#METABOLOMICS WORKBENCH Xiaodong_20211112_000203 DATATRACK_ID:2925 STUDY_ID:ST001987 ANALYSIS_ID:AN003239 PROJECT_ID:PR001263 VERSION 1 CREATED_ON November 12, 2021, 2:27 am #PROJECT PR:PROJECT_TITLE Transcriptomic and lipidomic analysis unravels the response of Faecalibacterium PR:PROJECT_TITLE prausnitzii to calcium palmitate PR:PROJECT_SUMMARY Infant formula is a suggested alternative to human milk if breastfeeding is not PR:PROJECT_SUMMARY an option; vegetable oil blends are commonly used in infant formula (IF) to PR:PROJECT_SUMMARY replace dairy fat, which can induce the formation of the poorly soluble soap PR:PROJECT_SUMMARY calcium palmitate (CP) in the infant’s gut. Previously, we observed that CP at PR:PROJECT_SUMMARY a low concentration of 0.01 mg/ml inhibits the growth of dominant infant PR:PROJECT_SUMMARY bacteria such as Faecalibacterium prausnitzii both during the exponential phase PR:PROJECT_SUMMARY as well as in the stationary phase. Here, we investigate the underlying PR:PROJECT_SUMMARY mechanism of the CP inhibition on infant-gut bacteria using F. prausnitzii as a PR:PROJECT_SUMMARY model by analysing its growth at a transcriptomic and lipidomic level. PR:INSTITUTE University of Groningen PR:LAST_NAME Horvatovich PR:FIRST_NAME Péter PR:ADDRESS Antonius Deusinglaan 1, 9713 AV Groningen, The Netherlands PR:EMAIL p.l.horvatovich@rug.nl PR:PHONE +31 (0)50 363 3341 #STUDY ST:STUDY_TITLE Transcriptomic and lipidomic analysis unravels the response of Faecalibacterium ST:STUDY_TITLE prausnitzii to calcium palmitate ST:STUDY_SUMMARY Infant formula is a suggested alternative to human milk if breastfeeding is not ST:STUDY_SUMMARY an option; vegetable oil blends are commonly used in infant formula (IF) to ST:STUDY_SUMMARY replace dairy fat, which can induce the formation of the poorly soluble soap ST:STUDY_SUMMARY calcium palmitate (CP) in the infant’s gut. Previously, we observed that CP at ST:STUDY_SUMMARY a low concentration of 0.01 mg/ml inhibits the growth of dominant infant ST:STUDY_SUMMARY bacteria such as Faecalibacterium prausnitzii both during the exponential phase ST:STUDY_SUMMARY as well as in the stationary phase. Here, we investigate the underlying ST:STUDY_SUMMARY mechanism of the CP inhibition on infant-gut bacteria using F. prausnitzii as a ST:STUDY_SUMMARY model by analysing its growth at a transcriptomic and lipidomic level. ST:INSTITUTE University of Groningen ST:LAST_NAME Horvatovich ST:FIRST_NAME Péter ST:ADDRESS Antonius Deusinglaan 1, 9713 AV Groningen, The Netherlands ST:EMAIL p.l.horvatovich@rug.nl ST:PHONE +31 (0)50 363 3341 #SUBJECT SU:SUBJECT_TYPE Bacteria SU:SUBJECT_SPECIES Faecalibacterium prausnitzii #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 - Neg5 Factor:CP0Hour5 Negative RAW_FILE_NAME=Neg5 SUBJECT_SAMPLE_FACTORS - Neg6 Factor:CP0Hour5 Negative RAW_FILE_NAME=Neg6 