Summary of Study ST002094

This data is available at the NIH Common Fund's National Metabolomics Data Repository (NMDR) website, the Metabolomics Workbench, https://www.metabolomicsworkbench.org, where it has been assigned Project ID PR001102. The data can be accessed directly via it's Project DOI: 10.21228/M8840R This work is supported by NIH grant, U2C- DK119886.

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Study IDST002094
Study TitleCommensal intestinal microbiota regulates host luminal proteolytic activity and intestinal barrier integrity through β-glucuronidase activity (Part 1)
Study TypeMS
Study SummaryProteases constitute the largest enzyme gene family in vertebrates with intracellular and secreted proteases having critical roles in cellular and organ physiology. Intestinal tract contains diverse set of proteases mediating digestion, microbial responses, epithelial and immune signaling. Transit of chyme through the intestinal tract results in significant suppression of proteases. Although endogenous protease inhibitors have been identified, the broader mechanisms underlying protease regulation in the intestinal tract remains unclear. The objective of this study was to determine microbial regulation of proteolytic activity in intestinal tract using phenotype of post-infection irritable bowel syndrome, a condition characterized by high fecal proteolytic activity. Proteases of host pancreatic origin (chymotrypsin like pancreatic elastase 2A, 3B and trypsin 2) drove proteolytic activity. Of the 14 differentially abundant taxa, high proteolytic activity state was characterized by complete absence of the commensal Alistipes putredinis. Germ free mice had very high proteolytic activity (10-fold of specific-pathogen free mice) which dropped significantly upon humanization with microbiota from healthy volunteers. In contrast, high proteolytic activity microbiota failed to inhibit it, a defect that corrected with fecal microbiota transplant as well as addition of A. putredinis. These mice also had increased intestinal permeability similar to that seen in patients. Microbiota β-glucuronidases mediate bilirubin deconjugation and unconjugated bilirubin is an inhibitor of serine proteases. We found that high proteolytic activity patients had lower urobilinogen levels, a product of bilirubin deconjugation. Mice colonized with β-glucuronidase overexpressing E. coli demonstrated significant inhibition of proteolytic activity and treatment with β-glucuronidase inhibitors increased it. The findings establish that specific commensal microbiota mediates effective inhibition of host pancreatic proteases and maintains intestinal barrier function through the production of β-glucuronidases. This suggests an important homeostatic role for commensal intestinal microbiota.
Institute
Mayo Clinic
DepartmentBiomedical Statistics and Informatics
Last NameDasari
First NameSurendra
Address200 First Street SW, Rochester, MN, 55905, USA
EmailDasari.Surendra@mayo.edu
Phone507-284-0513
Submit Date2022-02-17
Num Groups2
Total Subjects7
Raw Data AvailableYes
Raw Data File Type(s)d
Analysis Type DetailLC-MS
Release Date2022-03-09
Release Version1
Surendra Dasari Surendra Dasari
https://dx.doi.org/10.21228/M8840R
ftp://www.metabolomicsworkbench.org/Studies/ application/zip

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Project:

