#METABOLOMICS WORKBENCH sakanaka_20230721_023123 DATATRACK_ID:4178 STUDY_ID:ST002793 ANALYSIS_ID:AN004544 PROJECT_ID:PR001741 VERSION 1 CREATED_ON July 24, 2023, 9:36 am #PROJECT PR:PROJECT_TITLE Interspecies metabolite transfer fuels methionine metabolism of Fusobacterium PR:PROJECT_TITLE nucleatum to stimulate volatile methyl mercaptan production PR:PROJECT_SUMMARY Using 13C/15N-labeled methionine, the fate of methionine in F. nucleatum cells PR:PROJECT_SUMMARY was examined to elucidate the intracellular metabolic dynamics underlying PR:PROJECT_SUMMARY enhanced CH3SH production in the presence of S. gordonii. PR:INSTITUTE Osaka University PR:LAST_NAME Kuboniwa PR:FIRST_NAME Masae PR:ADDRESS Yamadaoka 1-8, Suita. Osaka, Japan PR:EMAIL kuboniwa.masae.dent@osaka-u.ac.jp PR:PHONE +81668792922 #STUDY ST:STUDY_TITLE Interspecies metabolite transfer fuels methionine metabolism of Fusobacterium ST:STUDY_TITLE nucleatum to stimulate volatile methyl mercaptan production ST:STUDY_SUMMARY The major oral odor compound methyl mercaptan (CH3SH) is strongly associated ST:STUDY_SUMMARY with halitosis and periodontitis. CH3SH production stems from metabolism of ST:STUDY_SUMMARY polymicrobial communities in periodontal pockets and on the tongue dorsum. ST:STUDY_SUMMARY However, understanding of CH3SH-producing oral bacteria and their interactions ST:STUDY_SUMMARY is limited. This study aimed to investigate CH3SH production by major oral ST:STUDY_SUMMARY bacteria and the impact of interspecies interactions on its generation. Using a ST:STUDY_SUMMARY newly constructed large-volume anaerobic non-contact coculture system, ST:STUDY_SUMMARY Fusobacterium nucleatum was found to be a potent producer of CH3SH, with that ST:STUDY_SUMMARY production stimulated by metabolic interactions with Streptococcus gordonii, an ST:STUDY_SUMMARY early dental plaque colonizer. Furthermore, analysis of extracellular amino ST:STUDY_SUMMARY acids using an S. gordonii arginine-ornithine antiporter (ArcD) mutant ST:STUDY_SUMMARY demonstrated that ornithine excreted from S. gordonii is a key contributor to ST:STUDY_SUMMARY increased CH3SH production by F. nucleatum. Further study with 13C, ST:STUDY_SUMMARY 15N-methionine, as well as gene expression analysis, revealed that ornithine ST:STUDY_SUMMARY secreted by S. gordonii increased the demand for methionine through accelerated ST:STUDY_SUMMARY polyamine synthesis by F. nucleatum, leading to elevated methionine pathway ST:STUDY_SUMMARY activity and CH3SH production. Collectively, these findings suggest that ST:STUDY_SUMMARY interaction between S. gordonii and F. nucleatum plays a key role in CH3SH ST:STUDY_SUMMARY production, providing new insight into the mechanism of CH3SH generation in oral ST:STUDY_SUMMARY microbial communities. Better understanding of the underlying interactions among ST:STUDY_SUMMARY oral bacteria involved in CH3SH generation can lead