SUBJECT_SAMPLE_FACTORS - Neg43 Factor:CP0Hour5 Negative RAW_FILE_NAME=Neg43 SUBJECT_SAMPLE_FACTORS - Neg37 Factor:CP0Hour5 Negative RAW_FILE_NAME=Neg37 SUBJECT_SAMPLE_FACTORS - Neg36 Factor:CP0Hour5 Negative RAW_FILE_NAME=Neg36 SUBJECT_SAMPLE_FACTORS - Neg33 Factor:CP0Hour5 Negative RAW_FILE_NAME=Neg33 SUBJECT_SAMPLE_FACTORS - Neg29 Factor:CP0Hour5 Negative RAW_FILE_NAME=Neg29 SUBJECT_SAMPLE_FACTORS - Neg23 Factor:CP0Hour5 Negative RAW_FILE_NAME=Neg23 SUBJECT_SAMPLE_FACTORS - Neg22 Factor:CP0Hour5 Negative RAW_FILE_NAME=Neg22 SUBJECT_SAMPLE_FACTORS - Neg21 Factor:CP0Hour5 Negative RAW_FILE_NAME=Neg21 SUBJECT_SAMPLE_FACTORS - Neg17 Factor:CP0Hour5 Negative RAW_FILE_NAME=Neg17 SUBJECT_SAMPLE_FACTORS - Neg16 Factor:CP0Hour5 Negative RAW_FILE_NAME=Neg16 SUBJECT_SAMPLE_FACTORS - Neg7 Factor:CP0Hour16 Negative RAW_FILE_NAME=Neg7 SUBJECT_SAMPLE_FACTORS - Neg8 Factor:CP0Hour16 Negative RAW_FILE_NAME=Neg8 SUBJECT_SAMPLE_FACTORS - Neg42 Factor:CP0Hour16 Negative RAW_FILE_NAME=Neg42 SUBJECT_SAMPLE_FACTORS - Neg41 Factor:CP0Hour16 Negative RAW_FILE_NAME=Neg41 SUBJECT_SAMPLE_FACTORS - Neg38 Factor:CP0Hour16 Negative RAW_FILE_NAME=Neg38 SUBJECT_SAMPLE_FACTORS - Neg35 Factor:CP0Hour16 Negative RAW_FILE_NAME=Neg35 SUBJECT_SAMPLE_FACTORS - Neg30 Factor:CP0Hour16 Negative RAW_FILE_NAME=Neg30 SUBJECT_SAMPLE_FACTORS - Neg26 Factor:CP0Hour16 Negative RAW_FILE_NAME=Neg26 SUBJECT_SAMPLE_FACTORS - Neg20 Factor:CP0Hour16 Negative RAW_FILE_NAME=Neg20 SUBJECT_SAMPLE_FACTORS - Neg19 Factor:CP0Hour16 Negative RAW_FILE_NAME=Neg19 SUBJECT_SAMPLE_FACTORS - Neg18 Factor:CP0Hour16 Negative RAW_FILE_NAME=Neg18 SUBJECT_SAMPLE_FACTORS - Neg13 Factor:CP0Hour16 Negative RAW_FILE_NAME=Neg13 SUBJECT_SAMPLE_FACTORS - Neg9 Factor:CP2Hour5 Negative RAW_FILE_NAME=Neg9 SUBJECT_SAMPLE_FACTORS - Neg46 Factor:CP2Hour5 Negative RAW_FILE_NAME=Neg46 SUBJECT_SAMPLE_FACTORS - Neg44 Factor:CP2Hour5 Negative RAW_FILE_NAME=Neg44 SUBJECT_SAMPLE_FACTORS - Neg40 Factor:CP2Hour5 Negative RAW_FILE_NAME=Neg40 SUBJECT_SAMPLE_FACTORS - Neg34 Factor:CP2Hour5 Negative RAW_FILE_NAME=Neg34 SUBJECT_SAMPLE_FACTORS - Neg3 Factor:CP2Hour5 Negative RAW_FILE_NAME=Neg3 SUBJECT_SAMPLE_FACTORS - Neg28 Factor:CP2Hour5 Negative RAW_FILE_NAME=Neg28 SUBJECT_SAMPLE_FACTORS - Neg24 Factor:CP2Hour5 Negative RAW_FILE_NAME=Neg24 SUBJECT_SAMPLE_FACTORS - Neg2 Factor:CP2Hour5 Negative RAW_FILE_NAME=Neg2 SUBJECT_SAMPLE_FACTORS - Neg14 Factor:CP2Hour5 Negative RAW_FILE_NAME=Neg14 SUBJECT_SAMPLE_FACTORS - Neg11 Factor:CP2Hour5 Negative RAW_FILE_NAME=Neg11 SUBJECT_SAMPLE_FACTORS - Neg1 Factor:CP2Hour5 Negative RAW_FILE_NAME=Neg1 SUBJECT_SAMPLE_FACTORS - Neg48 Factor:CP2Hour16 Negative RAW_FILE_NAME=Neg48 SUBJECT_SAMPLE_FACTORS - Neg47 Factor:CP2Hour16 Negative RAW_FILE_NAME=Neg47 SUBJECT_SAMPLE_FACTORS - Neg45 Factor:CP2Hour16 Negative RAW_FILE_NAME=Neg45 SUBJECT_SAMPLE_FACTORS - Neg4 Factor:CP2Hour16 Negative RAW_FILE_NAME=Neg4 