The NLRP6 Inflammasome Recognizes Lipoteichoic Acid and Regulates Gram-Positive Pathogen Infection.

The activator and composition of the NLRP6 inflammasome remain poorly understood. We find that lipoteichoic acid (LTA), a molecule produced by Gram-positive bacteria, binds and activates NLRP6. In response to cytosolic LTA or infection with Listeria monocytogenes, NLRP6 recruited caspase-11 and caspase-1 via the adaptor ASC. NLRP6 activation by LTA induced processing of caspase-11, which promoted caspase-1 activation and interleukin-1ß (IL-1ß)/IL-18 maturation in macrophages. Nlrp6-/- and Casp11-/- mice were less susceptible to L. monocytogenes infection, which was associated with reduced pathogen loads and impaired IL-18 production. Administration of IL-18 to Nlrp6-/- or Casp11-/- mice restored the susceptibility of mutant mice to L. monocytogenes infection. These results reveal a previously unrecognized innate immunity pathway triggered by cytosolic LTA that is sensed by NLRP6 and exacerbates systemic Gram-positive pathogen infection via the production of IL-18.

NITR12+ NK Cells Release Perforin to Mediate IgMhi B Cell Killing in Turbot (Scophthalmus maximus).

B lymphocytes engaged in humoral immunity play a critical role in combating pathogenic infections; however, the mechanisms of NK cells in regulating the responses of B cells remain largely unknown. In the present study, we established an Edwardsiella piscicida infection model in turbot (Scophthalmus maximus) and found that the production of IgM was decreased. Meanwhile, through establishing the head kidney-derived lymphocyte infection model, we revealed that the impairment of IgMhi B cells was associated with bacterial infection-induced perforin production. Interestingly, we reveal that perforin production in NK cells is tightly regulated by an inhibitory novel immune-type receptor, NITR12. Moreover, we confirm that inhibiting NITR12 can result in elevated perforin production, engaging the impairment of IgMhi B cells. Taken together, these findings demonstrate an innovative strategy of NK cells in mediating B lymphocyte killing in turbot and suggest that relieving NK cells through NITR12 might be the target for the development of efficacious vaccines.

ERF4 and MYB52 transcription factors play antagonistic roles in regulating homogalacturonan de-methylesterification in Arabidopsis seed coat mucilage.

Homogalacturonan (HG), a component of pectin, is synthesized in the Golgi apparatus in its fully methylesterified form. It is then secreted into the apoplast where it is typically de-methylesterified by pectin methylesterases (PME). Secretion and de-esterification are critical for normal pectin function, yet the underlying transcriptional regulation mechanisms remain largely unknown. Here, we uncovered a mechanism that fine-tunes the degree of HG de-methylesterification (DM) in the mucilage that surrounds Arabidopsis thaliana seeds. We demonstrate that the APETALA2/ETHYLENE RESPONSE FACTOR (AP2/ERF) transcription factor (TF) ERF4 is a transcriptional repressor that positively regulates HG DM. ERF4 expression is confined to epidermal cells in the early stages of seed coat development. The adhesiveness of the erf4 mutant mucilage was decreased as a result of an increased DM caused by a decrease in PME activity. Molecular and genetic analyses revealed that ERF4 positively regulates HG DM by suppressing the expression of three PME INHIBITOR genes (PMEIs) and SUBTILISIN-LIKE SERINE PROTEASE 1.7 (SBT1.7). ERF4 shares common targets with the TF MYB52, which also regulates pectin DM. Nevertheless, the erf4-2 myb52 double mutant seeds have a wild-type mucilage phenotype. We provide evidence that ERF4 and MYB52 regulate downstream gene expression in an opposite manner by antagonizing each other's DNA-binding ability through a physical interaction. Together, our findings reveal that pectin DM in the seed coat is fine-tuned by an ERF4-MYB52 transcriptional complex.

Caspase-11 Requires the Pannexin-1 Channel and the Purinergic P2X7 Pore to Mediate Pyroptosis and Endotoxic Shock.