to development of more ST:STUDY_SUMMARY appropriate prophylactic approaches to treat halitosis and periodontitis. ST:INSTITUTE Osaka University ST:LAST_NAME Kuboniwa ST:FIRST_NAME Masae ST:ADDRESS 1-8 Yamadaoka, Suita. Osaka. Japan ST:EMAIL kuboniwa.masae.dent@osaka-u.ac.jp ST:PHONE +81668792922 #SUBJECT SU:SUBJECT_TYPE Bacteria SU:SUBJECT_SPECIES Fusobacterium nucleatum SU:TAXONOMY_ID 190304 #FACTORS #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 - 0h-2 Hour:0 O.D.mL=20.2749 SUBJECT_SAMPLE_FACTORS - 0h-3 Hour:0 O.D.mL=20.1343 SUBJECT_SAMPLE_FACTORS - 0h-4 Hour:0 O.D.mL=20.0906 SUBJECT_SAMPLE_FACTORS - 1h-1 Hour:1 O.D.mL=19.8189 SUBJECT_SAMPLE_FACTORS - 1h-3 Hour:1 O.D.mL=19.8512 SUBJECT_SAMPLE_FACTORS - 1h-4 Hour:1 O.D.mL=19.7961 SUBJECT_SAMPLE_FACTORS - 2h-2 Hour:2 O.D.mL=21.2230 SUBJECT_SAMPLE_FACTORS - 2h-3 Hour:2 O.D.mL=21.3104 SUBJECT_SAMPLE_FACTORS - 2h-4 Hour:2 O.D.mL=21.3769 SUBJECT_SAMPLE_FACTORS - 3h-1 Hour:3 O.D.mL=20.4193 SUBJECT_SAMPLE_FACTORS - 3h-3 Hour:3 O.D.mL=21.0596 SUBJECT_SAMPLE_FACTORS - 3h-4 Hour:3 O.D.mL=21.2553 SUBJECT_SAMPLE_FACTORS - 6h-2 Hour:6 O.D.mL=20.8772 SUBJECT_SAMPLE_FACTORS - 6h-3 Hour:6 O.D.mL=20.9418 SUBJECT_SAMPLE_FACTORS - 6h-4 Hour:6 O.D.mL=20.8012 #COLLECTION CO:COLLECTION_SUMMARY To investigate the time course of changes in intracellular metabolites of F. CO:COLLECTION_SUMMARY nucleatum cells after coculturing with S. gordonii WT, F. nucleatum cells were CO:COLLECTION_SUMMARY collected by centrifugation (7670 × g for 7 min at 4°C), washed twice with 1 CO:COLLECTION_SUMMARY ml of ultra-pure water, and then immediately treated twice with 1 ml of 100% CO:COLLECTION_SUMMARY methanol containing the internal standards (H3304-1002, Human Metabolome CO:COLLECTION_SUMMARY Technologies, Inc., Tsuruoka, Japan). CO:SAMPLE_TYPE Bacterial cells #TREATMENT TR:TREATMENT_SUMMARY [13C5, 15N] L-Methionine (13C5, 15N, 98%; Taiyo Nippon Sanso Corp., Tokyo, TR:TREATMENT_SUMMARY Japan) was added at a final concentration of 10 mM to mCDM, with the following TR:TREATMENT_SUMMARY modified amino acid concentrations; 40 mM L-glutamic acid, 10 mM L-arginine-HCl, TR:TREATMENT_SUMMARY 1.0 mM L-tryptophan. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY At the mid exponential growth phase (0.5 to 1.0 OD units ml-1), bacterial cells SP:SAMPLEPREP_SUMMARY were harvested by centrifugation (7670 × g for 7 min at 4°C), washed twice SP:SAMPLEPREP_SUMMARY with PBS, and finally resuspended at 20 OD units ml-1 in mCDM containing 10 mM SP:SAMPLEPREP_SUMMARY [13C5, 15N] L-methionine. F. nucleatum and S. gordonii cells were then SP:SAMPLEPREP_SUMMARY inoculated at a density of 1.5 × 1010 CFU/well into a Transwell outer chamber SP:SAMPLEPREP_SUMMARY (2.6 cm3) and inner chamber (1.5 cm3), respectively. Thereafter anaerobic SP:SAMPLEPREP_SUMMARY incubation was performed at 37°C for 0, 1, 2, 3, or 6 h. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_TYPE CE CH:INSTRUMENT_NAME Agilent CE Capillary Electrophoresis System CH:COLUMN_NAME Agilent Fused silica capillary (800 mm x 50 um) CH:SOLVENT_A Solution ID: H3301-1001 for cation analysis CH:SOLVENT_B Solution ID: H3302-1021 for anion analysis CH:FLOW_GRADIENT NA CH:FLOW_RATE The sample was injected at a pressure of 50 mbar for 10 s (approximately 10 nL) CH:FLOW_RATE for cation analysis and 25 s (approximately 25 nL) for anion analysis CH:COLUMN_TEMPERATURE NA #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Agilent 6210 TOF MS:INSTRUMENT_TYPE Other MS:MS_TYPE ESI MS:ION_MODE POSITIVE MS:MS_COMMENTS The systems were controlled by the Agilent G2201AA ChemStation software package, MS:MS_COMMENTS version B.03.01, for CE (Agilent Technologies, Waldbronn, Germany). #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS pmol/ODml MS_METABOLITE_DATA_START Samples 0h-2 0h-3 0h-4 1h-1 1h-3 1h-4 2h-2 2h-3 2h-4 3h-1 3h-3 3h-4 6h-2 6h-3 6h-4 Factors Hour:0 Hour:0 Hour:0 Hour:1 Hour:1 Hour:1 Hour:2 Hour:2 Hour:2 Hour:3 Hour:3 Hour:3 Hour:6 Hour:6 Hour:6 5'-Deoxy-5'-methylthioadenosine_m+0 8.373811184 10.91290569 10.15726767 2.901546112 2.476862724 2.521916551 1.492412367 1.219700163 1.15092162 0.931426843 0.645690287 1.041937318 0.311552237 0.148310372 0.28161877 5'-Deoxy-5'-methylthioadenosine_m+1 0.127836108 0.117400234 0.093083896 5.41682585 3.904930492 4.612993323 7.218624663 4.029296138 5.982636718 5.422901472 4.944951238 7.425849017 2.773520153 3.652014723 4.494789294 5'-Deoxy-5'-methylthioadenosine_m+2 0 0 0.074049468 0.101363157 0 0.201379967 0 0.18883855 0 0.27724457 0.213943419 0.221170645 0.312614695 0.416481291 0.651374174 5'-Deoxy-5'-methylthioadenosine_m+3 0.104591561 0.124575868 0.10061283 0.047092376 0.189725543 0.104571107 0.151999282 0.073683126 0.288073327 0.061388903 0.021023327 0.038404852 0.032628666 0.042499477 0.039637602 Acetylcadaverine_m+3 0.088565211 0.071299245 0.051619182 0.218270741 0.083441598 0.187720456 0.262459405 0.049115115 0.050983707 0.07218514 0.040580759 0.10571312 0.042085173 0.041038294 0.041315681 Agmatine_m+0 4 2.952854754 10 23 3.99782198 10 10 1.891670894 2.910482637 3 2 3.928845356 1 1 1 Agmatine_m+1 0 0.047145246 0 0 0.00217802 0 0 0.108329106 0.089517363 0 0 0.071154644 0 0 0 Arg_m+0 5019.354227 4385.50956 5377.554944 4137.844285 3505.618631 4051.938827 3136.051051 2341.061558 2791.03306 2688.505831 2394.125405 2849.805067 1704.229525 1878.368125 1858.698189 Cadaverine_m+0 0.261784963 0.214219147 0.752115637 0 0 0 0 0 0 0 0 0 0 0 0 Lys_m+0 157.3412034 145.62101 185.023943 64.13750161 62.0751748 68.91190734 51.18621358 41.96694099 55.53689457 44.11143295 43.41800713 42.34655219 22.65914377 31.88859648 30.82322815 Lys_m+1 0 0 0 0 0 0 0.253612781 0 0 0 0 0.207170253 0.248467358 0 0.137910435 Lys_m+2 0 0.135063116 0 0.057794576 0 0 0.102175473 0.034522557 0 0 0.035365831 0.080770621 0 0.020115188 0.241552764 