#METABOLOMICS WORKBENCH khuus_20210711_060907 DATATRACK_ID:2742 STUDY_ID:ST001862 ANALYSIS_ID:AN003019 PROJECT_ID:PR001175 VERSION 1 CREATED_ON July 17, 2021, 9:25 am #PROJECT PR:PROJECT_TITLE Cross-feeding between intestinal pathobionts promotes their overgrowth during PR:PROJECT_TITLE undernutrition PR:PROJECT_SUMMARY Child undernutrition is a global health issue associated with a high burden of PR:PROJECT_SUMMARY infectious disease. Undernourished children display an overabundance of PR:PROJECT_SUMMARY intestinal pathogens and pathobionts, and these bacteria induce enteric PR:PROJECT_SUMMARY dysfunction in undernourished mice; however, the cause of their overgrowth PR:PROJECT_SUMMARY remains poorly defined. Here, we show that disease-inducing human isolates of PR:PROJECT_SUMMARY Enterobacteriaceae and Bacteroidales spp. are capable of multi-species symbiotic PR:PROJECT_SUMMARY cross-feeding, resulting in synergistic growth of a mixed community in vitro. PR:PROJECT_SUMMARY Growth synergy occurs uniquely under malnourished conditions limited in protein PR:PROJECT_SUMMARY and iron: in this context, Bacteroidales spp. liberate diet- and mucin-derived PR:PROJECT_SUMMARY sugars and Enterobacteriaceae spp. enhance the bioavailability of iron. Analysis PR:PROJECT_SUMMARY of human microbiota datasets reveals that Bacteroidaceae and Enterobacteriaceae PR:PROJECT_SUMMARY are strongly correlated in undernourished children, but not in adequately PR:PROJECT_SUMMARY nourished children, consistent with a diet-dependent growth synergy in the human PR:PROJECT_SUMMARY gut. Together these data suggest that dietary cross-feeding fuels the overgrowth PR:PROJECT_SUMMARY of pathobionts in undernutrition. PR:INSTITUTE University of British Columbia PR:DEPARTMENT Michael Smith Laboratories PR:LAST_NAME Huus PR:FIRST_NAME Kelsey PR:ADDRESS 3125 East Mall, Vancouver, British Columbia, V6T 1Z4, Canada PR:EMAIL khuus@msl.ubc.ca PR:PHONE +1-604-822-2210 #STUDY ST:STUDY_TITLE Cross-feeding between intestinal pathobionts promotes their overgrowth during ST:STUDY_TITLE undernutrition ST:STUDY_SUMMARY Child undernutrition is a global health issue associated with a high burden of ST:STUDY_SUMMARY infectious disease. Undernourished children display an overabundance of ST:STUDY_SUMMARY intestinal pathogens and pathobionts, and these bacteria induce enteric ST:STUDY_SUMMARY dysfunction in undernourished mice; however, the cause of their overgrowth ST:STUDY_SUMMARY remains poorly defined. Here, we show that disease-inducing human isolates of ST:STUDY_SUMMARY Enterobacteriaceae and Bacteroidales spp. are capable of multi-species symbiotic ST:STUDY_SUMMARY cross-feeding, resulting in synergistic growth of a mixed community in