Unpacking the Unknown: Characterisation of Citrobacter rodentium extracellular vesicles

Abstract

An emerging field in microbiology is the study of extracellular vesicles (EVs), which are suggested to play a role in the survival and pathogenesis of bacteria. Pathogenic strains of Escherichia coli are an important global health issue, and are known to produce extracellular vesicles. Enteropathogenic E. coli (EPEC) causes diarrhoea and high mortality in the developing world and enterohaemorrhagic E. coli (EHEC) causes sporadic outbreaks of bloody diarrhoea in the developed world. Citrobacter rodentium is a murine enteropathogen which infects mice using the same ‘modus operandi’ as EPEC and EHEC, allowing many aspects of pathogenesis to be explored in vivo. As prior studies have shown EV production by E. coli, this begs the question of whether the same is true for C. rodentium, which would provide a useful model for exploring the role of EVs in pathogenicity. In this project, I investigated the production and composition of EVs by a bioluminescent derivative of wild type C. rodentium (ICC180) when grown in rich and defined media. I hypothesise that there will be differences between the EVs produced during different growth conditions. I also compared EV production between ICC180 and a hyper-transmissible derivative of ICC180 (N4P20) to explore whether EVs may be involved in the hyper-transmissible phenotype of this isolate. I measured EV production throughout growth, and analysed the concentration, size, protein and DNA and RNA composition of the EVs following sample concentration. My results show that the concentration of EVs produced by C. rodentium in both rich and defined media is low compared to that reported for other bacterial species. I observed that there were differences between the protein profiles of C. rodentium grown in rich and defined media, suggesting a change in protein expression or packaging in response to nutrient availability. I did not oberve any differences in the concentration or composition of EVs between the two C. rodentium strains I studied. The work I present in this thesis lays the foundations for future studies of the role of EVs in vivo, using infection of mice by C. rodentium as a convenient model system for EPEC and EHEC.