#METABOLOMICS WORKBENCH sdasari_20210311_105822 DATATRACK_ID:2521 STUDY_ID:ST001718 ANALYSIS_ID:AN002800 PROJECT_ID:PR001102 VERSION 1 CREATED_ON March 12, 2021, 9:47 am #PROJECT PR:PROJECT_TITLE Fecal Metabolomics PR:PROJECT_TYPE Untargeted MS of mice fecal samples PR:PROJECT_SUMMARY Proteases constitute the largest enzyme gene family in vertebrates with PR:PROJECT_SUMMARY intracellular and secreted proteases having critical roles in cellular and organ PR:PROJECT_SUMMARY physiology. Intestinal tract contains diverse set of proteases mediating PR:PROJECT_SUMMARY digestion, microbial responses, epithelial and immune signaling. Transit of PR:PROJECT_SUMMARY chyme through the intestinal tract results in significant suppression of PR:PROJECT_SUMMARY proteases. Although endogenous protease inhibitors have been identified, the PR:PROJECT_SUMMARY broader mechanisms underlying protease regulation in the intestinal tract PR:PROJECT_SUMMARY remains unclear. The objective of this study was to determine microbial PR:PROJECT_SUMMARY regulation of proteolytic activity in intestinal tract using phenotype of PR:PROJECT_SUMMARY post-infection irritable bowel syndrome, a condition characterized by high fecal PR:PROJECT_SUMMARY proteolytic activity. Proteases of host pancreatic origin (chymotrypsin like PR:PROJECT_SUMMARY pancreatic elastase 2A, 3B and trypsin 2) drove proteolytic activity. Of the 14 PR:PROJECT_SUMMARY differentially abundant taxa, high proteolytic activity state was characterized PR:PROJECT_SUMMARY by complete absence of the commensal Alistipes putredinis. Germ free mice had PR:PROJECT_SUMMARY very high proteolytic activity (10-fold of specific-pathogen free mice) which PR:PROJECT_SUMMARY dropped significantly upon humanization with microbiota from healthy volunteers. PR:PROJECT_SUMMARY In contrast, high proteolytic activity microbiota failed to inhibit it, a defect PR:PROJECT_SUMMARY that corrected with fecal microbiota transplant as well as addition of A. PR:PROJECT_SUMMARY putredinis. These mice also had increased intestinal permeability similar to PR:PROJECT_SUMMARY that seen in patients. Microbiota β-glucuronidases mediate bilirubin PR:PROJECT_SUMMARY deconjugation and unconjugated bilirubin is an inhibitor of serine proteases. We PR:PROJECT_SUMMARY found that high proteolytic activity patients had lower urobilinogen levels, a PR:PROJECT_SUMMARY product of bilirubin deconjugation. Mice colonized with β-glucuronidase PR:PROJECT_SUMMARY overexpressing E. coli demonstrated significant inhibition of proteolytic PR:PROJECT_SUMMARY activity and treatment with β-glucuronidase inhibitors increased it. The PR:PROJECT_SUMMARY findings establish that specific commensal microbiota mediates effective PR:PROJECT_SUMMARY inhibition of host pancreatic proteases and maintains intestinal barrier PR:PROJECT_SUMMARY function through the production of β-glucuronidases. This suggests an important PR:PROJECT_SUMMARY homeostatic role for commensal intestinal microbiota. PR:INSTITUTE Mayo Clinic PR:DEPARTMENT Biomedical Statistics and Informatics PR:LABORATORY ENSP PR:LAST_NAME Grover PR:FIRST_NAME Madhu PR:ADDRESS 200 First Street SW, Rochester, MN, 55905, USA PR:EMAIL Dasari.Surendra@mayo.edu PR:PHONE 507-284-0513 #STUDY ST:STUDY_TITLE Commensal