Met_m+0 11.24870948 10.1027479 10.89464938 4.30411112 5.091782455 4.891334301 2.705397511 3.933592584 3.496979917 2.617564864 3.910164303 3.415005663 2.089691273 2.129354914 2.182621648 Met_m+1 0.204735273 0 0.150190196 0 0.163216973 0.166170679 0 0 0 0.27797664 0 0 0 0.04515535 0.182120832 Met_m+2 0.224593457 0 0.102801504 0 0 0 0 0 0 0 0 0 0 0 0 Met_m+5 2.020363705 1.655115666 2.18381049 1.029756106 1.268299773 1.140386719 0.901872851 0.678909548 1.110886408 0.805832374 0.580531132 0.980982511 0.425535219 0.494834055 0.657122123 Met_m+6 58.2176688 40.85194456 57.63700775 19.72692032 21.19498337 18.19426706 8.932032077 12.8040544 11.5430431 9.775132702 6.866579124 6.774857976 2.131454996 3.003992907 3.65408593 N1-Acetylspermidine_m+0 0.213000433 0.080878963 0.672791714 1.197584697 0.088064179 0.33630115 0.850387197 0.134850587 0.161690726 0.445456716 0.187248683 0.445862334 0.110501337 0.185633731 0.268244439 N-Acetylputrescine_m+0 0.585655385 0.483027371 0.549693133 1.58827059 1.229243222 1.596731099 1.745247311 0.727261817 0.918240955 1.042377896 0.938938771 1.041603253 0.534061377 0.804497463 0.892967928 N-Acetylputrescine_m+1 0.134964955 0.296965673 0.6574376 0.292516192 0.015932635 0.326346874 0.043694388 0 0 0.311913682 0.002544353 0 0.047342832 0.031465539 0.029490448 Ornithine_m+0 24.19769103 21.53592047 29.43275563 340.8812784 251.4736627 324.5393278 545.0110101 380.389774 401.758552 539.4386841 424.9379361 454.1012315 362.1519104 345.2261857 377.6253854 Ornithine_m+1 0 0.301739907 0 0 0 0 0 0 0 0 0 0 0 0 0 Ornithine_m+2 0 0 0 0.223308552 0.624895857 0 0 0 0.451318892 0.345688168 0 0.415960044 0.14073149 0.720819331 0.338084166 Putrescine_m+0 170.1205369 78.75189008 424.7531642 716.8529861 81.69655388 319.6486774 398.2675115 39.10714314 50.29025481 107.5569768 43.40347831 99.53509306 38.29691129 31.02159584 38.13656664 Putrescine_m+1 0 0 0 0 0 0 0 0 0.03805817 0 0 0 0 0 0 S-Adenosylhomocysteine_m+0 0.422162788 0.567356284 0.582438556 0.303056428 0.417158151 0.364582595 0.286090948 0.239607261 0.360589572 0.31418265 0.234689579 0.425789428 0.109747027 0.1156604 0.142405632 S-Adenosylmethionine_m+0 67.9420312 63.19513364 85.21423725 23.6729664 20.38459358 24.21319155 9.147728809 9.517003083 8.308234019 4.885596851 3.590303769 5.036583461 0.697432931 0.648466637 0.618220829 S-Adenosylmethionine_m+1 0.54214232 0 0 0 0.049458348 0 0.536370713 0.05114201 0 0.035681096 0.260244871 0.02111394 0.175170625 0.096460272 0.226636925 S-Adenosylmethionine_m+2 0 0 0 0.297997246 0 0 0.3550948 0.308057766 0 0.343467289 0.661569273 0.074285275 0.096717902 0.022607 0.089256782 S-Adenosylmethionine_m+3 0.155752892 0.076709089 0 0.109271705 0.208195578 0 0.030996452 0.045584974 0.224167483 0.231130912 0.083472552 0.172302716 0.07498408 0.042829482 0.119439363 S-Adenosylmethionine_m+4 2.788184096 2.044212224 1.81651651 1.437509169 1.512809602 2.418662344 4.378783773 