vitro. ST:STUDY_SUMMARY Growth synergy occurs uniquely under malnourished conditions limited in protein ST:STUDY_SUMMARY and iron: in this context, Bacteroidales spp. liberate diet- and mucin-derived ST:STUDY_SUMMARY sugars and Enterobacteriaceae spp. enhance the bioavailability of iron. Analysis ST:STUDY_SUMMARY of human microbiota datasets reveals that Bacteroidaceae and Enterobacteriaceae ST:STUDY_SUMMARY are strongly correlated in undernourished children, but not in adequately ST:STUDY_SUMMARY nourished children, consistent with a diet-dependent growth synergy in the human ST:STUDY_SUMMARY gut. Together these data suggest that dietary cross-feeding fuels the overgrowth ST:STUDY_SUMMARY of pathobionts in undernutrition. ST:INSTITUTE University of British Columbia ST:DEPARTMENT Michael Smith Laboratories ST:LAST_NAME Huus ST:FIRST_NAME Kelsey ST:ADDRESS 3125 East Mall ST:EMAIL khuus@msl.ubc.ca ST:PHONE +1-604-822-2210 #SUBJECT SU:SUBJECT_TYPE Bacteria SU:SUBJECT_SPECIES Bacteroides spp. and Escherichia spp. (mixed communities) SU:TAXONOMY_ID B. fragilis 3_1_12; B. vulgatus 3_1_40A; B. ovatus 3_8_47; B. dorei 5_1_36; P. SU:TAXONOMY_ID distasonis 2_1_33B; E. coli 3_1_53; E. coli 4_1_47 #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 Bacteroidales_mix 01_B1-16h Factor:16h RAW_FILE_NAME=01_B1-16h.d; Replicate=1; Analyte=Sugars SUBJECT_SAMPLE_FACTORS Bacteroidales_mix 02_B2-16h Factor:16h RAW_FILE_NAME=02_B2-16h.d; Replicate=2; Analyte=Sugars SUBJECT_SAMPLE_FACTORS Bacteroidales_mix 03_B3-16h Factor:16h RAW_FILE_NAME=03_B3-16h.d; Replicate=3; Analyte=Sugars SUBJECT_SAMPLE_FACTORS Bacteroides_ovatus 04_BO1-16h Factor:16h RAW_FILE_NAME=04_BO1-16h.d; Replicate=1; Analyte=Sugars SUBJECT_SAMPLE_FACTORS Bacteroides_ovatus 05_BO2-16h Factor:16h RAW_FILE_NAME=05_BO2-16h.d; Replicate=2; Analyte=Sugars SUBJECT_SAMPLE_FACTORS Bacteroides_ovatus 06_BO3-16h Factor:16h RAW_FILE_NAME=06_BO3-16h.d; Replicate=3; Analyte=Sugars SUBJECT_SAMPLE_FACTORS E_coli 07_E1-16h Factor:16h RAW_FILE_NAME=07_E1-16h.d; Replicate=1; Analyte=Sugars SUBJECT_SAMPLE_FACTORS E_coli 08_E2-16h Factor:16h RAW_FILE_NAME=08_E2-16h.d; Replicate=2; Analyte=Sugars SUBJECT_SAMPLE_FACTORS E_coli 09_E3-16h Factor:16h RAW_FILE_NAME=09_E3-16h.d; Replicate=3; Analyte=Sugars SUBJECT_SAMPLE_FACTORS Bacteroidales_Ecoli_mix 10_BE1-16h Factor:16h RAW_FILE_NAME=10_BE1-16h.d; Replicate=1; Analyte=Sugars SUBJECT_SAMPLE_FACTORS Bacteroidales_Ecoli_mix 11_BE2-16h Factor:16h RAW_FILE_NAME=11_BE2-16h.d; Replicate=2; Analyte=Sugars SUBJECT_SAMPLE_FACTORS Bacteroidales_Ecoli_mix 12_BE3-16h Factor:16h RAW_FILE_NAME=12_BE3-16h.d; Replicate=3; Analyte=Sugars SUBJECT_SAMPLE_FACTORS Bacteroidales_mix 13_B1-24h Factor:24h RAW_FILE_NAME=13_B1-24h.d; Replicate=1; Analyte=Sugars