Summary of Study ST002090

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.

See: https://www.metabolomicsworkbench.org/about/howtocite.php

Perform statistical analysis  |  Show all samples  |  Show named metabolites  |  Download named metabolite data  
Download mwTab file (text)   |  Download mwTab file(JSON)
Study IDST002090
Study TitleCommensal intestinal microbiota regulates host luminal proteolytic activity and intestinal barrier integrity through β-glucuronidase activity (Part 2)
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 Groups4
Total Subjects4
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

Select appropriate tab below to view additional metadata details:


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:SU002174
Subject Type:Mammal
Subject Species:Mus musculus
Taxonomy ID:10090
Gender:Female

Factors:

Subject type: Mammal; Subject species: Mus musculus (Factor headings shown in green)

mb_sample_id local_sample_id SAMPLE_DESCRIPTION
SA200579MAYO-03806Mouse; cecal post D Glucaro 1,4 lactone Female
SA200580MAYO-03809Mouse; cecal post D Glucaro 1,4 lactone Female Control
SA200577MAYO-03789Mouse; Control mono E. coli Cecum
SA200578MAYO-03792Mouse; GUS+ mono E. coli Cecum
Showing results 1 to 4 of 4

Collection:

Collection ID:CO002167
Collection Summary:Mice fecal material was collected for analysis.
Sample Type:Feces

Treatment:

Treatment ID:TR002186
Treatment Summary:Mouse: Control mono E. coli Cecum Mouse: GUS+ mono E. coli Cecum Mouse: cecal post D Glucaro 1,4 lactone Female Mouse: cecal post D Glucaro 1,4 lactone Female Control

Sample Preparation:

Sampleprep ID:SP002180
Sampleprep Summary:Fecal material was processed by metabolon as per their protocols

Combined analysis:

Analysis ID AN003414
Analysis type MS
Chromatography type Reversed phase
Chromatography system Waters Acquity
Column Waters CSH C18 (100 x 2.1mm, 1.7um)
MS Type ESI
MS instrument type Orbitrap
MS instrument name Thermo Q Exactive Orbitrap
Ion Mode POSITIVE
Units raw intensity

Chromatography:

Chromatography ID:CH002525
Chromatography Summary:Ultrahigh Performance Liquid Chromatography-Tandem Mass Spectroscopy (UPLC-MS/MS): All methods utilized a Waters ACQUITY ultra-performance liquid chromatography (UPLC) and a Thermo Scientific Q-Exactive high resolution/accurate mass spectrometer interfaced with a heated electrospray ionization (HESI-II) source and Orbitrap mass analyzer operated at 35,000 mass resolution. The sample extract was dried then reconstituted in solvents compatible to each of the four methods. Each reconstitution solvent contained a series of standards at fixed concentrations to ensure injection and chromatographic consistency. One aliquot was analyzed using acidic positive ion conditions, chromatographically optimized for more hydrophilic compounds. In this method, the extract was gradient eluted from a C18 column (Waters UPLC BEH C18-2.1x100 mm, 1.7 µm) using water and methanol, containing 0.05% perfluoropentanoic acid (PFPA) and 0.1% formic acid (FA). Another aliquot was also analyzed using acidic positive ion conditions, however it was chromatographically optimized for more hydrophobic compounds. In this method, the extract was gradient eluted from the same afore mentioned C18 column using methanol, acetonitrile, water, 0.05% PFPA and 0.01% FA and was operated at an overall higher organic content. Another aliquot was analyzed using basic negative ion optimized conditions using a separate dedicated C18 column. The basic extracts were gradient eluted from the column using methanol and water, however with 6.5mM Ammonium Bicarbonate at pH 8. The fourth aliquot was analyzed via negative ionization following elution from a HILIC column (Waters UPLC BEH Amide 2.1x150 mm, 1.7 µm) using a gradient consisting of water and acetonitrile with 10mM Ammonium Formate, pH 10.8. The MS analysis alternated between MS and data-dependent MSn scans using dynamic exclusion. The scan range varied slighted between methods but covered 70-1000 m/z. Raw data files are archived and extracted as described below.
Instrument Name:Waters Acquity
Column Name:Waters CSH C18 (100 x 2.1mm, 1.7um)
Chromatography Type:Reversed phase

MS:

MS ID:MS003179
Analysis ID:AN003414
Instrument Name:Thermo Q Exactive Orbitrap
Instrument Type:Orbitrap
MS Type:ESI
MS Comments:Ultrahigh Performance Liquid Chromatography-Tandem Mass Spectroscopy (UPLC-MS/MS): All methods utilized a Waters ACQUITY ultra-performance liquid chromatography (UPLC) and a Thermo Scientific Q-Exactive high resolution/accurate mass spectrometer interfaced with a heated electrospray ionization (HESI-II) source and Orbitrap mass analyzer operated at 35,000 mass resolution. The sample extract was dried then reconstituted in solvents compatible to each of the four methods. Each reconstitution solvent contained a series of standards at fixed concentrations to ensure injection and chromatographic consistency. One aliquot was analyzed using acidic positive ion conditions, chromatographically optimized for more hydrophilic compounds. In this method, the extract was gradient eluted from a C18 column (Waters UPLC BEH C18-2.1x100 mm, 1.7 µm) using water and methanol, containing 0.05% perfluoropentanoic acid (PFPA) and 0.1% formic acid (FA). Another aliquot was also analyzed using acidic positive ion conditions, however it was chromatographically optimized for more hydrophobic compounds. In this method, the extract was gradient eluted from the same afore mentioned C18 column using methanol, acetonitrile, water, 0.05% PFPA and 0.01% FA and was operated at an overall higher organic content. Another aliquot was analyzed using basic negative ion optimized conditions using a separate dedicated C18 column. The basic extracts were gradient eluted from the column using methanol and water, however with 6.5mM Ammonium Bicarbonate at pH 8. The fourth aliquot was analyzed via negative ionization following elution from a HILIC column (Waters UPLC BEH Amide 2.1x150 mm, 1.7 µm) using a gradient consisting of water and acetonitrile with 10mM Ammonium Formate, pH 10.8. The MS analysis alternated between MS and data-dependent MSn scans using dynamic exclusion. The scan range varied slighted between methods but covered 70-1000 m/z. Raw data files are archived and extracted as described below.
Ion Mode:POSITIVE
  logo