intestinal microbiota regulates host luminal proteolytic activity and ST:STUDY_TITLE intestinal barrier integrity through β-glucuronidase activity ST:STUDY_TYPE Complex ST:STUDY_SUMMARY Proteases constitute the largest enzyme gene family in vertebrates with ST:STUDY_SUMMARY intracellular and secreted proteases having critical roles in cellular and organ ST:STUDY_SUMMARY physiology. Intestinal tract contains diverse set of proteases mediating ST:STUDY_SUMMARY digestion, microbial responses, epithelial and immune signaling. Transit of ST:STUDY_SUMMARY chyme through the intestinal tract results in significant suppression of ST:STUDY_SUMMARY proteases. Although endogenous protease inhibitors have been identified, the ST:STUDY_SUMMARY broader mechanisms underlying protease regulation in the intestinal tract ST:STUDY_SUMMARY remains unclear. The objective of this study was to determine microbial ST:STUDY_SUMMARY regulation of proteolytic activity in intestinal tract using phenotype of ST:STUDY_SUMMARY post-infection irritable bowel syndrome, a condition characterized by high fecal ST:STUDY_SUMMARY proteolytic activity. Proteases of host pancreatic origin (chymotrypsin like ST:STUDY_SUMMARY pancreatic elastase 2A, 3B and trypsin 2) drove proteolytic activity. Of the 14 ST:STUDY_SUMMARY differentially abundant taxa, high proteolytic activity state was characterized ST:STUDY_SUMMARY by complete absence of the commensal Alistipes putredinis. Germ free mice had ST:STUDY_SUMMARY very high proteolytic activity (10-fold of specific-pathogen free mice) which ST:STUDY_SUMMARY dropped significantly upon humanization with microbiota from healthy volunteers. ST:STUDY_SUMMARY In contrast, high proteolytic activity microbiota failed to inhibit it, a defect ST:STUDY_SUMMARY that corrected with fecal microbiota transplant as well as addition of A. ST:STUDY_SUMMARY putredinis. These mice also had increased intestinal permeability similar to ST:STUDY_SUMMARY that seen in patients. Microbiota β-glucuronidases mediate bilirubin ST:STUDY_SUMMARY deconjugation and unconjugated bilirubin is an inhibitor of serine proteases. We ST:STUDY_SUMMARY found that high proteolytic activity patients had lower urobilinogen levels, a ST:STUDY_SUMMARY product of bilirubin deconjugation. Mice colonized with β-glucuronidase ST:STUDY_SUMMARY overexpressing E. coli demonstrated significant inhibition of proteolytic ST:STUDY_SUMMARY activity and treatment with β-glucuronidase inhibitors increased it. The ST:STUDY_SUMMARY findings establish that specific commensal microbiota mediates effective ST:STUDY_SUMMARY inhibition of host pancreatic proteases and maintains intestinal barrier ST:STUDY_SUMMARY function through the production of β-glucuronidases. This suggests an important ST:STUDY_SUMMARY homeostatic role for commensal intestinal microbiota. ST:INSTITUTE Mayo Clinic ST:DEPARTMENT Gastroenterology ST:LAST_NAME Grover ST:FIRST_NAME Madhu ST:ADDRESS 200 First Street SW, Rochester, MN ST:EMAIL dasari.surendra@mayo.edu ST:PHONE 5072840513 ST:NUM_GROUPS 2 ST:TOTAL_SUBJECTS 16 ST:NUM_MALES 8 ST:NUM_FEMALES 8 #SUBJECT SU:SUBJECT_TYPE Mammal SU:SUBJECT_SPECIES Homo sapiens SU:TAXONOMY_ID 9606 SU:AGE_OR_AGE_RANGE 16-60 SU:WEIGHT_OR_WEIGHT_RANGE NA SU:HEIGHT_OR_HEIGHT_RANGE NA SU:GENDER