SUBJECT_SAMPLE_FACTORS Bacteroidales_mix 14_B2-24h Factor:24h RAW_FILE_NAME=14_B2-24h.d; Replicate=2; Analyte=Sugars SUBJECT_SAMPLE_FACTORS Bacteroidales_mix 15_B3-24h Factor:24h RAW_FILE_NAME=15_B3-24h.d; Replicate=3; Analyte=Sugars SUBJECT_SAMPLE_FACTORS Bacteroides_ovatus 16_BO1-24h Factor:24h RAW_FILE_NAME=16_BO1-24h.d; Replicate=1; Analyte=Sugars SUBJECT_SAMPLE_FACTORS Bacteroides_ovatus 17_BO2-24h Factor:24h RAW_FILE_NAME=17_BO2-24h.d; Replicate=2; Analyte=Sugars SUBJECT_SAMPLE_FACTORS Bacteroides_ovatus 18_BO3-24h Factor:24h RAW_FILE_NAME=18_BO3-24h.d; Replicate=3; Analyte=Sugars SUBJECT_SAMPLE_FACTORS E_coli 19_E1-24h Factor:24h RAW_FILE_NAME=19_E1-24h.d; Replicate=1; Analyte=Sugars SUBJECT_SAMPLE_FACTORS E_coli 20_E2-24h Factor:24h RAW_FILE_NAME=20_E2-24h.d; Replicate=2; Analyte=Sugars SUBJECT_SAMPLE_FACTORS E_coli 21_E3-24h Factor:24h RAW_FILE_NAME=21_E3-24h.d; Replicate=3; Analyte=Sugars SUBJECT_SAMPLE_FACTORS Bacteroidales_Ecoli_mix 22_BE1-24h Factor:24h RAW_FILE_NAME=22_BE1-24h.d; Replicate=1; Analyte=Sugars SUBJECT_SAMPLE_FACTORS Bacteroidales_Ecoli_mix 23_BE2-24h Factor:24h RAW_FILE_NAME=23_BE2-24h.d; Replicate=2; Analyte=Sugars SUBJECT_SAMPLE_FACTORS Bacteroidales_Ecoli_mix 24_BE3-24h Factor:24h RAW_FILE_NAME=24_BE3-24h.d; Replicate=3; Analyte=Sugars SUBJECT_SAMPLE_FACTORS Blank 25_Blank1 Factor:0h RAW_FILE_NAME=25_Blank1.d; Replicate=1; Analyte=Sugars SUBJECT_SAMPLE_FACTORS Blank 26_Blank2 Factor:0h RAW_FILE_NAME=26_Blank2.d; Replicate=2; Analyte=Sugars SUBJECT_SAMPLE_FACTORS Blank 27_Blank3 Factor:0h RAW_FILE_NAME=27_Blank3.d; Replicate=3; Analyte=Sugars SUBJECT_SAMPLE_FACTORS Blank 28_Blank4 Factor:0h RAW_FILE_NAME=28_Blank4.d; Replicate=4; Analyte=Sugars SUBJECT_SAMPLE_FACTORS Bacteroidales_mix B4_16h Factor:16h RAW_FILE_NAME=B4_16H_OK.d; Replicate=4; Analyte=SCFA SUBJECT_SAMPLE_FACTORS Bacteroidales_mix B5_16h Factor:16h RAW_FILE_NAME=B5_16H_3.d; Replicate=4; Analyte=SCFA SUBJECT_SAMPLE_FACTORS Bacteroidales_mix B6_16h Factor:16h RAW_FILE_NAME=B6_16H_3.d; Replicate=4; Analyte=SCFA SUBJECT_SAMPLE_FACTORS Bacteroides_ovatus BO4_16h Factor:16h RAW_FILE_NAME=B04_16H_OK.d; Replicate=4; Analyte=SCFA SUBJECT_SAMPLE_FACTORS Bacteroides_ovatus BO5_16h Factor:16h RAW_FILE_NAME=B05_16H_3.d; Replicate=4; Analyte=SCFA SUBJECT_SAMPLE_FACTORS Bacteroides_ovatus BO6_16h Factor:16h RAW_FILE_NAME=B06_16H_3.d; Replicate=4; Analyte=SCFA SUBJECT_SAMPLE_FACTORS E_coli E4_16h Factor:16h RAW_FILE_NAME=E4_16H_OK.d; Replicate=4; Analyte=SCFA SUBJECT_SAMPLE_FACTORS E_coli E5_16h Factor:16h RAW_FILE_NAME=E5_16H_3.d; Replicate=4; Analyte=SCFA SUBJECT_SAMPLE_FACTORS E_coli E6_16h Factor:16h RAW_FILE_NAME=E6_16H_3.d; Replicate=5; Analyte=SCFA SUBJECT_SAMPLE_FACTORS Bacteroidales_Ecoli_mix BE4_16h