#METABOLOMICS WORKBENCH jravilap_20190117_205305_mwtab.txt DATATRACK_ID:1607 STUDY_ID:ST001128 ANALYSIS_ID:AN001853 PROJECT_ID:PR000754 VERSION 1 CREATED_ON February 4, 2019, 12:36 pm #PROJECT PR:PROJECT_TITLE Bacteroides-derived sphingolipids are critical for maintaining intestinal PR:PROJECT_TITLE homeostasis and symbiosis PR:PROJECT_SUMMARY Sphingolipids are structural membrane components and important eukaryotic PR:PROJECT_SUMMARY signaling molecules. We hypothesized that sphingolipids mediate intestinal PR:PROJECT_SUMMARY health as they were identified as the most upregulated metabolite feature in PR:PROJECT_SUMMARY stool of inflammatory bowel disease (IBD) patients. Commensal Bacteroidetes also PR:PROJECT_SUMMARY produce sphingolipids, but the impact of these metabolites on host pathways is PR:PROJECT_SUMMARY largely uncharacterized. To study Bacteroidetes sphingolipids in intestinal PR:PROJECT_SUMMARY health, we colonized germ-free mice with a sphingolipid-deficient Bacteroides PR:PROJECT_SUMMARY thetaiotaomicron strain. A lack of Bacteroides-derived sphingolipids increased PR:PROJECT_SUMMARY intestinal inflammation, dysregulated innate immunity and altered the host PR:PROJECT_SUMMARY ceramide pool. Using metabolomic analysis, we described the Bacteroides PR:PROJECT_SUMMARY sphingolipid biosynthesis pathway and revealed a greater variety of PR:PROJECT_SUMMARY Bacteroides-derived sphingolipids than previously recognized, including ceramide PR:PROJECT_SUMMARY phosphoinositol and deoxy-sphingolipids. We annotated Bacteroides sphingolipids PR:PROJECT_SUMMARY in an IBD metabolomic dataset, discovering lower abundances in IBD and negative PR:PROJECT_SUMMARY correlations with gut inflammation and host sphingolipid production. These data PR:PROJECT_SUMMARY highlight the role of sphingolipids in maintaining host-bacterial symbiosis and PR:PROJECT_SUMMARY intestinal homeostasis. PR:INSTITUTE Broad Institute of MIT and Harvard PR:LAST_NAME Avila-Pacheco PR:FIRST_NAME Julian PR:ADDRESS 415 Main Street, Cambridge MA PR:EMAIL jravilap@broadinstitute.org PR:PHONE 617-714-8264 PR:FUNDING_SOURCE National Institutes of Health (P30 DK043351 and R01 AT009708) PR:CONTRIBUTORS Eric M. Brown, Xiaobo Ke, Daniel Hitchcock, Timothy D. Arthur, Toru Nakata, PR:CONTRIBUTORS Nadine Fornelos, Cortney Heim, Eric A. Franzosa1,4, Curtis Huttenhower1,4, Henry PR:CONTRIBUTORS J. Haiser3, Glen 6 Dillow3, Daniel B. Graham1, B. Brett Finlay, Aleksandar D. PR:CONTRIBUTORS Kostic, Jeffrey A. Porter, Hera Vlamakis, Sarah Jeanfavre, Julian Avila-Pacheco, PR:CONTRIBUTORS Clary B. Clish, and Ramnik J. Xavier #STUDY ST:STUDY_TITLE WT and ΔSPT cultures of B. thetaiotaomicron grown in Minimal Media with or ST:STUDY_TITLE without d4-alanine (part IV) ST:STUDY_SUMMARY Lipid profiling was applied on WT and ΔSPT cultures of B. thetaiotaomicron ST:STUDY_SUMMARY grown in minimal liquid media supplemented with or without deuterium ST:STUDY_SUMMARY (D4)-labelled alanine, in order to elucidate the production pathways of ST:STUDY_SUMMARY Bacteroides-derived sphingolipids. ST:INSTITUTE Broad Institute of MIT and Harvard ST:LAST_NAME Avila-Pacheco ST:FIRST_NAME Julian ST:ADDRESS 415 Main Street ST:EMAIL jravilap@broadinstitute.org ST:PHONE 617-714-8264 ST:NUM_GROUPS 3 ST:TOTAL_SUBJECTS 9 #SUBJECT SU:SUBJECT_TYPE Bacteria SU:SUBJECT_SPECIES Bacteroides thetaiotaomicron VPI-5482 SU:TAXONOMY_ID 226186 SU:GENOTYPE_STRAIN WT (VPI-5482), SPT K.O. (BT_0870) SU:CELL_BIOSOURCE_OR_SUPPLIER ATCC SU:CELL_STRAIN_DETAILS Wild type Bacteroides tethaiotamicron strains and serine palmitoyltransferase SU:CELL_STRAIN_DETAILS (SPT) knockouts SU:SUBJECT_COMMENTS Grown in Minimal Media with or without d4-alanine #SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Additional sample data SUBJECT_SAMPLE_FACTORS - MM1 Strain:WT (VPI-5482) Type=Bacterial Cell extract; Species=Bacteroides thetaiotaomicron; Raw File=MM1.raw SUBJECT_SAMPLE_FACTORS - MM2 Strain:WT (VPI-5482) Type=Bacterial Cell extract; Species=Bacteroides thetaiotaomicron; Raw File=MM2.raw SUBJECT_SAMPLE_FACTORS - MM3 Strain:WT (VPI-5482) Type=Bacterial Cell extract; Species=Bacteroides thetaiotaomicron; Raw File=MM3.raw SUBJECT_SAMPLE_FACTORS - SPT1 Strain:SPT K.O. (BT_0870) + d4-Ala Type=Bacterial Cell extract; Species=Bacteroides thetaiotaomicron; Raw File=SPT1.raw SUBJECT_SAMPLE_FACTORS - SPT2 Strain:SPT K.O. (BT_0870) + d4-Ala Type=Bacterial Cell extract; Species=Bacteroides thetaiotaomicron; Raw File=SPT2.raw SUBJECT_SAMPLE_FACTORS - SPT3 Strain:SPT K.O. (BT_0870) + d4-Ala Type=Bacterial Cell extract; Species=Bacteroides thetaiotaomicron; Raw File=SPT3.raw SUBJECT_SAMPLE_FACTORS - WT1 Strain:WT (VPI-5482) + d4-Ala Type=Bacterial Cell extract; Species=Bacteroides thetaiotaomicron; Raw File=WT1.raw SUBJECT_SAMPLE_FACTORS - WT2 Strain:WT (VPI-5482) + d4-Ala Type=Bacterial Cell extract; Species=Bacteroides thetaiotaomicron; Raw File=WT2.raw SUBJECT_SAMPLE_FACTORS - WT3 Strain:WT (VPI-5482) + d4-Ala Type=Bacterial Cell extract; Species=Bacteroides thetaiotaomicron; Raw File=WT3.raw #COLLECTION CO:COLLECTION_SUMMARY After 48 hrs of labeling, resulting cultures were pelleted by centrifugation at CO:COLLECTION_SUMMARY 8000 rpm for 10 min and cell density was normalized. Lipids were extracted using CO:COLLECTION_SUMMARY isopropanol and stored at -80C for lipidomic analysis. CO:SAMPLE_TYPE Bacterial cells CO:STORAGE_CONDITIONS -80℃ #TREATMENT TR:TREATMENT_SUMMARY WT and ΔSPT cultures of B. thetaiotaomicron and B. ovatus were grown in 5 mL of TR:TREATMENT_SUMMARY minimal media (M9 salts (Teknova), 1% vitamin K1-hemin solution (Becton TR:TREATMENT_SUMMARY Dickinson), 1% trace mineral supplement (ATCC), 1% trace vitamin supplement TR:TREATMENT_SUMMARY (ATCC), 2% lactose), supplemented with 10μM of deuterium (D4)-labelled alanine TR:TREATMENT_SUMMARY (Sigma), or 10μM of -alanine (Sigma). TR:CELL_STORAGE -80C TR:CELL_GROWTH_CONTAINER anaerobic chamber (Coy Laboratory Products) with an atmosphere of 20% CO2, 5% TR:CELL_GROWTH_CONTAINER H2, and 75% N2 at 37°C. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY After 48 hrs, resulting cultures were pelleted by centrifugation at 8000 rpm for SP:SAMPLEPREP_SUMMARY 10 min and cell density was normalized. Lipids were extracted using isopropanol SP:SAMPLEPREP_SUMMARY and stored at -80C until ready for lipidomic analysis. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Shimadzu Nexera X2 CH:COLUMN_NAME Waters Acquity BEH C8 (100 x 2.1mm, 1.7um) #ANALYSIS AN:ANALYSIS_TYPE MS AN:LABORATORY_NAME Metabolomics Platform AN:OPERATOR_NAME Daniel Hitchcock #MS MS:INSTRUMENT_NAME Thermo Q Exactive Focus MS:INSTRUMENT_TYPE Orbitrap MS:MS_TYPE ESI MS:ION_MODE POSITIVE MS:MS_COMMENTS Raw data were processed using TraceFinder 3.1 (Thermo Fisher) and Progenesis QI MS:MS_COMMENTS (Nonlinear dynamics). Retention time, m/z, MS/MS fragmentation were used to MS:MS_COMMENTS confirm the identity of metabolite using authentic reference standards when MS:MS_COMMENTS available (level 1 identifications), in addition to monitoring the product ions MS:MS_COMMENTS produced and the retention time and mass of species belonging to the each lipid MS:MS_COMMENTS class (level 2-3 annotation). #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS Abundances MS_METABOLITE_DATA_START Samples MM1 MM2 MM3 SPT1 SPT2 SPT3 WT1 WT2 WT3 Factors Strain:WT (VPI-5482) Strain:WT (VPI-5482) Strain:WT (VPI-5482) Strain:SPT K.O. (BT_0870) + d4-Ala Strain:SPT K.O. (BT_0870) + d4-Ala Strain:SPT K.O. (BT_0870) + d4-Ala Strain:WT (VPI-5482) + d4-Ala Strain:WT (VPI-5482) + d4-Ala Strain:WT (VPI-5482) + d4-Ala CerPE C34 1038278 1002943 749549 258161 76059 119483 CerPE C33 202751 191542 131209 44974 5186 16163 CerPE C35 986278 950804 753309 393671 153066 166218 CerPI C34 155828 242094 82796 62920 9113 16304 CerPI C35 152707 277226 89122 126068 30216 28048 1-deoxysphinganine iso-C16 74549 68849 57409 61203 31488 38737 1-deoxysphinganine iso-C16-d3 113648 72454 83261 1-deoxysphinganine iso-C17 195468 185885 160449 5360 5774 4628 204534 142175 149646 1-deoxysphinganine iso-C17-d3 506034 440537 452343 1-deoxysphinganine iso-C18 245213 223439 211644 342064 252364 210840 1-deoxysphinganine iso-C18-d3 982181 874945 695978 sphinganine iso-C15 10125 9093 8259 9053 9206 9250 9677 9576 8857 sphinganine iso-C16 51015 44389 37245 