The molecular basis of bacteria adhesion to gastrointestinal mucus
Lead Research Organisation:
QUADRAM INSTITUTE BIOSCIENCE
Department Name: UNLISTED
Abstract
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Technical Summary
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.
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.
Planned Impact
unavailable
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ORCID iD |
| Nathalie Juge (Principal Investigator) |