The noncanonical inflammasome induced by intracellular lipopolysaccharide (LPS) leads to caspase-11-dependent pyroptosis, which is critical for induction of endotoxic shock in mice. However, the signaling pathway downstream of caspase-11 is unknown. We found that cytosolic LPS stimulation induced caspase-11-dependent cleavage of the pannexin-1 channel followed up by ATP release, which in turn activated the purinergic P2X7 receptor to mediate cytotoxicity. In the absence of P2X7 or pannexin-1, pyroptosis induced by cytosolic LPS was abrogated. Cleavage of pannexin-1 required the catalytic activity of caspase-11 and was essential for ATP release and P2X7-mediated pyroptosis. Priming the caspase-11 pathway in vivo with LPS or Toll-like receptor-3 (TLR3) agonist resulted in high mortality in wild-type mice after secondary LPS challenge, but not in Casp11(-/-), Panx1(-/-), or P2x7(-/-) mice. These results reveal a critical role for pannexin-1 and P2X7 downstream of caspase-11 for pyroptosis and susceptibility to sepsis induced by the noncanonical inflammasome.

Poly(I:C) induces anti-inflammatory response against secondary LPS challenge in zebrafish larvae.

Poly(I:C) is known as an agonist of the TLR3 receptor which could prime inflammation and elicit the host immune response, which is widely applied as adjuvant or antivirus treatment. However, the negative effects of poly(I:C) on regulating immune response to protect the host from inflammatory diseases remain largely unknown. Here, we establish an in vivo model to pre-treat zebrafish larvae with poly(I:C) at 2 dpf, then challenge them with LPS at 6 dpf, and find that poly(I:C) training could significantly alleviate the LPS challenge-induced septic shock and inflammatory phenotypes. Moreover, the poly(I:C)-trained larvae exhibit decreased number of macrophages, but not neutrophils, after secondary LPS challenge. Furthermore, training the larvae with poly(I:C) could elevate the transcripts of mTOR signaling and heighten the H3K4me3-mediated epigenetic modifications. And interestingly, we find that inhibiting the H3K4me3 modification, rather than mTOR signaling, could recover the number of macrophages in poly(I:C)-trained larvae, which is consistent with the observations of inflammatory phenotypes. Taken together, these results suggest that poly(I:C) training could induce epigenetic rewiring to mediate the anti-inflammatory response against secondary LPS challenge-induced septic shock through decreasing macrophages' number in vivo, which might expand our understanding of poly(I:C) in regulating fish immune response.

Single-Cell Transcriptomic Analysis Reveals Neutrophil as Orchestrator during ß-Glucan-Induced Trained Immunity in a Teleost Fish.

Trained immunity defines long-term memory of innate immunity based on transcriptional, epigenetic, and metabolic modifications of myeloid cells, which are characterized by elevated proinflammatory responses toward homologous or heterologous secondary stimuli in mammals. However, the evidence of trained immunity-associated immune cells and its molecular mechanism in teleost fish remains largely unknown. In this study, we established a trained immunity activation model in turbot (Scophthalmus maximus) and found that administration with ß-glucan induces protection against a bacterial infection. Through single-cell RNA sequencing to annotate 14 clusters of innate and adaptive immune cells, as well as two clusters of blood cells, from head kidney and spleen, respectively, we characterized that neutrophil displays cardinal features of trained immunity by analyzing the expression abundance of trained immunity database-related genes at the single-cell level. Subsequently, through establishing an in vivo training and in vitro neutrophil challenge model, we found that the trained neutrophils exhibit a significant elevation of the IL-1R signaling pathway after Edwardsiella piscicida infection. Furthermore, inhibition of neutrophil's IL-1R signaling pathway through anakinra treatment impaired the heightened production of reactive oxygen, nitrogen species, lactate, as well as the neutrophil extracellular traps formation and bacterial killing ability. Taken together, these findings characterized neutrophil as the orchestrator to express features of trained immunity, and revealed that the IL-1R signaling pathway plays a critical role in induction of trained immunity for bacterial clearance in teleost fish.