Project ID:PR001102
Project DOI:doi: 10.21228/M8840R
Project Title:Fecal Metabolomics
Project Type:Untargeted MS of mice fecal samples
Project Summary:Proteases constitute the largest enzyme gene family in vertebrates with intracellular and secreted proteases having critical roles in cellular and organ physiology. Intestinal tract contains diverse set of proteases mediating digestion, microbial responses, epithelial and immune signaling. Transit of chyme through the intestinal tract results in significant suppression of proteases. Although endogenous protease inhibitors have been identified, the broader mechanisms underlying protease regulation in the intestinal tract remains unclear. The objective of this study was to determine microbial regulation of proteolytic activity in intestinal tract using phenotype of post-infection irritable bowel syndrome, a condition characterized by high fecal proteolytic activity. Proteases of host pancreatic origin (chymotrypsin like pancreatic elastase 2A, 3B and trypsin 2) drove proteolytic activity. Of the 14 differentially abundant taxa, high proteolytic activity state was characterized by complete absence of the commensal Alistipes putredinis. Germ free mice had very high proteolytic activity (10-fold of specific-pathogen free mice) which dropped significantly upon humanization with microbiota from healthy volunteers. In contrast, high proteolytic activity microbiota failed to inhibit it, a defect that corrected with fecal microbiota transplant as well as addition of A. putredinis. These mice also had increased intestinal permeability similar to that seen in patients. Microbiota β-glucuronidases mediate bilirubin deconjugation and unconjugated bilirubin is an inhibitor of serine proteases. We found that high proteolytic activity patients had lower urobilinogen levels, a product of bilirubin deconjugation. Mice colonized with β-glucuronidase overexpressing E. coli demonstrated significant inhibition of proteolytic activity and treatment with β-glucuronidase inhibitors increased it. The findings establish that specific commensal microbiota mediates effective inhibition of host pancreatic proteases and maintains intestinal barrier function through the production of β-glucuronidases. This suggests an important homeostatic role for commensal intestinal microbiota.
Institute:Mayo Clinic
Department:Biomedical Statistics and Informatics
Laboratory:ENSP
Last Name:Grover
First Name:Madhu
Address:200 First Street SW, Rochester, MN, 55905, USA
Email:Dasari.Surendra@mayo.edu
Phone:507-284-0513

Subject:

Subject ID:SU002178
Subject Type:Human
Subject Species:Homo sapiens
Taxonomy ID:9606
Gender:Male and female

Factors:

Subject type: Human; Subject species: Homo sapiens (Factor headings shown in green)

mb_sample_id local_sample_id Group
SA200941ms5520-9Case-High PA PI-IBS
SA200942ms5520-7Case-High PA PI-IBS
SA200943ms5520-5Case-High PA PI-IBS
SA200944ms5520-4Case-Low PA PI-IBS
SA200945ms5520-13Case-Low PA PI-IBS
SA200946ms5520-11Case-Low PA PI-IBS
SA200947ms5520-3Case-Low PA PI-IBS
Showing results 1 to 7 of 7

Collection:

Collection ID:CO002171
Collection Summary:Fecal supernatants (FSNs) were made fresh prior to each experiment. Feces from patients (0.1g) or mice (1 pellet) was added to 0.8mL of phosphate buffered saline (PBS) and subsequently homogenized with a pellet pestle for 5-10 seconds (Sigma-Aldrich, St. Louis, MO, USA). Homogenates were spun twice at 5,000 g for 10 min at 4°C and then added to a 0.22 µm Spin-X tube filter (Corning Life Sciences, Durham, NC, USA). Samples were filtered at 4°C, 10,000 g for 5 min and FSN was stored on ice until use.
Sample Type:Feces

Treatment:

Treatment ID:TR002190
Treatment Summary:A total of 52 PI-IBS patients defined by Rome III criteria and 38 healthy volunteers were recruited. Those with a history of abdominal surgery (except hernia, C-section, hysterectomy, appendectomy or cholecystectomy), inflammatory bowel disease, microscopic colitis, or celiac disease were excluded. Additionally, recruited volunteers were not pregnant at the time of the study. Use of tobacco or alcohol for the duration of the study was prohibited. Following medications were prohibited 7 days prior to study participation: those affecting gastrointestinal transit, serotonergic agents, anti-cholinergic agents, antimuscarinics, narcotics, peppermint oil, antibiotics or new probiotics. Ingestion of artificial sweeteners such as SplendaTM (sucralose), Nutrasweet TM (aspartame), lactulose or mannitol was prohibited for 2 days before the start and during the study. All subjects taking part in the study were asked to complete the Hospital Anxiety and Depression Scale (HADS) and a 7-day bowel diary. All participants completed the Hospital anxiety and depression scale (HADS). PI-IBS patients also completed the Symptom Checklist-90 (SCL-90), IBS Symptom severity scale (IBS-SSS), IBS-quality of life (IBS-QoL) questionnaire as well as the Long Bowel Disease questionnaire (BDQ). Mayo Clinic Institutional Review Board approved human studies and all participants provided a written informed consent (IRB protocol: 12-006529; ClinicalTrials.gov identifier: NCT03266068).

