.72 8653.69 144606.48 alpha-D-Glucosamine 1-phosphate 131167.62 125335.2 213224.3 0 0 1767.72 UDP-N-acetyl-D-glucosamine 1676212.38 1673444.88 1979703.62 32286.17 35330.11 20769.4 Pantothenate 1979638.5 1967572.88 1798268.38 0 0 0 Pantetheine 4--phosphate 142141.27 135852.77 138963.11 0 0 0 2-Oxoadipate 587103.38 537480.69 630806.06 3558.45 5519.09 7560.9 Glycerol 3-phosphate 800978.44 1092431.88 136238.08 109808.66 109616.15 71760.73 Heptanoic acid 342009.5 399712.5 285969.75 416236.53 428394.75 355998.59 Octanoic acid (caprylate) 1422911.62 1521505 1251098.88 1504949.62 1495772.62 1445953.38 Nonanoic acid (pelargonate) 12168096 13313001 10390859 12632688 13774148 12729704 Decanoic acid (caprate) 13577924 13796421 12800744 13585168 13120849 13226937 Dodecanoic acid 1717667 1739987 1811522.12 1995914.88 1958127.38 1615198.5 Tetradecanoic acid 1542287.12 1727739.5 1431568 5095260 3344786.25 3648100.25 Hexadecanoic acid 7668288.5 9908994 6918688 54665388 41633180 41320012 Octadecanoic acid (stearic acid) 4886990 5888602 4432776 10910413 7979126.5 7184710.5 Hexadecenoic acid 1051318.5 1056869.25 1203661.5 19724606 14294729 14602151 Octadecenoic acid (oleic acid) 354757.78 357468.59 520992.16 13303312 10801480 9408129 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name KEGG ID m/z r.t. Aspartate C00049 132.0303 0.57 Glutamate C00025 146.0459 0.58 Glutamine C00064 145.0619 0.61 Proline C00148 114.0561 0.67 ATP C00002 505.9886 0.55 ADP C00008 426.0222 0.56 AMP C00020 346.0558 0.62 dAMP C00360 330.0612 0.66 GDP C00035 442.0172 0.55 GMP C00144 362.0508 0.59 CTP C00063 481.9773 0.54 CDP C00112 402.0110 0.55 CMP C00055 322.0447 0.57 dCMP C00239 306.0492 0.57 UTP C00075 482.9614 0.55 UDP C00015 402.9950 0.55 UMP C00105 323.0287 0.58 Uracil C00106 111.0200 0.62 IMP C00130 347.0392 0.59 Urate C00366 167.0210 0.61 3',5'-Cyclic AMP C00575 328.0454 0.66 2',3'-Cyclic CMP C02354 304.0341 0.61 Adenylosuccinic acid C03794 462.0670 0.56 UDP-glucose C00029 565.0479 0.53 NADP+ C00006 742.0683 0.53 Phosphate C00009 96.9696 0.61 Diphosphate C00013 176.9360 0.57 D-Glucose C00031 179.0561 0.59 Hexose phosphate C02965 259.0225 0.56 D-Fructose 1-6-bisphosphate C00354 338.9889 0.57 D-Glyceraldehyde 3-phosphate/Glycerone phosphate C00118 168.9909 0.57 1-3-Bisphosphoglycerate C00236 264.9521 0.57 2/3-Phospho-D-glycerate C00631 184.9857 0.57 Phosphoenolpyruvate C00074 166.9751 0.57 Pyruvate C00022 87.0087 0.59 Lactate C01432 89.0244 0.59 Mannitol/Sorbitol/Glucitol/Iditol C00392 181.0718 0.63 Ribose/Ribulose/Arabinose/Xylose/Xylulose C00121 149.0458 0.59 Citrate C00158 191.0198 0.57 2-Oxoglutarate C00026 145.0148 0.57 Succinate C00042 117.0195 0.56 Fumarate C00122 115.0037 0.55 Malate C00149 133.0143 0.56 2-Hydroxyglutarate/Citramalate C02630 147.0302 0.58 Itaconate C00490 129.0193 0.55 D-Erythrose 4-phosphate C00279 199.0011 0.57 Sedoheptulose 1-phosphate C06222 289.0331 0.56 alpha-D-Ribose 1-phosphate C00620 229.0119 0.57 Glutathione (GSH) C00051 306.0766 0.59 Glutathione disulfide C00127 611.1463 0.53 gamma-L-Glutamyl-L-cysteine C00669 249.0560 0.60 gamma-L-Glutamyl-D-alanine C03738 217.0836 0.59 gamma-Glutamyl-gamma-aminobutyrate C15767 231.0986 0.57 (5-L-Glutamyl)-L-glutamine C05283 274.1045 0.58 Dimethylglycine C01026 102.0561 0.59 3-Phosphonooxypyruvate C03232 182.9677 0.70 N-Acetylneuraminate C00270 308.0988 0.58 alpha-D-Glucosamine 1-phosphate C06156 258.0384 0.58 UDP-N-acetyl-D-glucosamine C00043 606.0745 0.53 Pantothenate C00864 218.1033 0.66 Pantetheine 4--phosphate C01134 357.0890 0.66 2-Oxoadipate C00322 159.0299 0.58 Glycerol 3-phosphate C00093 171.0064 0.57 Heptanoic acid C17714 129.0921 1.93 Octanoic acid (caprylate) C06423 143.1078 2.01 Nonanoic acid (pelargonate) C01601 157.1234 2.10 Decanoic acid (caprate) C01571 171.1391 2.19 Dodecanoic acid C02679 199.1704 2.39 Tetradecanoic acid C06424 227.2017 2.64 Hexadecanoic acid C00249 255.2330 3.06 Octadecanoic acid (stearic acid) C01530 283.2643 3.58 Hexadecenoic acid C08362 253.2173 2.69 Octadecenoic acid (oleic acid) C00712 281.2486 3.07 METABOLITES_END #END