SUBJECT_SAMPLE_FACTORS - Neg39 Factor:CP2Hour16 Negative RAW_FILE_NAME=Neg39 SUBJECT_SAMPLE_FACTORS - Neg32 Factor:CP2Hour16 Negative RAW_FILE_NAME=Neg32 SUBJECT_SAMPLE_FACTORS - Neg31 Factor:CP2Hour16 Negative RAW_FILE_NAME=Neg31 SUBJECT_SAMPLE_FACTORS - Neg27 Factor:CP2Hour16 Negative RAW_FILE_NAME=Neg27 SUBJECT_SAMPLE_FACTORS - Neg25 Factor:CP2Hour16 Negative RAW_FILE_NAME=Neg25 SUBJECT_SAMPLE_FACTORS - Neg15 Factor:CP2Hour16 Negative RAW_FILE_NAME=Neg15 SUBJECT_SAMPLE_FACTORS - Neg12 Factor:CP2Hour16 Negative RAW_FILE_NAME=Neg12 SUBJECT_SAMPLE_FACTORS - Neg10 Factor:CP2Hour16 Negative RAW_FILE_NAME=Neg10 SUBJECT_SAMPLE_FACTORS - Pos5 Factor:CP0Hour5 Positive RAW_FILE_NAME=Pos5 SUBJECT_SAMPLE_FACTORS - Pos6 Factor:CP0Hour5 Positive RAW_FILE_NAME=Pos6 SUBJECT_SAMPLE_FACTORS - Pos43 Factor:CP0Hour5 Positive RAW_FILE_NAME=Pos43 SUBJECT_SAMPLE_FACTORS - Pos37 Factor:CP0Hour5 Positive RAW_FILE_NAME=Pos37 SUBJECT_SAMPLE_FACTORS - Pos36 Factor:CP0Hour5 Positive RAW_FILE_NAME=Pos36 SUBJECT_SAMPLE_FACTORS - Pos33 Factor:CP0Hour5 Positive RAW_FILE_NAME=Pos33 SUBJECT_SAMPLE_FACTORS - Pos29 Factor:CP0Hour5 Positive RAW_FILE_NAME=Pos29 SUBJECT_SAMPLE_FACTORS - Pos23 Factor:CP0Hour5 Positive RAW_FILE_NAME=Pos23 SUBJECT_SAMPLE_FACTORS - Pos22 Factor:CP0Hour5 Positive RAW_FILE_NAME=Pos22 SUBJECT_SAMPLE_FACTORS - Pos21 Factor:CP0Hour5 Positive RAW_FILE_NAME=Pos21 SUBJECT_SAMPLE_FACTORS - Pos17 Factor:CP0Hour5 Positive RAW_FILE_NAME=Pos17 SUBJECT_SAMPLE_FACTORS - Pos16 Factor:CP0Hour5 Positive RAW_FILE_NAME=Pos16 SUBJECT_SAMPLE_FACTORS - Pos7 Factor:CP0Hour16 Positive RAW_FILE_NAME=Pos7 SUBJECT_SAMPLE_FACTORS - Pos8 Factor:CP0Hour16 Positive RAW_FILE_NAME=Pos8 SUBJECT_SAMPLE_FACTORS - Pos42 Factor:CP0Hour16 Positive RAW_FILE_NAME=Pos42 SUBJECT_SAMPLE_FACTORS - Pos41 Factor:CP0Hour16 Positive RAW_FILE_NAME=Pos41 SUBJECT_SAMPLE_FACTORS - Pos38 Factor:CP0Hour16 Positive RAW_FILE_NAME=Pos38 SUBJECT_SAMPLE_FACTORS - Pos35 Factor:CP0Hour16 Positive RAW_FILE_NAME=Pos35 SUBJECT_SAMPLE_FACTORS - Pos30 Factor:CP0Hour16 Positive RAW_FILE_NAME=Pos30 SUBJECT_SAMPLE_FACTORS - Pos26 Factor:CP0Hour16 Positive RAW_FILE_NAME=Pos26 SUBJECT_SAMPLE_FACTORS - Pos20 Factor:CP0Hour16 Positive RAW_FILE_NAME=Pos20 SUBJECT_SAMPLE_FACTORS - Pos19 Factor:CP0Hour16 Positive RAW_FILE_NAME=Pos19 SUBJECT_SAMPLE_FACTORS - Pos18 Factor:CP0Hour16 Positive RAW_FILE_NAME=Pos18 SUBJECT_SAMPLE_FACTORS - Pos13 Factor:CP0Hour16 Positive RAW_FILE_NAME=Pos13 SUBJECT_SAMPLE_FACTORS - Pos9 Factor:CP2Hour5 Positive RAW_FILE_NAME=Pos9 SUBJECT_SAMPLE_FACTORS - Pos46 Factor:CP2Hour5 Positive RAW_FILE_NAME=Pos46 SUBJECT_SAMPLE_FACTORS - Pos44 Factor:CP2Hour5 Positive RAW_FILE_NAME=Pos44 SUBJECT_SAMPLE_FACTORS - Pos40 Factor:CP2Hour5 Positive RAW_FILE_NAME=Pos40 SUBJECT_SAMPLE_FACTORS - Pos34 Factor:CP2Hour5 Positive RAW_FILE_NAME=Pos34 SUBJECT_SAMPLE_FACTORS - Pos3 Factor:CP2Hour5 