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Genotype-to-structure: analysis of the glycosylation of the flagella of Campylobacter jejuni, a major cause of food-borne gastroenteritis


Project Description

Campylobacter jejuni is the major causative agent of foodborne gastroenteritis across Europe. Contaminated chicken meat is the main source of infections and hence control of this pathogen is critical to food security in the poultry industry.

The three major virulence determinants of C. jejuni are capsule, lipooligosaccharide and flagella. The flagella controls the motility of this bacterial pathogen enabling movement towards nutrients and away from damaging agents. The flagella is decorated with sugar molecules. Some of these glycans have known virulence phenotypes such as serum resistance and autoimmunity. These glycans vary due to switches in gene expression caused by hypermutable sequences.

This project will involve a collaboration between three groups with complementary expertise in analysis of this bacterial pathogen. Dr. Bayliss’ group has worked extensively on the hypermutation phenomenon (see below). Prof. Cooper’s group have utilised FAIMS mass spectrometry to demonstrate that C. jejuni flagellin is heavily glycosylated at multiple locations with a variety of glycan types. Prof. Ketley’s group has dissected links between motility and chemotaxis, known determinants of C. jejuni virulence. Some flagella glycans have known roles in aggregation of bacterial cells, suggesting that glycans may directly influence virulence phenotypes. Equally C. jejuni flagellin is a signal for induction of innate immunity and a target of adaptive immunity, phenotypes that can be modified by the flagellin glycans. Some correlations have been established between specific genes and particular glycans and between specific glycans and phenotypes. There are however major gaps with many uncharacterised genes and limited understanding of the amount of glycan present on the flagella.

Phase variation refers to high frequency, reversible ON and OFF switching of specific phenotypes mediated by simple sequence repeats (microsatellites), which are DNA sequences with very high mutation rates. Multiple genes of C. jejuni are subject to phase variation due to mutations in polyG tracts including multiple genes encoding putative glycosylation genes. Recent unpublished data from Dr. Bayliss group indicates that alterations in expression of the phase-variable genes in the flagella locus is frequent during persistence of C. jejuni in chickens. Most of the phase-variable genes are of unknown function and have not been linked to specific glycan structures or biological phenotypes.

This project will combine genomics, molecular genetics and mass spectometry to link genotype, structural type and phenotype for glycosylation genes. A major output will be BIG data sets for use in systems biology for elucidation of a microbal glycome.

Timeline
Objective 1. Examine the strain-to-strain variation in flagella glycosylation
Year 1. 0-6 months. Interrogate Campylobacter genomic databases for distribution of the phase-variable and non-variable flagella glycosylation genes. Analyse structure of flagella glycans from 3-4 strains with diverse patterns of genes.

Objective 2. Determine genotype-to-structural type for flagellin glycosylation genes
Year 1. 7-12 months. Construct a series of mutants in flagellin glycosylation genes and in the phase-variable glycosylation genes including locked-ON and locked-OFF mutants.
Year 2. Perform structural analyses of flagellin glycosylation in mutant strains.

Objective 3. Investigate virulence phenotypes of flagellin glycosylation genes
Year 3. Compare motility and aggregation phenotypes of mutant strains. Compare adhesion and invasion phenotypes in cultured cells. Compare colonisation and disease phenotypes in chicken and mouse models. Perform a direct analysis of glycan patterns in tissue, caecal and faecal samples from model infections.

Supervision
Dr. Bayliss, genomic studies and molecular genetics; Prof. Cooper, mass spectrometry and other structural studies; Prof Ketley, motility and infection studies. Dr. Bayliss and Prof. Ketley are recipients of £410,0406 BBSRC grant for genotypic studies of C. jejuni virulence whilst Prof. Cooper has received £425,000 for a Fourier transform ion cyclotron resonance mass spectrometer from the BBSRC and is funded by EPSRC via an Established Career Fellowship

Techniques that will be undertaken during the project
Molecular Microbiological Techniques. Experimental skills will include multiplex PCR and GeneScan analyses, cloning and site-directed modification of DNA sequences, and bacterial growth. Utilisation of models of infection including cell culture and mouse/chicken models.

Genomics and Data Analysis Techniques
Genomic approaches will include analysis of large genome databases available for Campylobacter strains in collaboration with researchers in Oxford, Swansea and Aberdeen.

Structural Techniques
High resolution mass spectrometry, high field asymmetric wave from ion mobility spectrometry (FAIMS), tandem mass spectrometry, liquid chromatography, proteomics.

Available to UK/EU applicants only

Application information
https://www2.le.ac.uk/research-degrees/doctoral-training-partnerships/bbsrc
(see Food Security)

Funding Notes

4 year funded BBSRC Midlands Innovative Biosciences Training Partnership Studentship (MIBTP)
The funding provides a stipend at RCUK rates and UK/EU tuition fees for 4 years

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