Multi-wavelength diffractive neural network with the weighting method.

Recently, the diffractive deep neural network (D2NN) has demonstrated the advantages to achieve large-scale computational tasks in terms of high speed, low power consumption, parallelism, and scalability. A typical D2NN with cascaded diffractive elements is designed for monochromatic illumination. Here, we propose a framework to achieve the multi-wavelength D2NN (MW-D2NN) based on the method of weight coefficients. In training, each wavelength is assigned a specific weighting and their output planes construct the wavelength weighting loss function. The trained MW-D2NN can implement the classification of images of handwritten digits at multi-wavelength incident beams. The designed 3-layers MW-D2NN achieves a simulation classification accuracy of 83.3%. We designed a 1-layer MW-D2NN. The simulation and experiment classification accuracy are 71.4% and 67.5% at RGB wavelengths. Furthermore, the proposed MW-D2NN can be extended to intelligent machine vision systems for multi-wavelength and incoherent illumination.

Characterization of ccl20a.3 and ccl20l as gene markers for Th17 cell in turbot.

T-helper 17 lymphocytes (Th17) are the most common inflammatory cells identified in mammals. However, the identification of Th17 cells and the clarification of their function in teleost fish remain largely unknown. In this study, we took advantage of the single-cell RNA sequencing-based immune cell atlas that was identified in turbot (Scophthalmus maximus), and revealed two chemokine-related genes, ccl20a.3 and ccl20l, that were specifically expressed in Th17 cells. Moreover, through immuno-fluorescence analysis, we found that CCL20a.3 or CCL20l was co-expressed with the classical makers in Th17 cells, including IL17a/f1 and IL22. Furthermore, through a Th17 lineage-specific transcription factor RORc inhibitor GSK805 treatment, we found that the expression of ccl20a.3 and ccl20l was significantly impaired, compared with other T cell markers. Besides, we also found that ccl20a.3 and ccl20l exhibited the same dynamic response with the classical markers that were identified in Th17 cells during bacterial infection. Taken together, these results provide potential gene markers for better understanding of the dynamic immune responses of Th17 cells in teleost fish.

Characterization of T-cell receptors and immunoglobulin heavy chains loci and identification of T/B cell clusters in teleost.

Bacterial disease is one of the important factors leading to economic losses in the turbot (Scophthalmus maximus) cultivation industry. T lymphocytes are major components of cellular immunity, whereas B lymphocytes produce immunoglobulins (Ig) that are key elements of humoral immune responses against infection. However, the genomic organization of genes encoding T-cell receptors (TCR) and immunoglobulin heavy chains (IgHs) in turbot remains largely unknown. In this study, abundant full-length transcripts of TCRs and IgHs were sequenced by Isoform-sequencing (Iso-seq), and we investigated and annotated the V, D, J and C gene loci of TCRα, TCRß, IgT, IgM and IgD in turbot. Furthermore, through single-cell RNA sequencing (scRNA-seq) of blood leukocytes, we confirmed that these identified TCRs and IgHs were highly expressed in T/B cell clusters, respectively. Meanwhile, we also identified the IgM+IgD+ B and IgT+ B cells with differential gene expression profiles and potential functions. Taken together, our results provide a comprehensive understanding of TCRs and IgHs loci in turbot, which will contribute to evolutionary and functional characterization of T and B lymphocytes in teleost.

Pyroptosis Mediates Neutrophil Extracellular Trap Formation during Bacterial Infection in Zebrafish.

