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The molecular basis of bacteria adhesion to gastrointestinal mucus

Reference	BBS/E/F/00042698
Principal Investigator / Supervisor	Professor Nathalie Juge
Co-Investigators / Co-Supervisors
Institution	Quadram Institute Bioscience
Department	Quadram Institute Bioscience Department
Funding type	Research
Value (£)	143,971
Status	Completed
Type	Institute Project
Start date	06/12/2013
End date	05/12/2016
Duration	35 months

Abstract

The overall aim of the work is to provide a mechanistic understanding of how commensal gut bacteria bind to mucus and communicate with intestinal cells underlying the mucus layer, focusing on the role of mucus binding proteins (MUB) from Lactobacillus reuteri in this process. In Objective 1, we will gain detailed ligand recognition of MUB-mucus interaction in vitro using heterologously expressed Mub repeats. Following screening of mucin and glycan microarrays, we will use surface plasmon resonance and isothermal calorimetry to define the thermodynamics and kinetics of the interactions. We will determine the glycan structures recognised by MUB and Mub repeats by mass spectrometry. Other MUB interactions important for intestinal homeostasis are with mucosal IgA. Here X-ray crystallography and mutagenesis will be used to gain structural details and identify key residues of the interaction. In Objective 2, we will extend the analysis of MUB recognition to cell and tissue culture models to determine whether binding is dependent on sugar-binding activity and of tissue and host specificity. Here we will utilise native MUB isolated from bacteria and immunofluorescence to assess binding to mucus from animal and mouse intestinal tissue sections and human intestinal cell models. Specificity of binding will be carried out following differential chemical or enzymatic treatment altering mucin glycosylation, as determined using lectin-mapping. In Objective 3, we will move to whole bacteria adhesion assays to help determine the contribution of MUB-mediated mucin and IgA to the interaction of the bacteria to mucus. In vitro and ex-vivo adhesion assays will be performed using L. reuteri wild-type and mutant strains for MUB cell-surface expression. We will determine the consequence of MUB and IgA on the bacteria adhesion to mucus, biofilm formation and host responses (cytokine and mucins) by fluorescence-based assays and quantitative PCR.

Summary

unavailable

Committee	Not funded via Committee
Research Topics	Diet and Health, Microbiology, Structural Biology
Research Priority	X – Research Priority information not available
Research Initiative	X - not in an Initiative
Funding Scheme	X – not Funded via a specific Funding Scheme

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