Male and female #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 12 ms5520-1 Factor:control RAW_FILE_NAME=16apr15_004-r001.d (Negative HILIC), 10jun15_004.d (Positive C18), 11apr15_004-r001.d (Positive HILIC) SUBJECT_SAMPLE_FACTORS 10 ms5520-2 Factor:control RAW_FILE_NAME=16apr15_005-r001.d (Negative HILIC), 10jun15_005.d (Positive C18), 11apr15_005-r001.d (Positive HILIC) SUBJECT_SAMPLE_FACTORS 8 ms5520-3 Factor:case RAW_FILE_NAME=16apr15_006-r001.d (Negative HILIC), 10jun15_006.d (Positive C18), 11apr15_006-r001.d (Positive HILIC) SUBJECT_SAMPLE_FACTORS 5 ms5520-4 Factor:case RAW_FILE_NAME=16apr15_007-r001.d (Negative HILIC), 10jun15_007.d (Positive C18), 11apr15_007-r001.d (Positive HILIC) SUBJECT_SAMPLE_FACTORS 3 ms5520-5 Factor:case RAW_FILE_NAME=16apr15_008-r001.d (Negative HILIC), 10jun15_008.d (Positive C18), 11apr15_008-r001.d (Positive HILIC) SUBJECT_SAMPLE_FACTORS 15 ms5520-6 Factor:control RAW_FILE_NAME=16apr15_009-r001.d (Negative HILIC), 10jun15_009.d (Positive C18), 11apr15_009-r001.d (Positive HILIC) SUBJECT_SAMPLE_FACTORS 1 ms5520-7 Factor:case RAW_FILE_NAME=16apr15_010-r001.d (Negative HILIC), 10jun15_010.d (Positive C18), 11apr15_010-r001.d (Positive HILIC) SUBJECT_SAMPLE_FACTORS 6 ms5520-8 Factor:control RAW_FILE_NAME=16apr15_011-r001.d (Negative HILIC), 10jun15_011.d (Positive C18), 11apr15_011-r001.d (Positive HILIC) SUBJECT_SAMPLE_FACTORS 2 ms5520-9 Factor:case RAW_FILE_NAME=16apr15_012-r001.d (Negative HILIC), 10jun15_012.d (Positive C18), 11apr15_012-r001.d (Positive HILIC) SUBJECT_SAMPLE_FACTORS 13 ms5520-10 Factor:control RAW_FILE_NAME=16apr15_013-r001.d (Negative HILIC), 10jun15_013.d (Positive C18), 11apr15_013-r001.d (Positive HILIC) SUBJECT_SAMPLE_FACTORS 4 ms5520-11 Factor:case RAW_FILE_NAME=16apr15_014-r001.d (Negative HILIC), 10jun15_014.d (Positive C18), 11apr15_014-r001.d (Positive HILIC) SUBJECT_SAMPLE_FACTORS 7 ms5520-12 Factor:control RAW_FILE_NAME=16apr15_015-r001.d (Negative HILIC), 10jun15_015.d (Positive C18), 11apr15_015-r001.d (Positive HILIC) SUBJECT_SAMPLE_FACTORS 16 ms5520-13 Factor:case RAW_FILE_NAME=16apr15_016-r001.d (Negative HILIC), 10jun15_016.d (Positive C18), 11apr15_016-r001.d (Positive HILIC) SUBJECT_SAMPLE_FACTORS 17 ms5520-14 Factor:control RAW_FILE_NAME=16apr15_017-r001.d (Negative HILIC), 10jun15_017.d (Positive C18), 11apr15_017-r001.d (Positive HILIC) SUBJECT_SAMPLE_FACTORS 11 ms5520-15 Factor:control RAW_FILE_NAME=16apr15_018-r001.d (Negative HILIC), 10jun15_018.d (Positive C18), 11apr15_018-r001.d (Positive HILIC) SUBJECT_SAMPLE_FACTORS 9 ms5520-16 Factor:control RAW_FILE_NAME=16apr15_019-r001.d (Negative HILIC), 10jun15_019.d (Positive C18), 11apr15_019-r001.d (Positive HILIC) #COLLECTION CO:COLLECTION_SUMMARY Fecal supernatants (FSNs) were made fresh prior to each experiment. Feces from CO:COLLECTION_SUMMARY patients (0.1g) or mice (1 pellet) was added to 0.8mL of phosphate buffered CO:COLLECTION_SUMMARY saline (PBS) and subsequently homogenized with a pellet pestle for 5-10 seconds CO:COLLECTION_SUMMARY (Sigma-Aldrich, St. Louis, MO, USA). Homogenates were spun twice at 5,000 g for CO:COLLECTION_SUMMARY 10 min at 4°C and then added to a 0.22 µm Spin-X tube filter (Corning Life CO:COLLECTION_SUMMARY Sciences, Durham, NC, USA). Samples were filtered at 4°C, 