Mechanism Of Negative Regulation Of Host NLRP3 Inflammasome Activation By Edwardsiella Piscicida

Pathogens utilize a variety of mechanisms to invade and survive in the host to cause disease.Inflammasomes constitute multimeric intracellular complexes formed in response to a variety of physiological and pathogenic stimuli recognized by PRRs followed by recruiting downstream adaptors and effectors,which served as an essential component of the innate immune response critical for clearing pathogens or damaged cells.In particular,NLRP1,NLRP3,and NLRC4 play pivotal roles as surveillance machineries in microbial infection.Since inflammasome is an important anti-infection means of the host,increasing evidences indicated that pathogen has evolved multiple virulence mechanisms to inhibit the activation of inflammasome and thus promote infection in vivo.The fish pathogen Edwardsiella piscicida causes a range of diseases from ascites to immune organ dysfunction along with persistent systemic infection,and the edwardsiellasis caused by it has brought huge economic losses to the aquaculture industry.After the invasion of host cells via T3SS,replication occurs within an ECV,that is established and maintained by virulence effector delivery into the host cell cytosol.Whereas several virulence effectors can directly target MAP kinase signaling to promote systemic infection.Moreover,E.piscicida T6SS effector EvpP targets Ca2+-Jnk signaling to inhibit NLRP3 inflammasomes in macrophages.While the existence of additional putative NLRP3 inflammasome inhibitory mechanisms during pathogen infection remains largely unknown.We devised a screen to identify such modulators utilizing a transposon-based high-density insertion library of E.piscicida to infect nlrc4-/-BMDMs,and candidates were identified by LDH release relative to that of the wild-type E.piscicida strain.Of the top 20 candidates,four genes（ETAE₁881,ETAE₁934,ETAE₂963,and ETAE₃005）associated with bacterial arginine metabolism pathways were significantly clustered,they encode spermidine/putrescine transport system ATP-binding protein PotA,arginine:ornithine antiporter ArcD,arginine decarboxylase SpeA,and putrescine:ornithine antiporter PotE,respectively.The transcription of arginine metabolism-annotated genes was further determined in E.piscicida.Results indicated that E.piscicda arginine:ornithine antiporter ArcD,agmatinase SpeB and putrescine:ornithine antiporter PotE were upregulated during infection,which negatively regulated host NLRP3 inflammasome activation.The mechanism of negative regulation of NLRP3 inflammasome by E.piscicida was directed toward the co-localization of ECV with host arginine transporter mCAT-1 or putrescine transporter Oct-2.Furthermore,it was found that E.piscicida could capture and convert host cytosol arginine into spermine,causing intracellular spermine accumulation.Moreover,cytosolic spermine accumulation effectively inhibited E.piscicida infection-induced and K+efflux-dependent NLRP3 inflammasome activation but had no influence on K+ efflux-independent NLRP3 inflammasome activation.As a polycationic molecule,spermine could directly bind to the inward rectifying potassium ion channel,and thereby block the outflow of potassium ions without affecting their inflow.To further understand the effects of spermine accumulation on K+efflux,the intracellular K+concentration was analyzed during NLRP3 inflammasome activator treatment.Notably,elevated intracellular spermine concentration could significantly inhibit K+ efflux to restrict NLRP3 inflammasome activation.Comparing the levels of inflammasome activation and colonization in liver of E.piscicida wild-type strain as well as arginine metabolism mutant strains infected mice,the results showed that arginine metabolic reprogramming induced by E.piscicida infection promoted bacterial infection in vivo.Furthermore,when the spermine synthase gene Sms in the liver was knocked out via CRISPR/Cas9 technology,E.piscicida infection induced inflammasome activation was enhanced,accompanied by reduced bacterial colonization in liver.These results further proved that E.piscicida infection-induced arginine metabolic reprogramming and spermine accumulation could promote the survival and infection of E.piscicida in vivo.Prevention and treatment are two important factors for disease control.Revealing the pathogenic mechanism of pathogens plays an important role in antimicrobial drug screening and vaccine development.Significantly,timely detection of pathogens is also essential for disease prevention and surveillance.Based on recombinase polymerase amplification technology and lateral flow technology,we established an RPA-LF rapid detection system for E.piscicida,with well specificity and sensitivity,and the detection limit is 10 fg/μl bacterial genomic DNA.