The formation of neutrophil extracellular trap (NET) is a critical host defense when neutrophils migrate to infection sites. Pyroptosis is a newly identified programmed cell death, which is tightly regulated by inflammasome activation. However, the mechanism of pyroptotic signaling participating in NET production remains to be elucidated. In this study, the zebrafish larvae otic vesicle microinjection model was used to infect larvae with hemolysin-overexpressing Edwardsiella piscicida (EthA+), and a rapid migration of neutrophils to infection sites was observed. Intriguingly, EthA+ infection effectively induced significant neutrophil membrane rupture in vivo, which was dependent on caspase-B (caspy2) and gasdermin Eb (GSDMEb) but not caspase-A or gasdermin Ea. Specifically, the EthA+ E. piscicida infection induced pyroptosis along with NETosis in vitro, and depletion of either caspy2 or GSDMEb impaired NET formation in vivo. Consequently, inhibition of the caspy2-GSDMEb axis-gated NETosis impaired bacterial clearance in vivo. Altogether, these data provide evidence that teleost fish innate immune cells, including neutrophils, express features of pyroptosis that are critical for NETosis in teleost innate immunity.

The c-di-GMP signalling component YfiR regulates multiple bacterial phenotypes and virulence in Pseudomonas plecoglossicida.

AIMS: Pseudomonas plecoglossicida (P. plecoglossicida) is the causative agent of visceral granulomas disease in large yellow croaker (Larimichthys crocea) and it causes severe economic loss to its industry. Biofilm formation, related to intracellular cyclic bis (3'-5') diguanylic acid (c-di-GMP) levels, is essential for the lifestyle of P. plecoglossicida. This research aims to investigate the role of YfiR-a key regulator of the diguanylate cyclase YfiN to regulate c-di-GMP levels and reveal its regulatory function of bacterial virulence expression in P. plecoglossicida. METHODS AND RESULTS: A genetic analysis was carried out to identify the yfiBNR operon for c-di-GMP regulation in P. plecoglossicida. Then, we constructed a yfiR mutant and observed increased c-di-GMP levels, enhanced biofilm formation, increased exopolysaccharides, and diminished swimming and swarming motility in this strain. Moreover, through establishing a yolk sac microinjection infection model in zebrafish larvae, an attenuated phenotype of yfiR mutant that manifested as restored survival and lower bacterial colonization was found. CONCLUSIONS: YfiR is the key regulator of virulence in P. plecoglossicida, which contributes to c-di-GMP level, biofilm formation, exopolysaccharides production, swimming, swarming motility, and bacterial colonization in zebrafish model.

Bacterial infection induces pyroptotic signaling-mediated neutrophil extracellular traps (NETs) formation in turbot (Scophthalmus maximus).

Neutrophils can capture and kill pathogens by releasing neutrophils extracellular traps (NETs), which play critical roles in anti-microbial infection in mammals; however, the mechanisms involved in NETs formation and its role in anti-bacterial infection in teleost fish remains largely unknown. In this study, to explore the function of NETs in turbot, we established an in vitro bacterial infection model in head kidney derived neutrophils, and found that the haemolysin over-expressed Edwardsiella piscicida (ethA+) could induce a robust phenotype of NETs, compared with that in wild type or ethA mutant (ethA+ -ΔethA) strains. Besides, the NETosis was mediated by ethA+ -induced pyroptosis, and arms the ability of bacterial killing in neutrophils of turbot. Moreover, we found that neutrophils elastase (NE) might involves in this pyroptotic signaling, rather than inflammatory Smcaspase. Taken together, this study reveals the important role of pyroptosis in NETs formation in turbot neutrophils, suggesting that NETs formation is a critical immune response during bacterial infection in teleost fish.

Dietary supplementation of propolis enhanced the innate immune response against Edwardsiella piscicida challenge in turbot (Scophthalmus maximus).

Propolis is non-hazardous resinous substance mixture containing bioactive ingredients such as polyphenols, flavonoids and organic acid. It has been widely used as food supplement and immune adjuvant due to its benefits in anti-microbial and immunomodulation. Edwardsiella piscicida is a kind of threatening pathogen which could cause high mortality in turbot. However, whether propolis could enhance the innate immune response against E. piscicida infection in turbot remains unknown. In this study, we found dietary propolis addition could improve the expression of anti-oxidative stress related enzymes, e.g., SOD, CAT and GPT, and relieved the histopathological changes of juvenile turbot after E. piscicida infection. Moreover, propolis addition increased the expression of cytokines such as il-1ß, il-6 and tnf-α in different organs of juvenile turbot. Importantly, rescued survival and decreased bacterial loads were observed in propolis feeding group. Taken together, these findings suggest that the important roles of propolis in protecting juvenile turbot from E. piscicida infection, indicating propolis might be applied as a promising immunopotentiator candidate in aquaculture.

