Bile Salts and Bacterial Bile Salt Hydrolase Activity Influence Escherichia coli Colonization and FXR Signaling in Colorectal Cancer Organoids.

Bile salts are key regulators of intestinal homeostasis and host-microbe interactions in the intestine. While bile salt hydrolase (BSH) activity is known to modulate host metabolism, the direct interplay between bile salt signaling, microbial BSH activity, bile acid signaling, and epithelial responses in colorectal cancer remains poorly understood. Here, we established a controlled in vitro co-culture platform using isogenic murine healthy and tumor-derived colon organoids to dissect bile salt-driven epithelial-microbe responses at cellular resolution. We investigated the dynamics between the host and an engineered BSH-expressing Escherichia coli strain (EcAZ-2LsBSH) compared to its native counterpart (EcAZ-2). We found that while bile salts broadly impaired bacterial colonization, EcAZ-2LsBSH preferentially colonized tumor organoids over healthy controls. Crucially, microbial BSH activity functionally rescued host signaling in healthy organoids, driving a robust 1.5-fold upregulation of the farnesoid X receptor (FXR) protein and activating downstream cell lineage pathways. In contrast, tumor organoids exhibited an intrinsic resistance to this FXR-mediated metabolic rescue. Furthermore, we identified the epithelial surface attachment factor Hspg2 (Perlecan) as a dynamically regulated target at the host-microbe interface. While bile salt exposure increased Hspg2 expression, bacterial colonization triggered significant transcriptional suppression, revealing opposing regulatory mechanisms that govern bacterial adhesion. Together, this study demonstrates that microbial metabolism of bile acids exerts profound, context-dependent effects on epithelial signaling and barrier integrity, providing a mechanistic foundation for the development of engineered bacterial therapeutics in colorectal cancer.