3692 4226 3690 9044 8148 9645 sphinganine iso-C17 34898 33421 26738 2408 2156 2576 sphinganine iso-C18 38897 37930 32624 6037 5379 4087 sphinganine iso-C19 10784 8687 7634 459 124 deoxy dihydroceramide C34 43594 34708 34008 55752 27705 34448 deoxy dihydroceramide C34-d3 108959 74802 82103 deoxy dihydroceramide C35 208793 183263 185684 341591 263394 223418 deoxy dihydroceramide C35-d3 895211 825891 683285 DHCer C33 25172 27279 22693 12096 1096 6134 DHCer C34 209733 197268 186674 78464 31726 47909 DHCer C35 152736 115770 113522 48604 44003 51888 DHCer C36 707653 510888 561176 222488 243523 235880 DHCer C37 650022 470711 549536 261887 346872 241351 3-ketosphinganine iso-C16 4259 5176 4877 4457 3117 4850 3384 5549 4345 3-ketosphinganine iso-C18 7237 5001 3993 124 1375 2045 Ceramide (d18:1) C17:1 82768 58241 58861 31313 25499 28122 Ceramide (d18:1) C18:1 302540 249668 276951 167655 203212 200904 Ceramide (d18:1) C19:1 266683 221309 279693 281469 333235 256434 CerPE C19 613480 583445 498810 332160 142650 136008 CerPI C19 104158 170413 61350 121397 38787 22056 DAG C30:0 5980592 5648083 5473129 3868244 3236239 2520897 1863974 2168720 2578014 DAG C30:2 192163 177766 166905 40733 30174 23906 36252 49304 53127 DAG C31:0 2679115 2664879 2750763 2654400 2240359 1876510 1082930 1614468 1904991 DAG C31:2 722695 647345 670286 385352 292068 295838 336295 479935 398775 DAG C32:0 618969 581102 594768 1490276 1058985 894764 363534 437763 436412 DAG C32:2 14347 15495 14211 15582 10850 10309 4924 9154 6904 DAG C33:0 162334 149559 160414 129209 113344 108536 85803 97959 126667 DAG C33:2 6191 5590 5899 30509 22316 22341 3660 8157 2000 DAG C34:0 48690 49671 45520 47060 44670 50165 39938 42703 41415 DAG C36:0 38416 26180 23972 25160 28194 23540 22422 27062 26202 LPC C17:0 1309342 1212985 1193020 1192585 1250260 1188617 1199894 1218486 1222523 LPE C15:0 2103 5811 2877 1291 1182 686 40966 947 5614 LPE C15:0 isomer 7607 13928 7773 2440 7718 1675 139030 7083 28858 LPE C16:0 1744 2552 432 527 193 29997 2276 4895 MAG C14:0 24493 23726 22877 24002 23796 24477 22907 22662 21553 MAG C15:0 13958 14593 14179 13423 16585 14269 36702 13422 18961 MAG C16:0 369624 353628 349489 339029 351601 342707 360649 347196 356858 MAG C17:0 9164 9824 8175 9793 11257 9557 10852 5307 8765 MAG C18:0 752920 734890 700558 687628 657528 667323 626574 626140 619666 MAG C22:0 22125 24715 23018 22050 21868 21564 22623 22314 22525 MAG C22:1 15482 15573 15985 15501 13782 14050 12998 14947 12158 oleamide 52404 51081 45547 45701 43630 48347 44204 41967 41696 PC C24:0 2280575 2444730 2326815 2337128 2486297 2332759 2256909 2278721 2264791 PE C29:0 1934137 1930753 1635986 1420325 977714 802254 628598 550942 759985 PE C30:0 18966976 16259055 15855681 7607787 5701688 4900407 7202933 5569046 6259415 