Characterization of the Japanese flounder NLRP3 inflammasome in restricting Edwardsiella piscicida colonization in vivo.

NLRP3 inflammasome is one of the most well-known inflammasomes in mammals, which plays critical roles in innate immunity. However, knowledge about this inflammasome in non-mammalian species, especially in teleost fish, remains rarely known. Herein, we established an Edwardsiella piscicida-head-kidney macrophages (HKMs) infection model in Japanese flounder, and found a robust caspase-1 activation and IL-1ß maturation. To characterize the upstream receptor, we established a bioinformatic screening analysis, and found an NLRP3 homolog (JfNLRP3) from Japanese flounder, which shares an overall conservative structure architecture to human NLRP3. Moreover, the JfNLRP3 can assemble JfASC through PYD-PYD domain interaction and trigger JfCaspase-1 activation and JfIL-1ß maturation. Meanwhile, the classical inflammasome activation stimulators, including nigericin, ATP or MSU, can trigger the JfCaspase-1 activation and JfIL-1ß maturation in Japanese flounder HKMs. During intraperitoneal infection of E. piscicida in Japanese flounder, we found a dynamic up-regulated transcription of JfNLRP3 and JfCaspase-1 in vivo. Furthermore, knockdown of either JfNLRP3 or JfCaspase-1 reduces the serum JfIL-1ß level, and promotes the bacterial colonization in systemic immune organs at 2 day-post infection, while overexpression of JfNLRP3 or JfCaspase-1 hampers the bacterial colonization in these organs of Japanese flounder. Taken together, our results identified the NLRP3 inflammasome paradigm in Japanese flounder, which not only providing new insight into the molecular mechanisms of teleost NLRP3 inflammasome and revealing its role in restricting bacterial infection in vivo, but also shedding light on the evolutionary of NLRP3 inflammasome in teleost.

Characterization of the inflammasome component SmASC in turbot (Scophthalmus maximus).

Apoptosis-associated speck-like protein containing a C-terminal caspase recruit domain (ASC) is an important adapter protein in the inflammasome complex that mediates inflammatory caspase activation and host innate immunity in mammals. However, the function of inflammasome components in lower vertebrate remains poorly understood. In this study, full length of SmASC was cloned from turbot (Scophthalmus maximus). Through bioinformatic analysis, we found that SmASC shares relatively high identity with ASC in bony fish. Furthermore, we found that the intact SmASC can form an oligomeric speck-like structure, while the PYD segment of SmASC can form the filamentous structure. Moreover, expression of SmASC was induced after intraperitoneal injection of Edwardsiella piscicida (E. piscicida) in vivo. To further explore the role of SmASC during infection, we constructed SmASC knockdown and overexpression models by administration of siRNA and overexpression plasmids in vivo, respectively. Expression of SmASC decreased the propagation of E. piscicida in different immune organs. In summary, our results characterize the function of SmASC in S. maximus, suggesting that the SmASC plays a critical role in turbot immune responses.

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.

EvpP inhibits neutrophils recruitment via Jnk-caspy inflammasome signaling in vivo.

Innate immunity is regulated by phagocytic cells and is critical for host control of bacterial infection. In many bacteria, the type VI secretion system (T6SS) can affect bacterial virulence in certain environments, but little is known about the mechanisms underlying T6SS regulation of innate immune responses during infection in vivo. Here, we developed an infection model by microinjecting bacteria into the tail vein muscle of 3-day-post-fertilized zebrafish larvae, and found that both macrophages and neutrophils are essential for bacterial clearance. Further study revealed that EvpP plays a critical role in promoting the pathogenesis of Edwardsiella piscicida (E. piscicida) via inhibiting the phosphorylation of Jnk signaling to reduce the expression of chemokine (CXC motif) ligand 8 (cxcl8a), matrix metallopeptidase 13 (mmp13) and interleukin-1ß (IL-1ß) in vivo. Subsequently, by utilizing Tg (mpo:eGFP+/+) zebrafish larvae for E. piscicida infection, we found that the EvpP-inhibited Jnk-caspy (caspase-1 homolog) inflammasome signaling axis significantly suppressed the recruitment of neutrophils to infection sites, and the caspy- or IL-1ß-morpholino (MO) knockdown larvae were more susceptible to infection and failed to restrict bacterial colonization in vivo. taken together, this interaction improves our understanding about the complex and contextual role of a bacterial T6SS effector in modulating the action of neutrophils during infection, and offers new insights into the warfare between bacterial weapons and host immunological surveillance.

