UK-IND ISPG Studentship: How sweet are our gut beneficial microbes?
Lead Research Organisation:
QUADRAM INSTITUTE BIOSCIENCE
Department Name: UNLISTED
Abstract
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Technical Summary
The aim of the project is to gain direct biochemical information on the glycosylation of cell-surface associated proteins in beneficial bacteria using the gut symbiont Lactobacillus reuteri as a model system. We will test the hypothesis that large adhesins are glycosylated via L. reuteri strain-specific secretion system. This knowledge will allow us to test the biological importance of glycans in supporting health-promoting properties of microbes and support the rational design and selection of probiotic strains.
Objective 1: to determine the repertoire of L. reuteri cell-surface associated glycoproteins. We will first assess the protein glycosylation potential of a range L. reuteri strains isolated from different hosts by glycostaining and metabolic labelling. We will assess and compare the glycoprotein profile of L. reuteri wt and derived isogenic mutants by proteomics. FPLC-based methods will be applied to enrich in glycopeptides for further analysis.
Objective 2: to identify glycosylated L. reuteri proteins and characterise the glycan structures of L. reuteri glycoproteins. The enriched glycoproteins will be separated by electrophoresis and enzymatically cleaved by in-gel digestion for MS analysis. Additional sequence information of separated proteins will be generated by tandem MS (MS/MS). Global characterisation of glycans obtained after ß-elimination of cell-surface associated glycoproteins will be performed using GC-MS and MALDI-ToF of derivatized glycans.
Objective 3: to assess the importance of protein glycosylation in functional assays Structural information from Objective 2 together with genomic analysis of L. reuteri strains will inform on the glycosylation pathways in L. reuteri and provide molecular targets for mutations and glycoengineering. L. reuteri strains with altered glycosylation profile will be tested for adherence to mucus, mucin-producing intestinal cell lines, intestinal tissues, and for aggregation phenotype by flow-cytometry.
Objective 1: to determine the repertoire of L. reuteri cell-surface associated glycoproteins. We will first assess the protein glycosylation potential of a range L. reuteri strains isolated from different hosts by glycostaining and metabolic labelling. We will assess and compare the glycoprotein profile of L. reuteri wt and derived isogenic mutants by proteomics. FPLC-based methods will be applied to enrich in glycopeptides for further analysis.
Objective 2: to identify glycosylated L. reuteri proteins and characterise the glycan structures of L. reuteri glycoproteins. The enriched glycoproteins will be separated by electrophoresis and enzymatically cleaved by in-gel digestion for MS analysis. Additional sequence information of separated proteins will be generated by tandem MS (MS/MS). Global characterisation of glycans obtained after ß-elimination of cell-surface associated glycoproteins will be performed using GC-MS and MALDI-ToF of derivatized glycans.
Objective 3: to assess the importance of protein glycosylation in functional assays Structural information from Objective 2 together with genomic analysis of L. reuteri strains will inform on the glycosylation pathways in L. reuteri and provide molecular targets for mutations and glycoengineering. L. reuteri strains with altered glycosylation profile will be tested for adherence to mucus, mucin-producing intestinal cell lines, intestinal tissues, and for aggregation phenotype by flow-cytometry.
Planned Impact
unavailable
People |
ORCID iD |
| Nathalie Juge (Principal Investigator) |