Cholera is an acute diarrhoeal disease caused by infection with the Gram-negative bacterium Vibrio cholerae. Infection occurs upon ingestion of this bacterium via contaminated food or water, and if left untreated can be life-threatening. In terms of incidence cholera, remains a global health concern with the World Health Organisation estimating between 1.3 to 4 million cases of cholera worldwide each year, equating to between 21,000 and 143,000 deaths. The major virulence factor for pathogenic strains of V. cholerae is cholera toxin, the presence of which is believed to illicit the profuse diarrhoea synonymous with cholera. Recent genomic data has shown that different phylogenetic lineages of V. cholerae are linked to different burdens and patterns of disease ranging from geographically restricted lineages responsible for low level sporadic cases, we term endemic disease. To a single lineage, termed 7PET, that is linked to all the major epidemics in the ongoing pandemic 7, which began in the 1960’s. The use of animals has been invaluable in understanding key virulence factors associated with epidemic disease, and to date, much of the cholera research field has been based upon animal models for bacterial colonisation and disease. Despite the wealth of research using animal models, the only known natural host of V. cholerae are humans and no studies have framed differences in phylogenetic position to pathogenicity and outcome. The main objective of my PhD thesis is to add to the existing knowledge and understanding of V. cholerae infection dynamics by developing and validating a novel human model of infection. Human induced pluripotent stem cell (HuiPSC) derived intestinal organoids were developed to understand and differentiate disease potential between defined V. cholerae lineages occupying distinct positions within the species phylogeny. I confirm that all cell types present within the small intestine are represented in the organoids cultured, that the complex architecture comprising of a basal lateral surface and an inner lumen are recreated in this organoid system. During this investigation I observed two technical anomalies of the organoid system. Firstly, two morphologically distinct organoid variants are formed during the aging and passaging process that importantly respond differently to challenge with toxin. Secondly, cholera toxin administered externally to the basolateral surface or through microinjection into the organoid lumen and so through the apical membrane elicited a similar response. During the validation of the model, I found key similarities in how both animal models and the human intestine respond to cholera toxin. For example, confocal staining and imaging shows degranulation of goblet cells, leading to release of mucus that has been shown to be a response that rabbit ilium illicit following exposure to cholera toxin. Furthermore, transcriptomic analysis reveals biomarkers linked to administration of cholera toxin which have previously been shown to be differentially regulated during natural infection in cholera patients. To test the model in V. cholerae strains representative of the species, I selected three distinct pathogenic isolates including the archetypal reference 7PET strain N16961, which has been included in the majority of V. cholerae studies to date, a classical strain which pre-dates the seventh pandemic and a strain that harbours a type three secretion system - all strains situated on different lineages. All genome differences between these representative strains were catalogued and explored for phenotypic differences in the intestinal organoid model. Immunofluorescent staining and electron microscopy revealed differences in early colonisation patterns of the representative strains with the classical strain showing distinct patterns of association with fucose residues within the intestinal organoids. Having established the model, future work would include microinjection of further pathogenic and non-pathogenic isolates and ultimately comparing both the host and pathogen response within the HuiPSC derived intestinal organoid.