PE C31:0 7898877 7277688 7027301 4704389 3545128 3428196 3619282 3675490 3908805 PE C32:0 1301038 1151554 1240642 2447568 1604707 1456696 586215 581139 530052 PE C33:0 437712 364800 372632 173757 133243 135816 180451 185365 195702 PE C33:1 14870 12366 10616 1140 383 406 2302 452 377 PE C34:0 10122 6123 5707 1516 987 2915 4714 6540 PS C30:0 247163 316798 180519 37677 21067 5856 143154 53106 58163 PS C32:0 11484 12914 7318 1370 490 5694 1981 1793 sphingosine 20619 19184 19292 17750 17493 16351 16125 17979 17859 TAG C42:0 82242 84096 83682 77021 89365 77025 74251 79394 71767 TAG C43:0 65337 59165 56157 60371 60803 57561 55894 51369 45737 TAG C44:0 178739 187281 167032 170791 181246 165423 171560 167043 164678 TAG C44:1 22476 21258 13672 19282 23859 12154 9872 15089 13162 TAG C45:0 221427 213332 219223 232078 240471 217097 191184 194784 193076 TAG C45:1 5843 6293 3338 2897 7389 4395 974 1551 1216 TAG C46:0 320346 353148 349903 346633 334754 332634 319006 314519 319330 TAG C46:1 52592 44880 50616 46283 62885 35478 33563 31515 35286 TAG C47:0 320211 316814 311792 311540 309230 320423 299168 286176 291449 TAG C47:1 34691 26467 29676 27553 36774 17405 17993 14732 16249 TAG C48:0 396961 392056 406690 379583 401367 366540 344015 374753 349769 TAG C48:1 70003 81565 67760 65781 103445 60570 59003 53172 54515 TAG C48:2 37236 31675 28702 17793 38295 16294 11700 15972 16361 TAG C49:0 162937 167671 157975 169593 155597 155291 165246 154487 155403 TAG C49:1 20600 19099 12530 13567 22466 10586 7360 14861 3347 TAG C50:0 168237 189844 169552 170935 187153 163365 160603 159092 158201 TAG C50:1 77015 82701 82978 76627 158964 60957 68878 63016 63474 TAG C50:2 32346 34508 33857 19653 62803 20147 20989 19719 18544 TAG C52:0 67248 70165 64492 71431 70058 79161 74528 69244 68569 TAG C52:1 13701 20503 16207 15982 47852 15187 16120 12372 6464 TAG C52:2 55882 63423 47381 50014 143107 36031 43127 35257 26943 TAG C52:3 15754 17160 9700 11814 52744 10144 3872 5346 6321 TAG C54:3 21258 17369 7500 12531 45746 6418 11468 8409 8416 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name quantified m/z rtimes (mins) Adduct Notes CerPE C34 679.5379076 8.45 [M+H]+ CerPE C33 665.5226654 8.19 [M+H]+ CerPE C35 693.5535544 8.66 [M+H]+ CerPI C34 798.5487152 8.09 [M+H]+ CerPI C35 812.5641022 8.36 [M+H]+ 1-deoxysphinganine iso-C16 272.2947938 4.69 [M+H]+ 1-deoxysphinganine iso-C16-d3 275.313575 4.69 [M+H]+ Labeled 1-deoxysphinganine iso-C17 286.3103625 4.97 [M+H]+ 1-deoxysphinganine iso-C17-d3 289.3291023 4.97 [M+H]+ Labeled 1-deoxysphinganine iso-C18 300.3259754 5.19 [M+H]+ 1-deoxysphinganine iso-C18-d3 303.344672 5.19 [M+H]+ Labeled sphinganine iso-C15 274.2740823 4.03 [M+H]+ sphinganine iso-C16 288.2894982 4.5 [M+H]+ sphinganine iso-C17 302.305179 4.8 [M+H]+ sphinganine