Toxin-triggered activation of regulated exocytosis enhances bacterial egress from the intestinal layer

Bacterial exit from host cells is essential for dissemination yet remains poorly understood. Here, we define a non-lytic egress pathway exploited by the enteric pathogen Yersinia pseudotuberculosis that co-opts the host exocytosis machinery in intestinal epithelial cells. The bacteria secrete a CNF-family toxin that activates the Cdc42-PLC {gamma}1-IP3-IP3R signaling cascade, triggering SNARE-dependent fusion of the Yersinia-containing vacuoles with the cell membrane via VAMP7, Stx4, and SNAP23. This controlled exocytotic release preserves epithelial barrier integrity and occurs infrequently, representing a key rate-limiting step in systemic spread. These findings establish the host exocytotic machinery as an active determinant of bacterial egress, uncover a conserved vesicle trafficking pathway hijacked by intracellular pathogens for dissemination, and assign a new role for bacterial toxins in regulating host cell exit. Authors SummaryMany bacterial pathogens penetrate and cross protective host cell barriers to spread within the body. While the mechanisms by which enteric bacteria enter host cells are well characterized, far less is known about how they exit host cells after invasion. In this study, we investigated how the enteric pathogen Yersinia pseudotuberculosis exits from human gut epithelial cells after crossing them. We found that they egress at the basolateral side of the intestinal cells through a controlled, non-destructive process that preserves the integrity of the epithelial barrier. To achieve this, the bacteria co-opt host signaling pathways involved in host cell vesicle release (exocytosis) to promote controlled egress. These exit events are rare, suggesting that bacterial escape from cells is a major bottleneck during infection. Importantly, we show that this process is enhanced by a secreted toxin of the Cytotoxic Necrotizing Factor (CNF) family (CNFY), which promotes the fusion of the bacteria-containing vacuole with the basolateral cell membrane. This finding uncovers a previously unrecognized role of bacterial toxins in facilitating bacterial cell egress.