Dysregulated hemolysin liberates bacterial outer membrane vesicles for cytosolic lipopolysaccharide sensing.

Inflammatory caspase-11/4/5 recognize cytosolic LPS from invading Gram-negative bacteria and induce pyroptosis and cytokine release， forming rapid innate antibacterial defenses. Since extracellular or vacuole-constrained bacteria are thought to rarely access the cytoplasm， how their LPS are exposed to the cytosolic sensors is a critical event for pathogen recognition. Hemolysin is a pore-forming bacterial toxin， which was generally accepted to rupture cell membrane， leading to cell lysis. Whether and how hemolysin participates in non-canonical inflammasome signaling remains undiscovered. Here， we show that hemolysin-overexpressed enterobacteria triggered significantly increased caspase-4 activation in human intestinal epithelial cell lines. Hemolysin promoted LPS cytosolic delivery from extracellular bacteria through dynamin-dependent endocytosis. Further， we revealed that hemolysin was largely associated with bacterial outer membrane vesicles (OMVs) and induced rupture of OMV-containing vacuoles， subsequently increasing LPS exposure to the cytosolic sensor. Accordingly， overexpression of hemolysin promoted caspase-11 dependent IL-18 secretion and gut inflammation in mice， which was associated with restricting bacterial colonization in vivo. Together， our work reveals a concept that hemolysin promotes noncanonical inflammasome activation via liberating OMVs for cytosolic LPS sensing， which offers insights into innate immune surveillance of dysregulated hemolysin via caspase-11/4 in intestinal antibacterial defenses.