The small intestine is an essential organ against harmful agents and pathogens. Enteropathogenic Escherichia coli (EPEC) infects the small intestine, causing diarrheal disease, which represents a major public health issue for infants in developing countries. Extensive research using immortalized cell lines has shown EPEC disrupts host cells. However, the impact of EPEC infection on intestinal stem cells remains poorly understood. As immortalized cell lines do not replicate the cellular complexity of the human gastrointestinal epithelium, in this project, we use human intestinal organoids to investigate how EPEC affects intestinal stem cell function.
AIM: To understand changes to intestinal stem cells after infection by establishing a co-culture system with organoid-derived monolayers and EPEC.
METHODS: A co-culture system was established by growing human intestinal organoids as a 2-dimensional monolayer and carrying out infections with wild-type EPEC O127:H6 strain E2348/69 or EPEC mutants (ΔPP4/IE6 and ΔescN). Immunostaining and imaging of infected monolayers as well as an organoid formation assay were conducted to confirm infection and assess stem cell function following infection.
RESULTS: Infection with wild type EPEC or ΔPP4/IE6 mutant showed attachment to the organoid derived monolayer. ΔescN lacks the type 3 secretion system and does not adhere intimately to the cell surface. Propidium Iodide (PI) staining suggested that infection with ΔPP4/IE6 leads more cell death than wild type EPEC, which induced more cell death than ΔescN. Organoids re-established from the ΔPP4/IE6-infected monolayers exhibited worse cell viability compared to those from the ΔescN and wild type EPEC groups, suggesting their recovery was impaired.
CONCLUSION: Wild type EPEC and EPEC mutants have different effects on epithelial and stem cell function and the degree of cell death after infection was associated with reduced viability after recovery. This co-culture system provides the ability to study the response and recovery of intestinal epithelial cell populations following infection.