iso-C18 316.3207703 5 [M+H]+ sphinganine iso-C19 330.3366411 5.36 [M+H]+ deoxy dihydroceramide C34 540.5351856 8.78 [M+H]+ deoxy dihydroceramide C34-d3 543.5536722 8.77 [M+H]+ Labeled deoxy dihydroceramide C35 554.5505981 8.98 [M+H]+ deoxy dihydroceramide C35-d3 557.5692378 8.98 [M+H]+ Labeled DHCer C33 542.5146001 8.33 [M+H]+ DHCer C34 556.5300642 8.53 [M+H]+ DHCer C35 570.5455547 8.55 [M+H]+ DHCer C36 584.5609793 8.75 [M+H]+ DHCer C37 598.5765333 8.98 [M+H]+ 3-ketosphinganine iso-C16 286.2740364 4.39 [M+H]+ 3-ketosphinganine iso-C18 314.3052493 4.86 [M+H]+ Ceramide (d18:1) C17:1 550.5192375 8.68 [M+H]+ Ceramide (d18:1) C18:1 564.5348753 8.91 [M+H]+ Ceramide (d18:1) C19:1 578.5494447 9.11 [M+H]+ CerPE C19 707.5692413 8.9 [M+H]+ CerPI C19 826.5801017 8.61 [M+H]+ DAG C30:0 558.5092016 9.28 [M+NH4]+ DAG C30:2 554.477919 8.71 [M+NH4]+ DAG C31:0 572.5246446 9.5 [M+NH4]+ DAG C31:2 568.4933508 8.95 [M+NH4]+ DAG C32:0 586.5403156 9.67 [M+NH4]+ DAG C32:2 582.5090287 9.18 [M+NH4]+ DAG C33:0 600.5558703 9.91 [M+NH4]+ DAG C33:2 596.5244462 9.41 [M+NH4]+ DAG C34:0 614.5713577 10.14 [M+NH4]+ DAG C36:0 642.6026711 10.49 [M+NH4]+ LPC C17:0 510.3556769 5.66 [M+H]+ LPE C15:0 440.2774394 5.05 [M+H]+ LPE C15:0 isomer 440.2773419 4.85 [M+H]+ LPE C16:0 454.2920343 5.37 [M+H]+ MAG C14:0 325.2347016 5.12 [M+Na]+ MAG C15:0 339.2505671 5.42 [M+Na]+ MAG C16:0 353.2660274 5.76 [M+Na]+ MAG C17:0 367.2816692 6.08 [M+Na]+ MAG C18:0 381.2972484 6.46 [M+Na]+ MAG C22:0 437.3602397 7.67 [M+Na]+ MAG C22:1 435.3445505 7.77 [M+Na]+ oleamide 282.2791292 5.79 [M+H]+ PC C24:0 622.4437597 7.17 [M+H]+ PE C29:0 650.4750648 8.52 [M+H]+ PE C30:0 664.4905563 8.75 [M+H]+ PE C31:0 678.5061512 9 [M+H]+ PE C32:0 692.5218318 9.18 [M+H]+ PE C33:0 706.5376354 9.44 [M+H]+ PE C33:1 704.5221825 9.18 [M+H]+ PE C34:0 720.5521837 9.67 [M+H]+ PS C30:0 708.4806832 8.5 [M+H]+ PS C32:0 736.5116707 8.83 [M+H]+ sphingosine 300.2896121 5.52 [M+H]+ TAG C42:0 740.675648 11.32 [M+NH4]+ TAG C43:0 754.690936 11.44 [M+NH4]+ TAG C44:0 768.7068125 11.61 [M+NH4]+ TAG C44:1 766.6911524 11.42 [M+NH4]+ TAG C45:0 782.7224467 11.78 [M+NH4]+ TAG C45:1 780.7069064 11.56 [M+NH4]+ TAG C46:0 796.7381912 11.95 [M+NH4]+ TAG C46:1 794.7224953 11.73 [M+NH4]+ TAG C47:0 810.7537666 12.12 [M+NH4]+ TAG C47:1 808.7383418 11.91 [M+NH4]+ TAG C48:0 824.7691447 12.31 [M+NH4]+ TAG C48:1 822.7538005 12.07 [M+NH4]+ TAG C48:2 820.7382544 11.87 [M+NH4]+ TAG C49:0 838.7845588 12.49 [M+NH4]+ TAG C49:1 836.7699856 12.25 [M+NH4]+ TAG C50:0 852.8004991 12.69 [M+NH4]+ TAG C50:1 850.7851069 12.42 [M+NH4]+ TAG C50:2 848.7696644 12.17 [M+NH4]+ TAG C52:0 880.8317908 13.12 [M+NH4]+ TAG C52:1 878.8166846 12.79 [M+NH4]+ TAG C52:2 876.8004369 12.51 [M+NH4]+ TAG C52:3 874.7850038 12.23 [M+NH4]+ TAG C54:3 902.8164628 12.61 [M+NH4]+ METABOLITES_END #END