1.132427855 2.477392547 1.984416236 2.355854831 2.08482631 0.841938698 1.12267396 1.450826739 S-Adenosylmethionine_m+5 0.141721804 0.168068359 0.30665431 1.796346754 1.577577438 1.990594966 2.884237279 2.360263747 2.000531189 2.15065981 3.270906127 3.146644837 3.191456162 3.365641727 3.313305318 S-Adenosylmethionine_m+6 0.4485976 0.064098294 0.573603833 44.26092706 38.08929564 44.16459638 55.88066609 48.89487716 52.42099972 46.27579354 45.71413246 51.70567886 30.94059951 27.42305517 27.43422817 S-Adenosylmethionine_m+7 0 0.015980792 0 1.590665725 0.63849211 1.490477128 0.424166301 2.360729565 0.928719844 1.372983177 1.958261233 2.447664236 1.141836849 2.168560527 2.54501126 S-Adenosylmethionine_m+8 0.096367942 0.099581558 0.005151887 0 0 0 0.302991509 0 0 0.098568053 0.137026243 0 0.52038137 0.04719286 0.453811556 Spermidine_m+0 0.169096702 0.143018959 0.586647751 1.446689143 0.322073106 0.914181712 0.937030407 0.229446426 0.220730347 0.438141747 0.202395032 0.45390687 0.298733952 0.156223895 0.049342697 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name PubChem CID HMDB ID m/z RT 5'-Deoxy-5'-methylthioadenosine_m+0 1173 298.0972571 9.389684321 5'-Deoxy-5'-methylthioadenosine_m+1 1173 299.1004907 9.391577933 5'-Deoxy-5'-methylthioadenosine_m+2 1173 300.0995067 9.395299933 5'-Deoxy-5'-methylthioadenosine_m+3 1173 301.1003213 9.39290025 Acetylcadaverine_m+3 148.1427967 8.1761495 Agmatine_m+0 199 1432 131.12917 4.801698613 Agmatine_m+1 199 1432 132.131475 4.80091667 Arg_m+0 6322 517 175.118522 6.570808327 Cadaverine_m+0 273 2322 103.1231 4.631113 Lys_m+0 866 182 147.1127053 6.386735813 Lys_m+1 866 182 148.115288 6.38528866 Lys_m+2 866 182 149.1183625 6.3820796 Met_m+0 876 696 150.058134 9.995223533 Met_m+1 876 696 151.061896 10.007804 Met_m+2 876 696 152.0541867 9.983394333 Met_m+5 876 696 155.0696007 9.993227267 Met_m+6 876 696 156.072076 9.990053467 N1-Acetylspermidine_m+0 496 1276 188.1756629 5.880389643 N-Acetylputrescine_m+0 122356 2064 131.118056 7.86112624 N-Acetylputrescine_m+1 122356 2064 132.121806 7.84772924 Ornithine_m+0 389 214 133.0967293 6.324388573 Ornithine_m+1 389 214 134.0998413 6.323652173 Ornithine_m+2 389 214 135.1032671 6.3147929 Putrescine_m+0 1045 1414 89.10734713 4.364620093 Putrescine_m+1 1045 1414 90.110025 4.365366053 S-Adenosylhomocysteine_m+0 S-Adenosylmethionine_m+0 34755 1185 399.1444379 6.56428045 S-Adenosylmethionine_m+1 34755 1185 400.1479615 6.567068546 S-Adenosylmethionine_m+2 34755 1185 401.1457073 6.567533755 S-Adenosylmethionine_m+3 34755 1185 402.1448475 6.558399 S-Adenosylmethionine_m+4 34755 1185 403.1486287 6.574434633 S-Adenosylmethionine_m+5 34755 1185 404.1555592 6.569510846 S-Adenosylmethionine_m+6 34755 1185 405.1583023 6.566903169 S-Adenosylmethionine_m+7 34755 1185 406.16107 6.568388858 S-Adenosylmethionine_m+8 34755 1185 406.16107 6.568388858 Spermidine_m+0 1102 1257 146.16469 4.2416328 METABOLITES_END #END