Factor:16h RAW_FILE_NAME=BE4_16H_OK.d; Replicate=5; Analyte=SCFA SUBJECT_SAMPLE_FACTORS Bacteroidales_Ecoli_mix BE5_16h Factor:16h RAW_FILE_NAME=BE5_16H_3.d; Replicate=5; Analyte=SCFA SUBJECT_SAMPLE_FACTORS Bacteroidales_Ecoli_mix BE6_16h Factor:16h RAW_FILE_NAME=BE6_16H_3.d; Replicate=5; Analyte=SCFA SUBJECT_SAMPLE_FACTORS Bacteroidales_mix B4_24h Factor:24h RAW_FILE_NAME=B4_24H.d; Replicate=5; Analyte=SCFA SUBJECT_SAMPLE_FACTORS Bacteroidales_mix B5_24h Factor:24h RAW_FILE_NAME=B5_24H_3.d; Replicate=5; Analyte=SCFA SUBJECT_SAMPLE_FACTORS Bacteroidales_mix B6_24h Factor:24h RAW_FILE_NAME=B6_24H_3.d; Replicate=5; Analyte=SCFA SUBJECT_SAMPLE_FACTORS Bacteroides_ovatus BO4_24h Factor:24h RAW_FILE_NAME=B04_24H.d; Replicate=5; Analyte=SCFA SUBJECT_SAMPLE_FACTORS Bacteroides_ovatus BO5_24h Factor:24h RAW_FILE_NAME=B05_24H_3.d; Replicate=6; Analyte=SCFA SUBJECT_SAMPLE_FACTORS Bacteroides_ovatus BO6_24h Factor:24h RAW_FILE_NAME=B06_24H_3.d; Replicate=6; Analyte=SCFA SUBJECT_SAMPLE_FACTORS E_coli E4_24h Factor:24h RAW_FILE_NAME=E4_24H.d; Replicate=6; Analyte=SCFA SUBJECT_SAMPLE_FACTORS E_coli E5_24h Factor:24h RAW_FILE_NAME=E5_24H_3.d; Replicate=6; Analyte=SCFA SUBJECT_SAMPLE_FACTORS E_coli E6_24h Factor:24h RAW_FILE_NAME=E6_16H_3.d; Replicate=6; Analyte=SCFA SUBJECT_SAMPLE_FACTORS Bacteroidales_Ecoli_mix BE4_24h Factor:24h RAW_FILE_NAME=BE4_24H.d; Replicate=6; Analyte=SCFA SUBJECT_SAMPLE_FACTORS Bacteroidales_Ecoli_mix BE5_24h Factor:24h RAW_FILE_NAME=BE5_24H_3.d; Replicate=6; Analyte=SCFA SUBJECT_SAMPLE_FACTORS Bacteroidales_Ecoli_mix BE6_24h Factor:24h RAW_FILE_NAME=BE6_24H_3.d; Replicate=5; Analyte=SCFA SUBJECT_SAMPLE_FACTORS Blank blank_media1 Factor:0h RAW_FILE_NAME=blank_3.d; Replicate=4; Analyte=SCFA SUBJECT_SAMPLE_FACTORS Blank blank_media2 Factor:0h RAW_FILE_NAME=blank2_3.d; Replicate=5; Analyte=SCFA SUBJECT_SAMPLE_FACTORS Blank blank_media3 Factor:0h RAW_FILE_NAME=BLANK4_OK.d; Replicate=6; Analyte=SCFA #COLLECTION CO:COLLECTION_SUMMARY Bacteria were grown anaerobically at 37ºC in MAL-M medium. Culture supernatants CO:COLLECTION_SUMMARY at 0, 16 and 24h were collected by centrifugation at 16000 g for 20 minutes. CO:COLLECTION_SUMMARY Supernatants were filter sterilized at 0.22 µM and stored at -70ºC before CO:COLLECTION_SUMMARY analysis. CO:SAMPLE_TYPE Bacterial cells CO:STORAGE_CONDITIONS Described in summary #TREATMENT TR:TREATMENT_SUMMARY The groups differ by bacterial community composition as follows: B, TR:TREATMENT_SUMMARY Bacteroidales mix (B. fragilis, B. vulgatus B. ovatus, B. dorei, P. distasonis); TR:TREATMENT_SUMMARY E, E. coli mix (E. coli 3_1_53 and E. coli 4_1_47); BE, Bacteroidales-E.coli mix TR:TREATMENT_SUMMARY (all seven strains as above); BO, B. ovatus only. Strains were inoculated in TR:TREATMENT_SUMMARY equal proportions based on normalized O.D. from overnight input cultures. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Sugars Analysis Low molecular weight sugars and NAc-sugar amines were quantified SP:SAMPLEPREP_SUMMARY by LC-MRM/MS at The Metabolomics Innovation Centre (TMIC) commercial service SP:SAMPLEPREP_SUMMARY (University of Victoria , Genome BC Proteomics Centre), according to previously SP:SAMPLEPREP_SUMMARY published UPLC-MRM/MS methods (Han et al 2016, Electrophoresis), with necessary SP:SAMPLEPREP_SUMMARY modifications. In brief, a mixed stock solution of 13 low-MW sugars and 4 SP:SAMPLEPREP_SUMMARY N-acetyl sugar amines was prepared with the use of their standard substances in SP:SAMPLEPREP_SUMMARY 80% methanol at 500µM for each compound. This solution was then serially SP:SAMPLEPREP_SUMMARY diluted with the same solvent to prepare calibration solutions in a SP:SAMPLEPREP_SUMMARY concentration range of 0.002 to 125µM. For chemical derivatization, 20µL of SP:SAMPLEPREP_SUMMARY each medium sample or each calibration solution was mixed in turn with 20µL of SP:SAMPLEPREP_SUMMARY an internal standard solution containing 13C6-glucose, 13C6-mannose, SP:SAMPLEPREP_SUMMARY 13C6-fructose and 13C5-ribose in water, 40µL of 200-mM 3-nitrophenylhydrazine SP:SAMPLEPREP_SUMMARY hydrochloride solution in 60% methanol and 40µL of 200-mM EDC.HCl solution SP:SAMPLEPREP_SUMMARY prepared in a mixed solvent of methanol/water/pyridine (60:40:5, v/v/v). The SP:SAMPLEPREP_SUMMARY mixture was allowed to react at 50ºC for 90 min. SCFA Analysis Quantification SP:SAMPLEPREP_SUMMARY of short-chain fatty acids was performed in-house according to a method SP:SAMPLEPREP_SUMMARY developed by Han et al., with minor modifications(Han et al 2015, Analytica SP:SAMPLEPREP_SUMMARY Chimica Acta). Briefly, 500µL of supernatant were mixed with 500µL of 50 % SP:SAMPLEPREP_SUMMARY acetonitrile, then the mixture was vortexed for 5 minutes and centrifuged at SP:SAMPLEPREP_SUMMARY 7000 x g for 5 minutes. 50 µL of the organic phase were derivatized adding SP:SAMPLEPREP_SUMMARY 20µL of 200mM 3NPH in 50 % acetonitrile and 20 µL 120 mM EDC solution of 6 % SP:SAMPLEPREP_SUMMARY pyridine in 50 % acetonitrile. The mixture was left under agitation at 40ºC for SP:SAMPLEPREP_SUMMARY 30 minutes. After this time reaction was stopped adding 100 µL of 0.1 % formic SP:SAMPLEPREP_SUMMARY acid in 90 % acetonitrile. