Sandhu final complete dissertation.pdf (4.95 MB)

The transducer-like proteins of Campylobacter jejuni

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thesis
posted on 24.11.2011, 10:08 by Randeep Sandhu
Campylobacter jejuni is the leading cause of gastrointestinal disease in the developed world. Chemotactic motility is a pre-requisite for intestinal colonisation by C. jejuni. In silico analysis of the C. jejuni NCTC 11168 genome identified homologues of 10 chemotaxis receptor and two aerotaxis genes. Six of the ten putative Transducer-like proteins (Tlp 1, 2, 3, 4, 7 and 10) resemble chemoreceptors of Escherichia coli. The aim of this project is to characterise the C. jejuni Tlp1-4 chemoreceptors. The genes encoding the Tlps were inactivated using an insertional inactivation strategy. Isogenic mutants were made in tlp1, tlp2 and tlp4; a final mutant in tlp3 could not be obtained. A tlp1 complement was also constructed in this work. The tlp1 mutant showed reduced chicken colonisation ability when tested by our collaborators. Chemotactic phenotypes of the tlp mutants were determined in the swarm assay; the tlp mutants appeared defective for chemotaxis when compared with the wild-type and non-motile flaAB mutant. The Capillary assay and Hard-Agar Plug (HAP) assay were developed as methods to ascertain the ligand specificities of the Tlp chemoreceptors under study. Unfortunately, the Capillary assay proved to be insufficiently reproducible for effective use with C. jejuni. The HAP procedure was optimised using a C. jejuni wild-type motile variant. Positive chemoattractant responses were observed in NCTC 11168 for the first time towards a range of chemicals. Data derived from the modified HAP assay indicated that Tlp1 may be the receptor for serine. Chemotactic responses could not be detected in the tlp2 and tlp4 mutants in the HAP assay. The signalling domain of Tlp1 was purified using a polyhistidine tag and used to produce a polyclonal antibody. The Tlp1 primary antibody and immunofluorescence labeling has shown for the first time that the Tlps cluster at the cell poles in C. jejuni.
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Funding

Biotechnology and Biological Sciences Research Council (BBSRC)

History

Supervisor(s)

Ketley, Julian

Date of award

01/11/2011

Awarding institution

University of Leicester

Qualification level

Doctoral

Qualification name

PhD

Language

en

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