The human intestinal organoid as a model system for enteropathogenic Escherichia coli pathogenesis
Olshefsky, Stephen Christopher (Skip)
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Escherichia coli are Gram-negative, facultative anaerobic bacteria commonly found in the intestine of humans and warm-blooded animals. E. coli can be mutualistic or pathogenic that cause diarrheal disease (diarrheagenic E. coli, DEC). The colonization of E. coli begins with the successful adherence to intestinal epithelial cell (IECs); which is mediated by a variety of colonization factors on the bacterial cell surface. This is the first and most crucial step for E. coli colonization. Therefore, valuable model systems to study E. coli should recapitulate this adherence. Several model systems have been developed and successful reproduce adherence and other important aspects of EPEC pathogenesis, but have significant limitations. However, human intestinal organoids (HIOs) are a 3-dimensional tissue culture composed of a single layer of mature, differentiated, columnar epithelial cells that surround a lumen. When compared to traditional cell cultures, animal models and ligated intestines, HIOs have the potential to be more representative human physiology. Here, we begin to demonstrate the use of HIOs as an in vitro model to study E. coli. We hypothesized that HIOs could be used to study epithelial colonization dynamics of E. coli. In this study, we established working protocols for a novel experimental approach for investigating attachment factors involved in E. coli attachment. HIOs are generated from embryonic or pluripotent stem cells into definitive endoderm that gives rise to 3-dimensional structures. These structures were routinely cultured to a diameter of approximately 3.0 mm and embedding in matrigel. An overnight culture of the prototypic EPEC strain, e2348/69 (O127:H6) was transfected with a plasmid carrying a green fluorescent protein that provided visualization by fluorescent microscopy. Approximately, 2.4 x 10 6 CFU was injected into individual HIOs using a microinjector and incubated for 12 hours at 35°C and 5% CO 2. HIOs were then fixed and either stained for fluorescence imaging or processed in 1% osmium tetroxide for examination by field emission scanning electron microscopy (FE SEM). The observations that EPEC was intimately associated with the intestinal epithelium, supports the use HIO for E. coli colonization investigations.