10,000 g for 5 min CO:COLLECTION_SUMMARY and FSN was stored on ice until use. CO:SAMPLE_TYPE Feces #TREATMENT TR:TREATMENT_SUMMARY A total of 52 PI-IBS patients defined by Rome III criteria and 38 healthy TR:TREATMENT_SUMMARY volunteers were recruited. Those with a history of abdominal surgery (except TR:TREATMENT_SUMMARY hernia, C-section, hysterectomy, appendectomy or cholecystectomy), inflammatory TR:TREATMENT_SUMMARY bowel disease, microscopic colitis, or celiac disease were excluded. TR:TREATMENT_SUMMARY Additionally, recruited volunteers were not pregnant at the time of the study. TR:TREATMENT_SUMMARY Use of tobacco or alcohol for the duration of the study was prohibited. TR:TREATMENT_SUMMARY Following medications were prohibited 7 days prior to study participation: those TR:TREATMENT_SUMMARY affecting gastrointestinal transit, serotonergic agents, anti-cholinergic TR:TREATMENT_SUMMARY agents, antimuscarinics, narcotics, peppermint oil, antibiotics or new TR:TREATMENT_SUMMARY probiotics. Ingestion of artificial sweeteners such as SplendaTM (sucralose), TR:TREATMENT_SUMMARY Nutrasweet TM (aspartame), lactulose or mannitol was prohibited for 2 days TR:TREATMENT_SUMMARY before the start and during the study. All subjects taking part in the study TR:TREATMENT_SUMMARY were asked to complete the Hospital Anxiety and Depression Scale (HADS) and a TR:TREATMENT_SUMMARY 7-day bowel diary. All participants completed the Hospital anxiety and TR:TREATMENT_SUMMARY depression scale (HADS). PI-IBS patients also completed the Symptom Checklist-90 TR:TREATMENT_SUMMARY (SCL-90), IBS Symptom severity scale (IBS-SSS), IBS-quality of life (IBS-QoL) TR:TREATMENT_SUMMARY questionnaire as well as the Long Bowel Disease questionnaire (BDQ). Mayo Clinic TR:TREATMENT_SUMMARY Institutional Review Board approved human studies and all participants provided TR:TREATMENT_SUMMARY a written informed consent (IRB protocol: 12-006529; ClinicalTrials.gov TR:TREATMENT_SUMMARY identifier: NCT03266068). #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Fecal samples were deproteinized with six times volume of cold SP:SAMPLEPREP_SUMMARY acetonitrile:methanol (1:1 ratio), kept on ice with intermittent vortexing for SP:SAMPLEPREP_SUMMARY 30 minutes at 4C, then centrifuged at 18000xg. 13C6-phenylalanine (3 µl at SP:SAMPLEPREP_SUMMARY 250ng/µl) was added as internal standard to each sample prior to SP:SAMPLEPREP_SUMMARY deproteinization. The supernatants were divided into 2 aliquots and dried down SP:SAMPLEPREP_SUMMARY for analysis on a Quadrupole Time-of-Flight Mass Spectrometer (Agilent SP:SAMPLEPREP_SUMMARY Technologies 6550 Q-TOF) coupled with an Ultra High Pressure Liquid SP:SAMPLEPREP_SUMMARY Chromatograph (1290 Infinity UHPLC Agilent Technologies). Profiling data were SP:SAMPLEPREP_SUMMARY acquired under both positive and negative electrospray ionization conditions SP:SAMPLEPREP_SUMMARY over a mass range of 100 - 1200 m/z at a resolution of 10,000-35,000 (separate SP:SAMPLEPREP_SUMMARY runs). Metabolite separation was achieved using two columns of differing SP:SAMPLEPREP_SUMMARY polarity, a hydrophilic interaction column (HILIC, ethylene-bridged hybrid 2.1 x SP:SAMPLEPREP_SUMMARY 150 mm, 1.7 mm; Waters) and a reversed-phase C18 column (high-strength silica