Positive RAW_FILE_NAME=Pos3 SUBJECT_SAMPLE_FACTORS - Pos28 Factor:CP2Hour5 Positive RAW_FILE_NAME=Pos28 SUBJECT_SAMPLE_FACTORS - Pos24 Factor:CP2Hour5 Positive RAW_FILE_NAME=Pos24 SUBJECT_SAMPLE_FACTORS - Pos2 Factor:CP2Hour5 Positive RAW_FILE_NAME=Pos2 SUBJECT_SAMPLE_FACTORS - Pos14 Factor:CP2Hour5 Positive RAW_FILE_NAME=Pos14 SUBJECT_SAMPLE_FACTORS - Pos11 Factor:CP2Hour5 Positive RAW_FILE_NAME=Pos11 SUBJECT_SAMPLE_FACTORS - Pos1 Factor:CP2Hour5 Positive RAW_FILE_NAME=Pos1 SUBJECT_SAMPLE_FACTORS - Pos48 Factor:CP2Hour16 Positive RAW_FILE_NAME=Pos48 SUBJECT_SAMPLE_FACTORS - Pos47 Factor:CP2Hour16 Positive RAW_FILE_NAME=Pos47 SUBJECT_SAMPLE_FACTORS - Pos45 Factor:CP2Hour16 Positive RAW_FILE_NAME=Pos45 SUBJECT_SAMPLE_FACTORS - Pos4 Factor:CP2Hour16 Positive RAW_FILE_NAME=Pos4 SUBJECT_SAMPLE_FACTORS - Pos39 Factor:CP2Hour16 Positive RAW_FILE_NAME=Pos39 SUBJECT_SAMPLE_FACTORS - Pos32 Factor:CP2Hour16 Positive RAW_FILE_NAME=Pos32 SUBJECT_SAMPLE_FACTORS - Pos31 Factor:CP2Hour16 Positive RAW_FILE_NAME=Pos31 SUBJECT_SAMPLE_FACTORS - Pos27 Factor:CP2Hour16 Positive RAW_FILE_NAME=Pos27 SUBJECT_SAMPLE_FACTORS - Pos25 Factor:CP2Hour16 Positive RAW_FILE_NAME=Pos25 SUBJECT_SAMPLE_FACTORS - Pos15 Factor:CP2Hour16 Positive RAW_FILE_NAME=Pos15 SUBJECT_SAMPLE_FACTORS - Pos12 Factor:CP2Hour16 Positive RAW_FILE_NAME=Pos12 SUBJECT_SAMPLE_FACTORS - Pos10 Factor:CP2Hour16 Positive RAW_FILE_NAME=Pos10 #COLLECTION CO:COLLECTION_SUMMARY Lipids were extracted using the MTBE method (Gil et al., 2018). The bacterial CO:COLLECTION_SUMMARY cell pellet was resuspended into 75 μL water, then it was incubated with 200 CO:COLLECTION_SUMMARY μL methanol and 625 μL MTBE (methyl tert-butyl ether) for 1 h on a shaker. The CO:COLLECTION_SUMMARY bacteria/methanol/MTBE mixture was further incubated with 100 μL water to CO:COLLECTION_SUMMARY induce phase separation. After 10 min centrifugation at 1000 × g, the upper CO:COLLECTION_SUMMARY organic phase was collected. The lower phase was re-extracted with 250 μL CO:COLLECTION_SUMMARY MTBE/methanol/water 10:3:2.5 v/v/v and incubated on a shaker for 30 min. After 3 CO:COLLECTION_SUMMARY min centrifugation at 1000 × g, the upper organic phase was collected. The CO:COLLECTION_SUMMARY combined organic phases containing the lipids were dried in a vacuum centrifuge CO:COLLECTION_SUMMARY at 30 ºC and then dissolved in 25 μL CHCl3/methanol/water 60:30:4.5 v/v/v. 75 CO:COLLECTION_SUMMARY μL isopropanol/acetonitrile/water 2:1:1 v/v/v was added to dilute the lipid CO:COLLECTION_SUMMARY solution. CO:SAMPLE_TYPE Bacteria CO:COLLECTION_LOCATION University Medical Center Groningen (UMCG) #TREATMENT TR:TREATMENT_SUMMARY The diluted lipid solution was separated using a Zorbax Eclipse Plus C18 column TR:TREATMENT_SUMMARY (1.8 μm, 50 × 2.1 mm) on an Acquity UPLC system (Waters, Manchester, UK). TR:TREATMENT_SUMMARY Mobile phases consisted