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.

Highlights d E. piscicida reprograms its arginine metabolism for NLRP3 inflammasome evasion in BMDMs d E. piscicida infection increases host cytosolic arginine for polyamine synthesis d E. piscicida infection facilitates spermine accumulation in the cytosol of macrophages d Cytosolic spermine inhibits K + efflux-dependent NLRP3 inflammasome activation

Edwardsiella piscicida ( E. piscicida ) is an intracellular pathogen within a broad spectrum of hosts. Essential to E. piscicida virulence is its ability to invade and replicate inside host cells, yet the survival mechanisms and the nature of the replicative compartment remain unknown. Here, we characterized its intracellular lifestyle in non-phagocytic cells and showed that intracellular replication of E. piscicida in non-phagocytic cells is dependent on its type III secretion system but not type VI secretion system. Following internalization, E. piscicida is contained in vacuoles that transiently mature into early endosomes but subsequently bypasses the classical endosome pathway and fusion with lysosomes which depend on its T3SS. Following a rapid escape from the degradative pathway, E. piscicida was found to create a specialized replication-permissive niche characterized by endoplasmic reticulum (ER) markers. Furthermore, we found that a T3SS effector EseJ is responsible for intracellular replication of E. piscicida by preventing endosome/lysosome fusion. In vivo experiments also confirmed that EseJ is necessary for bacterial colonization of E. piscicida in the epithelial layer followed by systemic dissemination both in zebrafish and mice. Thus, this work elucidates the tactics used by E. piscicida to survive and proliferate within host non-phagocytic cells. IMPORTANCE E. piscicida is a facultative intracellular bacterium associated with septicemia and fatal infections in many animals, including fish and humans. However, little is known about its intracellular life, which is important for successful invasion of the host. The present study is the first comprehensive characterization of E. piscicida ’s intracellular life-style in host cells. Upon internalization, E. piscicida is transiently contained in Rab5-positive vacuoles, but the pathogen prevents further endosome maturation and fusion with lysosomes by utilizing a T3SS effector EseJ. In addition, the bacterium creates a specialized replication niche for rapid growth via an interaction with the ER. Our study provides new insights into the strategies used by E. piscicida to successfully establishes an intracellular lifestyle that contributes to its survival and dissemination during infection.

17Inflammatory caspase-11/4/5 recognize cytosolic LPS from invading Gram-negative bacteria and 18 induce pyroptosis and cytokine release, forming rapid innate antibacterial defenses. Since 19 extracellular or vacuole-constrained bacteria are thought to rarely access the cytoplasm, how their 20 LPS are exposed to the cytosolic sensors is a critical event for pathogen recognition. Hemolysin is 21 a pore-forming bacterial toxin, which was generally accepted to rupture cell membrane, leading to 22 cell lysis. Whether and how hemolysin participates in non-canonical inflammasome signaling 23 remains uncovered. Here, we show that hemolysin-overexpressed enterobacteria triggered 24 significantly increased caspase-4 activation in human intestinal epithelial cells (IECs). Hemolysin 25 promoted LPS cytosolic delivery from extracellular bacteria through dynamin-dependent 26 endocytosis. Further, we revealed that hemolysin was largely associated with bacterial outer 27 membrane vesicles (OMVs) and induced rupture of OMV-containing vacuoles, subsequently 28 increasing LPS exposure to the cytosolic sensor. Accordingly, overexpression of hemolysin 29 promoted caspase-11 dependent IL-18 secretion, gut inflammation, and enterocyte pyroptosis in 30 orally-infected mice, which was associated with restricting bacterial colonization in vivo. Together, 31 our work reveals a concept that hemolysin promotes noncanonical inflammasome activation via 32 liberating OMVs for cytosolic LPS sensing, which offers insights into innate immune surveillance 33 of dysregulated hemolysin via caspase-11/4 in intestinal antibacterial defenses. 34 Keywords: hemolysin, OMVs, noncanonical inflammasome, intestinal infection 35 36. CC-BY 4.0 International license It is made available under a (which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.The copyright holder for this preprint . http://dx.doi.org/10.1101/290445 doi: bioRxiv preprint first posted online Mar. 28, 2018; 3 Significance 37 Sensing of lipopolysaccharide (LPS) in the cytosol triggers non-canonical 38inflammasome-mediated innate responses. Recent work revealed that bacterial outer membrane 39 vesicles (OMVs) enables LPS to access the cytosol for extracellular bacteria. However, since 40 intracellular OMVs are generally constrained in endosomes, how OMV-derived LPS gain access 41 to the cytosol remains unknown. Here, we reported that hemolysin largely bound with OMVs and 42 entered cells through dynamin-dependent endocytosis. Intracellular hemolysin significantly 43 impaired OMVs-constrained vacuole integrity and increased OMV-derived LPS exposure to the 44 cytosolic sensor, which promoted non-canonical inflammasome activation and restricted bacterial 45 gut infections. This work reveals the role of hemolysin in promoting non-canonical inflammasome 46 activation and alerting host immune recognition, which provides insights into the more 47 sophisticated biological functions of hemolysin upon infection.

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