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY SCFA Analysis CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Agilent 1200 CH:COLUMN_NAME Agilent Zorbax 300 C18 (250x4.6mm) CH:METHODS_FILENAME methods_summary_ms.docx #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Agilent 6460 QQQ MS:INSTRUMENT_TYPE Triple quadrupole MS:MS_TYPE ESI MS:ION_MODE POSITIVE MS:MS_COMMENTS A collision energy of 10V was used for multiple reaction monitoring (MRM), and MS:MS_COMMENTS LC-MS/MS data were analysed by Mass Hunter Qualitative Analysis B.06.00 software MS:MS_COMMENTS (Agilent Technologies). The identification and quantification of the SCFAs were MS:MS_COMMENTS carried out based on the retention time and mass fragmentation pattern comparing MS:MS_COMMENTS with standards. Six-point calibration curves made by peak area vs concentration MS:MS_COMMENTS of the pure standards were used to quantify the different SCFA. The linearity of MS:MS_COMMENTS the curves was determined by the coefficient of determination (R2), being higher MS:MS_COMMENTS than 0.99 for all standards. Concentrations of the SCFAs were calculated by MS:MS_COMMENTS interpolating the calibration curves of individual compounds. #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS µM MS_METABOLITE_DATA_START Samples B4_16h B4_24h B5_16h B5_24h B6_16h B6_24h BE4_16h BE4_24h BE5_16h BE5_24h BE6_16h BE6_24h BO4_16h BO4_24h BO5_16h BO5_24h BO6_16h BO6_24h E4_16h E4_24h E5_16h E5_24h E6_16h E6_24h Factors Factor:16h Factor:24h Factor:16h Factor:24h Factor:16h Factor:24h Factor:16h Factor:24h Factor:16h Factor:24h Factor:16h Factor:24h Factor:16h Factor:24h Factor:16h Factor:24h Factor:16h Factor:24h Factor:16h Factor:24h Factor:16h Factor:24h Factor:16h Factor:24h lactic acid 0.216319416 0.418668411 0.303744653 0.615087838 0.285882789 0.590861276 0.19137349 0.02518339 0.266537968 0.493279732 0.279849295 0.549332319 0.135810055 1.674187613 0.155498577 1.771797891 0.264811608 2.173965954 0.013144378 0.416259143 0.052295855 0.059590868 0.065745113 0.07129208 acetic acid 0.74411919 1.57920435 0.989406328 1.836486778 0.873085635 1.781273609 1.748542845 0.653692091 2.22383907 3.863670777 1.603206277 3.759617025 0.374867659 1.505910161 0.372199829 1.553801423 0.747727462 1.886878445 0.253109441 3.605446462 0.416802039 0.654555461 0.404385706 0.648794291 propionic acid 0.451605923 1.309627347 0.615456023 1.468421931 0.487008961 1.402489588 0.897122871 0.08918136 1.125763006 1.669607071 1.081934774 1.519170725 0.080765955 0.876525888 0.067346729 0.801903089 0.165274511 0.980996107 0.043658204 1.575518629 0.085026975 0.088498554 0.083639819 0.091203232 isobutiric acid 0.003129913 0.012125603 0.002337934 0.01180457 0.003095037 0.011102356 0.004731226 1.94364E-05 0.005269822 0.009056482 0.0048503 0.007587345 0 0.002299794 0.000249466 0.001521177 0 0.002200253 0 0.010210403 0 0.000236845 0 0.000168933 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name lactic acid acetic acid propionic acid isobutiric acid METABOLITES_END #END