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.

炎症性半胱天冬酶-11/4/5（caspase-11/4/5）可识别入侵革兰氏阴性菌带来的胞质脂多糖（lipopolysaccharide，LPS），并诱导细胞焦亡与细胞因子释放，构成快速的天然抗菌防御体系。此前学界普遍认为胞外或被囊泡限制的细菌极少能进入细胞质，因此其脂多糖如何被胞质感受器识别，是病原体识别过程中的关键环节。溶血素是一类成孔细菌毒素，经典观点认为其通过破坏细胞膜引发细胞裂解。然而，溶血素是否以及如何参与非经典炎性体信号通路，目前仍未明确。本研究发现，过表达溶血素的肠杆菌可显著激活人类肠上皮细胞系中的半胱天冬酶-4。溶血素可通过动力蛋白依赖的内吞作用，促进胞外细菌的脂多糖向胞质递送。进一步研究表明，溶血素主要结合于细菌外膜囊泡（outer membrane vesicles，OMVs），并诱导携带外膜囊泡的囊泡破裂，进而增加脂多糖对胞质感受器的暴露。在小鼠实验中，过表达溶血素可促进半胱天冬酶-11依赖的IL-18分泌与肠道炎症，这一现象与体内细菌定植受抑制相关。综上，本研究揭示了一种全新机制：溶血素通过释放外膜囊泡以介导胞质脂多糖的感知，从而促进非经典炎性体活化，这为肠道抗菌防御中通过半胱天冬酶-11/4对失调溶血素的天然免疫监视提供了全新见解。