Simulation for multiwavelength large-aperture all-silicon metalenses in long-wave infrared.

Long-wave infrared imaging systems are widely used in the field of environmental monitoring and imaging guidance. As the core components, the long-wave infrared lenses suffer the conditions of less available materials, difficult processing, large volume and mass. Metalens composed of sub-wavelength structures is one of the most potential candidates to achieve a lightweight and planar optical imaging systems. Meanwhile, it is essential to obtain large-aperture infrared lenses with high power and high resolution. However, it is difficult to use the finite-difference time-domain method to simulate a large-aperture metalens with the diameter of 201 mm due to the large amount of computational memory and computational time required. Here, to solve the mentioned problem, we firstly propose a simulation method for designing a large-aperture metalens, which combines the finite-difference time-domain algorithm and diffraction integration. The finite-difference time-domain algorithm is used to simulate the meta-atom's transmitted complex amplitude and the one-dimensional simplification of the diffraction integral is to calculate the focused field distributions of the designed metalens. Furthermore, the meta-atom spatial multiplexing is applied to design the all-silicon metalenses with the aperture of 201 mm to realize dual-wavelength (10 and 11µm) achromatic focusing, super anomalous dispersion focusing and super normal dispersion focusing. The designed metalenses are numerically confirmed, which reveal the feasibility of all-silicon sub-wavelength structures to accomplish the multiwavelength dispersion control. The designed all-silicon metalenses have the advantage of lightweight and compact. The proposed method is effective for the development of large-aperture imaging systems in the long-wave infrared.

Zebrafish GSDMEb Cleavage-Gated Pyroptosis Drives Septic Acute Kidney Injury In Vivo.

The bacteria LPS is one of the leading endotoxins responsible for sepsis; its sensing pathway-induced pyroptosis plays an important role in innate immunity. However, excessive pyroptosis might cause immunological diseases, even multiple organ failure and death by undefined mechanisms. Given that the development of acute kidney injury (AKI) in patients with sepsis causes significant morbidity and mortality, the mechanism of pyroptosis in regulating septic AKI remains unknown. In this study, we establish a zebrafish crispant in vivo analysis model and reveal that both caspy2 and gasdermin Eb (GSDMEb) contribute to lethal LPS-induced septic shock. Meanwhile, the in vitro analysis reveals that caspy2 activation can specifically cleave GSDMEb to release its N terminus to mediate pyroptosis, which functions as GSDMD in mammals. Interestingly, we establish an in vivo propidium iodide-staining method and reveal that the caspy2-GSDMEb signaling cascade is essential for enhancing renal tubular damage during lethal LPS-induced septic shock, whereas administration of the zebrafish-specific GSDMEb-derived peptide inhibitor Ac-FEID-CMK can attenuate mortality and septic AKI in vivo. Moreover, we confirm that either caspase-11 or GSDMD deficiency decreases both inflammatory cytokines and kidney dysfunction enzyme release and prolongs survival in a murine model of septic shock. Taken together, these findings demonstrate an evolutionary executor for pyroptosis in zebrafish and reveal that the pyroptosis of renal tubular cells is a major cause of septic AKI, and also provide an ideal in vivo screening model for potential antisepsis therapeutic strategies.

NEK7 is an essential mediator of NLRP3 activation downstream of potassium efflux.

