#METABOLOMICS WORKBENCH juliehaines_20241126_071750 DATATRACK_ID:5403 STUDY_ID:ST003608 ANALYSIS_ID:AN005930 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 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 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 Alanine 114878.37 108881.05 111265.31 22260.5 20375.24 232035.08 Arginine 762783.31 722331.5 794167.5 54635.5 59602.2 63663.89 Cysteine 16852.55 12722.6 19497.71 0 1109.92 0 Histidine 565186.31 657166.19 690912.69 12649.49 17928.51 19028.98 Leucine+isoleucine 761242.5 777570.69 772277.31 33427.72 33102.36 699205.75 Lysine 5061588.5 5146916.5 5517652 36458.71 40144.17 501278.09 Phenylalanine 715745.19 688789.81 725820.19 15188.43 19623.51 349109.41 Serine 9308.99 18467.02 25786.76 10442.92 25040.04 1548511.88 Threonine 19179.9 14158.39 24811.78 10606.39 15220.41 412505.62 Tryptophan 50497.24 52883.92 75742.85 10887.05 14631.59 33892.04 Tyrosine 910909 955892.56 1044148.31 9482.68 14360.1 651311.44 Valine 15270025 12619543 16672992 307878.28 332494.97 531115 Adenosine 6571965.5 6997774 4500093.5 89287.27 71850.21 53775.45 Adenine 2031852.62 1450688.12 398820.72 116523.91 109355.84 94919.77 Guanosine 221163.7 222088.19 72395.16 4125.46 3833.25 2145.62 Guanine 1289261.88 1141353.5 1015083.06 56880.02 59032.33 27280.23 Cytidine 345512.94 399430.53 61280.11 7740.02 5229.24 8206.22 Cytosine 324700.88 339256.53 323938.72 40708.21 39451.41 26954.02 Inosine 99795.71 99608.93 131787.17 0 0 1342.94 Hypoxanthine 1395854.5 1282852.62 1088232.38 5249.63 7782.05 15365.88 Xanthine 376327.62 383238.88 433447.25 714.42 0 7757.92 Nicotinamide 339402.53 327724 430754.12 13952.22 9530.48 9880.34 NAD+ 2840583 3164908.75 4043726.75 13190.61 15151.3 11737.77 5-Oxoproline 348513.38 380599.72 372708.66 19521.34 20242.4 366034.72 Cys-Gly 378210.66 371972 461347.5 5775.37 8736.96 0 gamma-Glutamyl-Se-methylselenocysteine 22610.16 20067.73 28325.2 0 1407.21 0 Ornithine 16460.37 16341.96 29903.08 2655.01 3169.49 661102.38 L-Citrulline 0 2741.45 6192.11 2811.97 2215.28 62374.56 Putrescine 245804.17 215325.62 156778.86 1547.56 2367.94 3527.79 Spermidine 1025666.06 1118549.38 731952.69 1067.85 3481.71 8888.82 Spermine 44896.61 42913.53 54867.98 16206.56 31233.03 4487.33 Choline 1228220.5 1134884.38 1081547.75 20664.46 21416.88 232791.06 Acetylcholine 223293.5 197420.25 194543.64 0 1610.26 997.88 L-Carnitine 76010.99 68186.32 73326.86 3081 6589.55 55543.2 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name KEGG ID mz rt Alanine C00041 90.0551 0.62 Arginine C00062 175.1189 0.59 Cysteine C00097 122.0271 0.63 Histidine C00135 156.0768 0.58 Leucine+isoleucine C00123 132.1020 0.74 Lysine C00047 147.1129 0.57 Phenylalanine C00079 166.0863 1.38 Serine C00065 106.0501 0.60 Threonine C00188 120.0657 0.62 Tryptophan C00078 205.0972 1.71 Tyrosine C00082 182.0811 0.65 Valine C00183 118.0864 0.64 Adenosine C00212 268.1040 0.67 Adenine C00147 136.0619 0.67 Guanosine C00387 284.0990 0.66 Guanine C00242 152.0567 0.66 Cytidine C00475 244.0929 0.64 Cytosine C00380 112.0508 0.64 Inosine C00294 269.0884 0.63 Hypoxanthine C00262 137.0459 0.67 Xanthine C00385 153.0407 0.67 Nicotinamide C00153 123.0554 0.66 NAD+ C00003 664.1168 0.64 5-Oxoproline C01879 130.0500 0.63 Cys-Gly C01419 179.0485 0.65 gamma-Glutamyl-Se-methylselenocysteine C05695 313.0297 0.61 Ornithine C01602 133.0973 0.58 L-Citrulline C00327 176.1030 0.62 Putrescine C00134 89.1075 0.58 Spermidine C00315 146.1652 0.56 Spermine C00750 203.2231 0.58 Choline C00114 104.1072 0.61 Acetylcholine C01996 146.1176 1.71 L-Carnitine C00318 162.1125 0.62 METABOLITES_END #END