SP:SAMPLEPREP_SUMMARY 2.1 x 150 mm, 1.8 mm; Waters). For each column, the run time is 20 min using a SP:SAMPLEPREP_SUMMARY flow rate of 400 ul/min. A total of four runs per sample will be performed to SP:SAMPLEPREP_SUMMARY give maximum coverage of metabolites. Samples were injected in duplicate or SP:SAMPLEPREP_SUMMARY triplicate, and a quality control sample, made up of a subset of samples from SP:SAMPLEPREP_SUMMARY the study was injected several times during a run. All raw data files obtained SP:SAMPLEPREP_SUMMARY were converted to compound exchange file format using Masshunter DA reprocessor SP:SAMPLEPREP_SUMMARY software (Agilent). Mass Profiler Professional (Agilent) was used for data SP:SAMPLEPREP_SUMMARY alignment and to convert each metabolite feature (m/z x intensity x time) into a SP:SAMPLEPREP_SUMMARY matrix of detected peaks for compound identification. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY HILIC CH:CHROMATOGRAPHY_TYPE HILIC CH:INSTRUMENT_NAME Agilent 6550 CH:COLUMN_NAME Phenomenex Kinetex C18 (150 x 2.1mm, 2.6 um) #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Agilent 6550 QTOF MS:INSTRUMENT_TYPE QTOF MS:MS_TYPE ESI MS:ION_MODE POSITIVE MS:MS_COMMENTS Fecal samples were deproteinized with six times volume of cold MS:MS_COMMENTS acetonitrile:methanol (1:1 ratio), kept on ice with intermittent vortexing for MS:MS_COMMENTS 30 minutes at 4C, then centrifuged at 18000xg. 13C6-phenylalanine (3 µl at MS:MS_COMMENTS 250ng/µl) was added as internal standard to each sample prior to MS:MS_COMMENTS deproteinization. The supernatants were divided into 2 aliquots and dried down MS:MS_COMMENTS for analysis on a Quadrupole Time-of-Flight Mass Spectrometer (Agilent MS:MS_COMMENTS Technologies 6550 Q-TOF) coupled with an Ultra High Pressure Liquid MS:MS_COMMENTS Chromatograph (1290 Infinity UHPLC Agilent Technologies). Profiling data were MS:MS_COMMENTS acquired under both positive and negative electrospray ionization conditions MS:MS_COMMENTS over a mass range of 100 - 1200 m/z at a resolution of 10,000-35,000 (separate MS:MS_COMMENTS runs). Metabolite separation was achieved using two columns of differing MS:MS_COMMENTS polarity, a hydrophilic interaction column (HILIC, ethylene-bridged hybrid 2.1 x MS:MS_COMMENTS 150 mm, 1.7 mm; Waters) and a reversed-phase C18 column (high-strength silica MS:MS_COMMENTS 2.1 x 150 mm, 1.8 mm; Waters). For each column, the run time is 20 min using a MS:MS_COMMENTS flow rate of 400 ul/min. A total of four runs per sample will be performed to MS:MS_COMMENTS give maximum coverage of metabolites. Samples were injected in duplicate or MS:MS_COMMENTS triplicate, and a quality control sample, made up of a subset of samples from MS:MS_COMMENTS the study was injected several times during a run. All raw data files obtained MS:MS_COMMENTS were converted to compound exchange file format using Masshunter DA reprocessor MS:MS_COMMENTS software (Agilent). Mass Profiler Professional (Agilent) was used for data MS:MS_COMMENTS alignment and to convert each metabolite feature (m/z x intensity x time) into a MS:MS_COMMENTS matrix of detected peaks for compound identification. MS:MS_RESULTS_FILE ST001718_AN002800_Results.txt UNITS:intensity Has m/z:Neutral masses Has RT:Yes RT units:Seconds #END