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Abstract
Campylobacter ureolyticus and Campylobacter concisus are emerging pathogens that have been associated with gastrointestinal disease. Detection of these bacteria in faecal samples of patients with gastroenteritis, and intestinal biopsies and faecal samples of patients with Crohn’s disease, led us to investigate the pathogenic mechanisms used by both C. ureolyticus and C. concisus to infect host cells and cause gastrointestinal disease.
Investigation of the ability of C. ureolyticus to interact with intestinal epithelial cell lines showed that it was able to adhere to both Caco-2 and HT-29 cell lines. This was confirmed using scanning electron microscopy (ScEM), showing that C. ureolyticus UNSWCD employed a “sticky-end” mechanism to attach to the microvilli of Caco-2 cells resulting in cellular damage and microvilli degradation. In contrast, gentamicin protection assays revealed that C. ureolyticus was unable to invade either cell line, a finding confirmed by ScEM. Furthermore, addition of pro-inflammatory cytokines TNF-α and IFN-γ to these cell lines prior to infection had no effect on attachment or invasion. Investigation of the secretome of C. ureolyticus UNSWCD revealed the presence of putative virulence and colonisation factors.
Given that previous studies had demonstrated varying abilities of C. concisus strains to invade intestinal cell lines, this thesis explored the potential of C. concisus strains to survive within host cells by manipulating the autophagy process. Gentamicin protection assays revealed that autophagy inhibition resulted in 2-4 fold increases in intracellular levels of C. concisus UNSWCD within Caco-2 cells, whereas autophagy induction resulted in a reduction in intracellular levels or bacterial clearance. Confocal laser scanning microscopy showed co-localisation of the bacterium within autophagosomes, while an optimised transmission electron microscopy procedure identified intracellular bacteria persisting within autophagic vesicles. Furthermore, qPCR showed that 13 genes in the autophagy process were significantly regulated following C. concisus infection. Overall, these findings revealed that C. concisus UNSWCD elicits a dampening effect on the autophagy process.
The studies from this thesis have significantly increased our understanding of the pathogenic mechanisms used by these bacteria to interact with and manipulate host intestinal cells and potentially cause gastrointestinal disease.