The noncanonical inflammasome is critical for cytosolic sensing of Gram-negative pathogens. Here, we show that bacterial infection induces caspy2 activation in zebrafish fibroblasts, which mediates pyroptosis via a caspase-5-like activity. Zebrafish caspy2 binds directly to lipopolysaccharide via the N-terminal pyrin death domain, resulting in caspy2 oligomerization, which is critical for pyroptosis. Furthermore, we show that caspy2 is highly expressed in the zebrafish gut and is activated during infection. Knockdown of caspy2 expression impairs the ability of zebrafish to restrict bacterial invasion in vivo, and protects larvae from lethal sepsis. Collectively, our results identify a crucial event in the evolution of pattern recognition into the death domain superfamily-mediated intracellular lipopolysaccharide-sensing pathway in innate immunity.

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.

Bacterium usually utilises type III secretion systems (T3SS) to deliver effectors directly into host cells with the aids of chaperones. Hence, it is very important to identify bacterial T3SS effectors and chaperones for better understanding of host-pathogen interactions. Edwardsiella piscicida is an invasive enteric bacterium, which infects a wide range of hosts from fish to human. Given E. piscicida encodes a functional T3SS to promote infection, very few T3SS effectors and chaperones have been identified in this bacterium so far. Here, we reported that EseK is a new T3SS effector protein translocated by E. piscicida. Bioinformatic analysis indicated that escH and escS encode two putative class I T3SS chaperones. Further investigation indicated that EscH and EscS can enhance the secretion and translocation of EseK. EscH directly binds EseK through undetermined binding domains, whereas EscS binds EseK via its N-terminal α-helix. We also found that EseK has an N-terminal chaperone-binding domain, which binds EscH and EscS to form a ternary complex. Zebrafish infection experiments showed that EseK and its chaperones EscH and EscS are necessary for bacterial colonisation in zebrafish. This work identified a new T3SS effector, EseK, and its two T3SS chaperones, EscH and EscS, in E. piscicida, which enriches our knowledge of bacterial T3SS effector-chaperone interaction and contributes to our understanding of bacterial pathogenesis.

No abstract

Inflammasomes are important innate immune components in mammals. However, the bacterial factors modulating inflammasome activation in fish, and the mechanisms by which they alter fish immune defences, remain to be investigated. In this work, a mutant of the fish pathogen Edwardsiella piscicida (E. piscicida), called 0909I, was shown to overexpress haemolysin, which could induce a robust pyroptotic-like cell death dependent on caspase-5-like activity during infection in fish nonphagocyte cells. E. piscicida haemolysin was found to mainly associate with bacterial outer membrane vesicles (OMVs), which were internalised into the fish cells via a dynamindependent endocytosis and induced pyroptotic-like cell death. Importantly, bacterial immersion infection of both larvae and adult zebrafish suggested that dysregulated expression of haemolysin alerts the innate immune system and induces intestinal inflammation to restrict bacterial colonisation in vivo. Taken together, these results suggest a critical role of zebrafish innate immunity in monitoring invaded pathogens via detecting the bacterial haemolysin-associated OMVs and initiating pyroptoticlike cell death. These new additions to the understanding of haemolysin-mediated pathogenesis in vivo provide evidence for the existence of noncanonical inflammasome signalling in lower vertebrates. KEYWORDS bacterial outer membrane vesicles, Edwardsiella piscicida, haemolysin, pyroptotic-like cell death, zebrafish intestinal infection