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Title: The role of transducer-like proteins in Campylobacter jejuni
Authors: Elgamoudi, Bassam Abusalama
Supervisors: Ketley, Julian
Award date: 2-Mar-2016
Presented at: University of Leicester
Abstract: Campylobacter jejuni is a significant gastrointestinal pathogen of humans. Many proposed virulence factors in C. jejuni remain poorly characterised, although many studies have already identified chemotaxis as a significant virulence factor. The chemotaxis pathway allows motile bacteria to sense and migrate toward or away from different environmental signals. Transducers like Proteins (Tlps), also known as methyl accepting chemotaxis proteins (MCP), enable this pathogen to respond to changing nutrient concentrations in the intestinal environment through mediating taxis toward or away from chemoeffector stimuli. In spite of recent research to characterise the role of Tlps in chemotaxis in C. jejuni, these proteins are still not fully understood, in particular, the cytoplasmic group C Tlps (Tlp 5, 6, and 8) which have not been extensively investigated. Here, the role of group C Tlps of C. jejuni NCTC 11168 in chemotaxis, and biofilm formation were investigated. Also the localisation of those proteins was determined. The genes encoding Tlp 5, 6 and 8 were successfully mutated and complemented. Inactivation of tlps showed important phenotypes associated with motility, chemotaxis and biofilm formation. The tlp5-, Δtlp6, tlp8- mutants all showed an altered phenotype in the swarm assay. The tlp5- and tlp8- mutants swarmed significantly further than the wild-type motile-variant, whereas the Δtlp6 mutant showed no differences in spreading on plates. No significant growth differences were seen between mutants, complements and wild-type. The modified Hard-Agar Plug (t-HAP) assay and μ-slide chemotaxis assay were developed to improve the determination of ligand specificities of Tlp chemoreceptors. The t-HAP assay showed a promising quantitative result which can be used to compare different concentrations of chemoattractant in a short time. Data derived from the t-HAP assay indicated that mutation of tlp 5, 6 and 8 has a significant effect on the chemotactic response to L-serine, L-aspartate and L-proline compared to wild type. The mutant phenotypes in the μ-slide chemotaxis assay correlated with the result of the t-HAP assay, where both show that C. jejuni has preferential chemotactic patterns, with L-serine more preferred to L-proline, L-glutamate and lastly L-aspartate. The mutation of tlp5 and tlp6 change the chemotaxis patterns to L-serine and L- aspartate compared to wild type, which may be related to the adaptation of the methylation status involved in the response to chemoattracts. The tlp8- mutant showed less chemotactic response to all amino acids tested in this study compared to wild type which may relate to a link with energy taxis. For the first time the localisation of Tlps in C. jejuni using a fluorescent reporter, iLOV, has been examined where the iLOV system was expressed using different promoters. Tlp5 and Tlp8 were shown to be localised at the cell poles. In addition, this is the first report to demonstrate that c-di-GMP-responsive adaption is present in C. jejuni and that there is a link between biofilm dynamics and the level of c-di-GMP. Tlp8 was named CbdA for Campylobacter biofilm dispersion as it was found to be involved in the modulation of c-di-GMP levels to mediate biofilm dispersion. Biofilm dispersion in C. jejuni was also found to be responsive to D-amino acids. The findings in this thesis indicate that transducer like proteins (group C) have an important role not only in chemotaxis but also in biofilm formation, which may provide a basis for understanding the pathogenicity of this pathogen.
Type: Thesis
Level: Doctoral
Qualification: PhD
Rights: Copyright © the author. All rights reserved.
Appears in Collections:Leicester Theses
Theses, Dept. of Genetics

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