Lithocholic acid induces T3SS-dependent formation of invasion-competent Shigella flexneri aggregates

Shigella flexneri causes shigellosis, the second leading cause of diarrheal deaths worldwide. The pathogen invades colonic epithelial cells using a type III secretion system (T3SS) that delivers effector proteins to remodel host actin cytoskeleton. Following invasion, S. flexneri acquires actin-based motility and spreads cell to cell, driving epithelial destruction and bloody diarrhea. These intracellular infection processes have been investigated primarily using exponentially growing planktonic bacteria. However, recent animal studies revealed that S. flexneri also forms multicellular aggregates in the colonic lumen, yet the function of this extracellular phase remains unclear. Here, we show that lithocholic acid (LCA), an abundant secondary bile acid in the colon, acts as a potent signal that induces S. flexneri aggregation at physiological concentrations (≥ 50 μM). LCA-induced aggregation depends on the T3SS and its tip protein IpaD, which increases aggregate size. Compared to non-aggregating controls, LCA-induced aggregates initiate invasion by eliciting a more robust actin remodeling and rapid T3SS activation during early interactions with colonic epithelial HT-29 cells. These findings identify LCA as a luminal cue that links the extracellular aggregation with intracellular infection, through a new aggregate-mediated mode of epithelial invasion.