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.

Sensing of cytosolic LPS through caspy2 pyrin domain mediates noncanonical inflammasome activation in zebrafish.

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.

Edwardsiella piscicida Type III Secretion System Effector EseK Inhibits Mitogen-Activated Protein Kinase Phosphorylation and Promotes Bacterial Colonization in Zebrafish Larvae.

Bacteria utilize type III secretion systems (T3SS) to deliver effectors directly into host cells. Hence, it is very important to identify the functions of bacterial (T3SS) effectors to understand host-pathogen interactions. Edwardsiella piscicida encodes a functional T3SS effector, EseK, which can be translocated into host cells and affect bacterial loads. Here, it was demonstrated that an eseK mutant (the ΔeseK mutant) significantly increased the phosphorylation levels of p38α, c-Jun NH2-terminal kinases (JNK), and extracellular signal-regulated protein kinases 1/2 (ERK1/2) in HeLa cells. Overexpression of EseK directly inhibited mitogen-activated protein kinase (MAPK) signaling pathways in HEK293T cells. The ΔeseK mutant consistently promoted the phosphorylation of MAPKs in zebrafish larva infection models. Further, it was shown that the ΔeseK mutant increased the expression of tumor necrosis factor alpha (TNF-α) in an MAPK-dependent manner. Importantly, the EseK-mediated inhibition of MAPKs in vivo attenuated bacterial clearance in larvae. Taken together, this work reveals that the E. piscicida T3SS effector EseK promotes bacterial infection by inhibiting MAPK activation, which provides insights into the molecular pathogenesis of E. piscicida in fish.

Morphology Tuning of Aggregation-Induced Emission Probes by Flash Nanoprecipitation: Shape and Size Effects on in Vivo Imaging.

Aggregation-induced emission (AIE) imaging probes have recently received considerable attention because of their unique property of high performance in the aggregated state and their imaging capability. However, the tendency of AIE molecules to aggregate into micron long irregular shapes, which significantly limits their application in vivo, is becoming a serious issue that needs to be addressed. Here, we introduce a novel engineering strategy to tune the morphology and size of AIE nanoaggregates, based on flash nanoprecipitation (FNP). Quinolinemalononitrile (ED) is encapsulated inside properly selected amphiphilic block copolymers of varying concentration. This leads to a variety of ED particle morphologies with different sizes. The shape and size are found to have strong influences on tumor targeting both in vitro and in vivo. The current results therefore indicate that the FNP method together with optimal choice of an amphiphilic copolymer is a universal method to systematically control the aggregation state of AIE materials and hence tune the morphology and size of AIE nanoaggregates, which is potentially useful for precise imaging at specific tumor sites.

The Bacterial T6SS Effector EvpP Prevents NLRP3 Inflammasome Activation by Inhibiting the Ca2+-Dependent MAPK-Jnk Pathway.

Inflammasome activation is an important innate immune defense mechanism against bacterial infection, and in return, bacteria express virulence determinants that counteract inflammasome activation. Many such effectors are secreted into host cells via specialized bacterial secretion systems. Here, the intracellular pathogenic bacterium Edwardsiella tarda was demonstrated to activate NLRC4 and NLRP3 inflammasomes via a type III secretion system (T3SS), and to inhibit NLRP3 inflammasome via a type VI secretion system (T6SS), indicating the antagonistic roles of these systems in inflammasome signaling. Furthermore, a non-VgrG T6SS effector, EvpP, was identified that significantly inhibited NLRP3 inflammasome activation. Subsequent studies revealed that EvpP significantly suppressed Jnk activation, thus impairing oligomerization of the inflammasome adaptor ASC. Moreover, EvpP counteracted cytoplasmic Ca2+ increase, which works upstream of Jnk activation to regulate the NLRP3 inflammasome. Finally, EvpP-mediated inflammasome inhibition promoted bacterial colonization in vivo. This work expands our understanding of bacterial T6SS in counteracting host immune responses.