Bacterial serine protease EspP induces differentiation and expansion of enteroendocrine cells in human colonoids.

Extracellular serine protease, plasmid-encoded (EspP) is a potent serine protease that functions as a cytotoxin and is secreted by enterohemorrhagic Escherichia coli (EHEC). This bacterial toxin can cause significant colonic epithelial damage and mimic the pathophysiology of EHEC infection on human colonic organoids. However, transcriptomic changes within the intestinal epithelial cells and impacts on cell proliferation and differentiation are largely unknown. Here, using human colonic organoids, we defined the molecular and cellular changes that occur in response to acute EspP exposure, independent of microbiota, stroma, and immune cells. We performed droplet-based single-cell RNA sequencing to determine changes in epithelial cell lineages in response to EspP and demonstrated that there is a disruption in proliferation and a preferential differentiation of enteroendocrine cells (EECs). Higher-resolution clustering revealed that the de novo EECs were primarily enterochromaffin or EEC progenitors. Interestingly, EECs induced from EspP treatment displayed an immune profile, expressing cytokines and chemokines associated with macrophage or neutrophil recruitment. These findings demonstrate that EspP, a potent EHEC cytotoxin, stimulates transcriptomic changes previously attributed to the whole bug infection and highlights the significance of crypts-based proliferative cells and secretory cell differentiation as major colonic responses to bacterial pathogens.NEW & NOTEWORTHY EspP, a bacterial serine protease secreted by enterohemorrhagic E. coli, can mimic the intestinal pathophysiology of EHEC in the absence of the bacteria. Using single-cell RNA sequencing, this study identified enteroendocrine cells as early responders to EspP treatment, with preferential hyperplasia of enterochromaffin cells. EspP-induced enteroendocrine cells also expressed immune cytokines and chemokines. This highlights the importance of enteroendocrine cell responses to bacterial pathogens and intestinal pathophysiology during infection.