Inflammasomes are intracellular protein complexes that drive the activation of inflammatory caspases. So far, four inflammasomes involving NLRP1, NLRP3, NLRC4 and AIM2 have been described that recruit the common adaptor protein ASC to activate caspase-1, leading to the secretion of mature IL-1ß and IL-18 proteins. The NLRP3 inflammasome has been implicated in the pathogenesis of several acquired inflammatory diseases as well as cryopyrin-associated periodic fever syndromes (CAPS) caused by inherited NLRP3 mutations. Potassium efflux is a common step that is essential for NLRP3 inflammasome activation induced by many stimuli. Despite extensive investigation, the molecular mechanism leading to NLRP3 activation in response to potassium efflux remains unknown. Here we report the identification of NEK7, a member of the family of mammalian NIMA-related kinases (NEK proteins), as an NLRP3-binding protein that acts downstream of potassium efflux to regulate NLRP3 oligomerization and activation. In the absence of NEK7, caspase-1 activation and IL-1ß release were abrogated in response to signals that activate NLRP3, but not NLRC4 or AIM2 inflammasomes. NLRP3-activating stimuli promoted the NLRP3-NEK7 interaction in a process that was dependent on potassium efflux. NLRP3 associated with the catalytic domain of NEK7, but the catalytic activity of NEK7 was shown to be dispensable for activation of the NLRP3 inflammasome. Activated macrophages formed a high-molecular-mass NLRP3-NEK7 complex, which, along with ASC oligomerization and ASC speck formation, was abrogated in the absence of NEK7. NEK7 was required for macrophages containing the CAPS-associated NLRP3(R258W) activating mutation to activate caspase-1. Mouse chimaeras reconstituted with wild-type, Nek7(-/-) or Nlrp3(-/-) haematopoietic cells showed that NEK7 was required for NLRP3 inflammasome activation in vivo. These studies demonstrate that NEK7 is an essential protein that acts downstream of potassium efflux to mediate NLRP3 inflammasome assembly and activation.

Edwardsiella piscicida Interferes with Classical Endocytic Trafficking and Replicates in a Specialized Replication-Permissive Niche in Nonphagocytic Cells.

Edwardsiella piscicida is an intracellular pathogen within a broad spectrum of hosts. Essential to E. piscicida's 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 nonphagocytic cells and showed that the intracellular replication of E. piscicida in nonphagocytic cells is dependent on its type III secretion system (T3SS) but not its 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 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 the intracellular replication of E. piscicida by preventing endosome/lysosome fusion. In vivo experiments also confirmed that EseJ is necessary for bacterial colonization by E. piscicida in the epithelial layer, followed by systemic dissemination in both zebrafish and mice. Thus, this work elucidates the tactics used by E. piscicida to survive and proliferate within host nonphagocytic cells. IMPORTANCEE. 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 lifestyle 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 establish an intracellular lifestyle that contributes to its survival and dissemination during infection.

The Edwardsiella piscicida thioredoxin-like protein inhibits ASK1-MAPKs signaling cascades to promote pathogenesis during infection.

It is important that bacterium can coordinately deliver several effectors into host cells to disturb the cellular progress during infection, however, the precise role of effectors in host cell cytosol remains to be resolved. In this study, we identified a new bacterial virulence effector from pathogenic Edwardsiella piscicida, which presents conserved crystal structure to thioredoxin family members and is defined as a thioredoxin-like protein (Trxlp). Unlike the classical bacterial thioredoxins, Trxlp can be translocated into host cells, mimicking endogenous thioredoxin to abrogate ASK1 homophilic interaction and phosphorylation, then suppressing the phosphorylation of downstream Erk1/2- and p38-MAPK signaling cascades. Moreover, Trxlp-mediated inhibition of ASK1-Erk/p38-MAPK axis promotes the pathogenesis of E. piscicida in zebrafish larvae infection model. Taken together, these data provide insights into the mechanism underlying the bacterial thioredoxin as a virulence effector in downmodulating the innate immune responses during E. piscicida infection.

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.

Dysregulated haemolysin promotes bacterial outer membrane vesicles-induced pyroptotic-like cell death in zebrafish.

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 dynamin-dependent 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 pyroptotic-like 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.