Abstract Text Gut regeneration mechanisms remain an important, yet elusive, facet of intestinal injury research. The intestinal epithelium must be intact for critical gut functions to remain in place, yet injury via microbe interactions or environmental toxicants is common. EspP is a potent serine. EspP is a potent serine protease that functions as a cytotoxin and is secreted by enterohemorrhagic Escherichia coli. This bacterial toxin has been shown to cause significant colonic epithelial damage in human colonoids. Previous proteomics and single cell RNA-seq analysis of human colonoids exposed to EspP found that there is an expansion of enteroendocrine cells (EECs) at acute exposures. Therefore, our goals are to understand the effects of altered secretory lineage differentiation upon EspP exposure during EHEC infection and determine the downstream effect on intestinal macrophages. We hypothesize that EspP induced EEC differentiation leads to increased enterohormone secretion, which ultimately enhances macrophage migration and infiltration of the epithelial barrier. Human colonoids derived from healthy adult colonic biopsies (UNM IRB approved study 18-626 and 18-171) were established and shown to physiologically model the colonic epithelia. Colonoids were acutely exposed to EspP. Control and EspP-exposed colonoids (n=3 unique donors) were dissociated into single cells and processed for droplet-based single cell sequencing (scRNA-seq) or tandem mass spectrometry. The scRNA-seq data was analyzed using the Seurat pipeline and integrated with the proteomics dataset. scRNA-seq results identified a 4-fold expansion of EECs, with increases in EEC hormones 5-HT and PYY as well as a 2-fold expansion of VEGF expression. These transcriptomic changes seen were validated in colonoids (>5 biologically unique human donors) via RNA FISH and immunofluorescence. Our results demonstrate that acute EspP exposure directly induces transcriptomic and cellular changes in intestinal epithelia independent of microbiota, stroma, and immune cells. As EEC expansion is seen broadly in disease states from colorectal cancer to celiac disease, determining the role of enterohormones upon these changes, as well as the function of EECs during acute injury, will fill a gap in knowledge relevant to a wider problem of intestinal dysfunction. This work was supported by grants from the National Institutes of Health: K01DK106323, R56ES034400, P42ES025589; and AGA SURF. Date of Presentation October 17, 2024