Characterisation of Campylobacter jejuni toxins and their effects on cultured mammalian cells

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Spears, K., 2002. Characterisation of Campylobacter jejuni toxins and their effects on cultured mammalian cells. PhD, Nottingham Trent University.

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Abstract

Campylobacter jejuni and other related strains are important pathogens in human enterocolitis, the most common bacterial cause of diarrhoea in the industrialised countries. In order to achieve a better level of understanding towards the role of toxins in Campylobacter infection the aims of this thesis were; to determine the optimal toxin producing Campylobacter jejuni strain from the three strains made available (namely; 11351, 11322, 11168), to optimise bacterial culture conditions for toxin production; to initially characterise the sub lethal effects of culture extracts with regards to their effects on cell morphology and cytoskeletal networks; to purify a novel Campylobacter jejuni toxin for N-terminal sequencing and characterisation at the molecular level.

Studies of mammalian cytotoxicity suggested that the bacterial strain Campylobacter jejuni 11351 was the most consistently toxic compared to other strains. BHI broth culture supernatants and the blood agar cell sonicates produced the most consistent cytotoxic effects on cultured mammalian cell lines (N2a and ECV). Results of cytotoxicity assays indicated that two types of toxins were being produced in the two bacterial culture conditions. Firstly, there was a secreted heat/trypsin labile exotoxin produced by bacteria grown in BHI broth cultures, and secondly, a heat/trypsin stable cell-attached endotoxin produced by bacteria grown on blood agar plates.

Sub-cytotoxic effects levels of the bacterial broth culture supernatant caused elongation of cell bodies in CHO cells, suggesting the presence of a cytolethal distending toxin (CDT). When introduced to ECV cells the toxin induced cell body rounding, suggesting the presence of a cytolethal rounding toxin (CRT). Neurotoxic effects, as demonstrated by reduction in the outgrowth of axon like neurites, were observed in N2a cells treated with both BHI broth culture supernatant and blood agar cell sonicate. These effects were heat sensitive for the former but heat resistant for the latter, suggesting a secreted proteinaceous neurotoxic exotoxin and an endotoxin, respectively. Probing of western blots of cell extracts treated with sub-cytotoxic levels of toxins revealed changes in the levels of microtubule, intermediate filament and microfilament proteins. In the case of BHI extracts, toxicity was also associated with disruption of microtubule and microfilament networks, as determined by confocal fluorescence microscopy.

N-terminal sequencing a 50 kDa polypeptide purified from blood agar cell sonicates by a combination of ion exchange chromatography and electro elution revealed a sequence which, when compared with known protein sequences using BLAST and LALIGN databanks, showed a most likely match for the major outer membrane protein reported as a cytotoxic porin.

In order to fully determine the identity and mechanism of action of the toxin, further work is required in the purification process, along with further characterisation of the toxin activity at the molecular level.

Item Type:

Thesis

Creators:

Spears, K.

Date:

2002

ISBN:

9781369313864

Identifiers: