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Title: Hyperinvasiveness in the major food-borne pathogen Campylobacter jejuni
Author: Javed, Muhammad Afzal
ISNI:       0000 0004 2678 3437
Awarding Body: Nottingham Trent University
Current Institution: Nottingham Trent University
Date of Award: 2009
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Campylobacter jejuni is a common cause of human gastrointestinal infections. Invasion of host epithelial cells is believed to be an important virulence Mechanism of this bacterium. C. jejuni strains vary in their ability to invade the human epithelial cells and some of the strains are hyperinvasive. The aim of this work was to find the molecular basis of this hyperinvasive phenotype. The previously studied hyperinvasive phenotype of C. jejuni 01/51 in INT-407 cells was verified using Caco-2 cell based invasion assay and the assay was set up at Nottingham Trent University by selecting blood agar for 48 hours as the pre-assay bacterial growth conditions and 100 multiplicity of infection as the starting inoculum. Seven hundred and sixty eight mutants generated in this strain by random transposon insertional mutagenesis were screened for their ability to invade human intestinal epithelial cells in an in vitro model and 174 mutants were selected for further studies. The motility of selected mutants was determined and 40 mutants that showed more than 75% motility compared to wildtype strain, with a very low level of invasion were selected for reconfirmation of this reduced invasion phenotype using standard INT-407 and Caco-2 cells based invasion assays. Localisation of the transposon insertion site by plasmid rescue was attempted in 15 mutants that showed very low levels of invasion in both eukaryotic cell lines. Preliminary DNA sequencing data from these mutants has identified, amongst others: cipA; an anion-uptake ABC-transport system permease; a glycosyltransferase; a membrane protein; a capsule polysaccharide biosynthesis protein, a putative histidine triad (HIT) family protein; a putative restriction modification enzyme; a putA; a putative cytochrome C and 2 hypothetical proteins. DNA sequence from one mutant had no database match. Targeted mutagenesis was done in 6 genes to verify the reduced invasion phenotype observed in the transposon mutants and Caco-2 cell-based adhesion and invasion assays were performed. All the six targeted mutants showed reduced invasion of Caco-2 cells compared to the wildtype strain but interestingly adhesion was variable. Complementation of 4 mutated genes partially restored the reduced invasion phenotype in the mutants. The reduced invasion of the mutants into human intestinal epithelial cells was not due to a reduction in their growth, motility, atmospheric air survival, culture media or intracellular survival of the mutants. The Cj1136 mutant showed significantly reduced ability to colonise the chick gut and the gene was also found to be crucial for lipooligosaccharides biosynthesis in C. jejuni.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available