Year: 2011
Type: dissertation
Abstract: Campylobacter jejuni is the leading cause of gastrointestinal disease in the developed\nworld. Chemotactic motility is a pre-requisite for intestinal colonisation by C. jejuni.\nIn silico analysis of the C. jejuni NCTC 11168 genome identified homologues of 10\nchemotaxis receptor and two aerotaxis genes. Six of the ten putative Transducer-like\nproteins (Tlp 1, 2, 3, 4, 7 and 10) resemble chemoreceptors of Escherichia coli. The\naim of this project is to characterise the C. jejuni Tlp1-4 chemoreceptors. The genes\nencoding the Tlps were inactivated using an insertional inactivation strategy. Isogenic\nmutants were made in tlp1, tlp2 and tlp4; a final mutant in tlp3 could not be obtained.\nA tlp1 complement was also constructed in this work. The tlp1 mutant showed\nreduced chicken colonisation ability when tested by our collaborators. Chemotactic\nphenotypes of the tlp mutants were determined in the swarm assay; the tlp mutants\nappeared defective for chemotaxis when compared with the wild-type and non-motile\nflaAB mutant. The Capillary assay and Hard-Agar Plug (HAP) assay were developed\nas methods to ascertain the ligand specificities of the Tlp chemoreceptors under\nstudy. Unfortunately, the Capillary assay proved to be insufficiently reproducible for\neffective use with C. jejuni. The HAP procedure was optimised using a C. jejuni\nwild-type motile variant. Positive chemoattractant responses were observed in NCTC\n11168 for the first time towards a range of chemicals. Data derived from the modified\nHAP assay indicated that Tlp1 may be the receptor for serine. Chemotactic responses\ncould not be detected in the tlp2 and tlp4 mutants in the HAP assay. The signalling\ndomain of Tlp1 was purified using a polyhistidine tag and used to produce a\npolyclonal antibody. The Tlp1 primary antibody and immunofluorescence labeling\nhas shown for the first time that the Tlps cluster at the cell poles in C. jejuni.
Author: Randeep Sandhu
Language: English
Subfield: Food Science
Domain: Life Sciences
Open Access status: green
OpenAlex ID: W760241385