Sample Preparation:

Sampleprep ID:SP002184
Sampleprep Summary:Fecal samples were deproteinized with six times volume of cold acetonitrile:methanol (1:1 ratio), kept on ice with intermittent vortexing for 30 minutes at 4C, then centrifuged at 18000xg. 13C6-phenylalanine (3 µl at 250ng/µl) was added as internal standard to each sample prior to deproteinization. The supernatants were divided into 2 aliquots and dried down for analysis on a Quadrupole Time-of-Flight Mass Spectrometer (Agilent Technologies 6550 Q-TOF) coupled with an Ultra High Pressure Liquid Chromatograph (1290 Infinity UHPLC Agilent Technologies). Profiling data were acquired under both positive and negative electrospray ionization conditions over a mass range of 100 - 1200 m/z at a resolution of 10,000-35,000 (separate runs). Metabolite separation was achieved using two columns of differing polarity, a hydrophilic interaction column (HILIC, ethylene-bridged hybrid 2.1 x 150 mm, 1.7 mm; Waters) and a reversed-phase C18 column (high-strength silica 2.1 x 150 mm, 1.8 mm; Waters). For each column, the run time is 20 min using a flow rate of 400 ul/min. A total of four runs per sample will be performed to give maximum coverage of metabolites. Samples were injected in duplicate or triplicate, and a quality control sample, made up of a subset of samples from the study was injected several times during a run. All raw data files obtained were converted to compound exchange file format using Masshunter DA reprocessor software (Agilent). Mass Profiler Professional (Agilent) was used for data alignment and to convert each metabolite feature (m/z x intensity x time) into a matrix of detected peaks for compound identification.

Combined analysis:

Analysis ID AN003420 AN003421 AN003422
Analysis type MS MS MS
Chromatography type Reversed phase HILIC HILIC
Chromatography system Agilent 1290 Infinity Agilent 1290 Infinity Agilent 1290 Infinity
Column Waters Acquity BEH HSS T3 (100 x 2.1mm, 1.8um) Waters Acquity BEH HILIC (150 x 2.1mm, 1.7um) Waters Acquity BEH HILIC (150 x 2.1mm, 1.7um)
MS Type ESI ESI ESI
MS instrument type QTOF QTOF QTOF
MS instrument name Agilent 6550 QTOF Agilent 6550 QTOF Agilent 6550 QTOF
Ion Mode POSITIVE POSITIVE NEGATIVE
Units raw intensity raw intensity raw intensity

Chromatography:

Chromatography ID:CH002530
Chromatography Summary:Reversed phase
Instrument Name:Agilent 1290 Infinity
Column Name:Waters Acquity BEH HSS T3 (100 x 2.1mm, 1.8um)
Chromatography Type:Reversed phase
  
Chromatography ID:CH002531
Chromatography Summary:HILIC
Instrument Name:Agilent 1290 Infinity
Column Name:Waters Acquity BEH HILIC (150 x 2.1mm, 1.7um)
Chromatography Type:HILIC
  
Chromatography ID:CH002532
Chromatography Summary:HILIC
Instrument Name:Agilent 1290 Infinity
Column Name:Waters Acquity BEH HILIC (150 x 2.1mm, 1.7um)
Chromatography Type:HILIC

MS:

MS ID:MS003185
Analysis ID:AN003420
Instrument Name:Agilent 6550 QTOF
Instrument Type:QTOF
MS Type:ESI
MS Comments:None
Ion Mode:POSITIVE
  
MS ID:MS003186
Analysis ID:AN003421
Instrument Name:Agilent 6550 QTOF
Instrument Type:QTOF
MS Type:ESI
MS Comments:None
Ion Mode:POSITIVE
  
MS ID:MS003187
Analysis ID:AN003422
Instrument Name:Agilent 6550 QTOF
Instrument Type:QTOF
MS Type:ESI
MS Comments:None
Ion Mode:NEGATIVE
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