of 10 mM ammonium formate in water/acetonitrile/formic TR:TREATMENT_SUMMARY acid 2:3:0.005 v/v/v (eluent A) and 10 mM ammonium formate in TR:TREATMENT_SUMMARY isopropanol/acetonitrile/formic acid 9:1:0.01 v/v/v (eluent B). Linear gradient TR:TREATMENT_SUMMARY elution was as follows: 0– 2 min from 40 to 43% eluent B, 2–12 min from 50 TR:TREATMENT_SUMMARY to 54% eluent B, 12-18 min from 70 to 99% eluent B, and 18–20 min 40% eluent TR:TREATMENT_SUMMARY B.. The column temperature was set at 55 °C, and the flow rate was 0.4 mL/min. TR:TREATMENT_SUMMARY One μL of sample was loaded to MaXis plus high-resolution QTof mass TR:TREATMENT_SUMMARY spectrometer (Bruker, Bremen, Germany), Lipids were detected by electrospray TR:TREATMENT_SUMMARY ionization in positive (ESI+) and negative mode (ESI-). #SAMPLEPREP SP:SAMPLEPREP_SUMMARY F. prausnitzii A2-165 (DSM 17677) was inoculated in YCFAG medium (Lopez-Siles et SP:SAMPLEPREP_SUMMARY al, 2012), without (CP0) and with 0.01 mg/mL CP (CP2) (Wang et al., 2021) in an SP:SAMPLEPREP_SUMMARY anaerobic chamber (80% N2, 12% CO2, and 8% H2) and incubated in the same chamber SP:SAMPLEPREP_SUMMARY at 37°C. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY The diluted lipid solution was separated using a Zorbax Eclipse Plus C18 column CH:CHROMATOGRAPHY_SUMMARY (1.8 μm, 50 × 2.1 mm) on an Acquity UPLC system (Waters, Manchester, UK). CH:CHROMATOGRAPHY_SUMMARY Mobile phases consisted of 10 mM ammonium formate in water/acetonitrile/formic CH:CHROMATOGRAPHY_SUMMARY acid 2:3:0.005 v/v/v (eluent A) and 10 mM ammonium formate in CH:CHROMATOGRAPHY_SUMMARY isopropanol/acetonitrile/formic acid 9:1:0.01 v/v/v (eluent B). Linear gradient CH:CHROMATOGRAPHY_SUMMARY elution was as follows: 0– 2 min from 40 to 43% eluent B, 2–12 min from 50 CH:CHROMATOGRAPHY_SUMMARY to 54% eluent B, 12-18 min from 70 to 99% eluent B, and 18–20 min 40% eluent CH:CHROMATOGRAPHY_SUMMARY B.. The column temperature was set at 55 °C, and the flow rate was 0.4 mL/min. CH:CHROMATOGRAPHY_SUMMARY One μL of sample was loaded to MaXis plus high-resolution QTof mass CH:CHROMATOGRAPHY_SUMMARY spectrometer (Bruker, Bremen, Germany), Lipids were detected by electrospray CH:CHROMATOGRAPHY_SUMMARY ionization in positive (ESI+) and negative mode (ESI-). CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Waters Acquity CH:COLUMN_NAME Agilent Zorbax Eclipse Plus C18 (100 x 2.1mm, 1.8 um) #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Bruker maXis Impact qTOF MS:INSTRUMENT_TYPE QTOF MS:MS_TYPE ESI MS:ION_MODE NEGATIVE MS:MS_COMMENTS Peak detection and statistics. The MSConvert (Version: 3.0.18234) tool from the MS:MS_COMMENTS ProteoWizard package was used to convert the raw files from the Bruker QToF into MS:MS_COMMENTS .mzML files. MS:MS_RESULTS_FILE ST001987_AN003239_Results.txt UNITS:Peak area Has m/z:Yes Has RT:Yes RT units:Seconds #END