Effector Identification Of Edwardsiella Piscicida And Activation Mechanism Of Host NLRP3 Inflammasome

The pathogenic bacteria devise a variety of strategies to invade the host,cause tissue damage,and avoid the immune defense to achieve their infection and pathogenesis.Many bacterial pathogens can inject virulence factors which are referred to as effectors into the host cells to target a variety of host cellular processes and promote bacterial dissemination and survival.Identify the bacterial effectors and elucidate their functions are critical to understanding the molecular pathogenesis of these pathogens.Edwardsiella piscicida is an intracellular pathogen which causes serious loss in the mariculture industry.Previous studies have shown that T3SS and T6SS are essential for the virulence of E.piscicida,but very few of its effectors have been identified.We established a macrophage infection model for E.piscicida,in which a population of intracellularly-replicating E.piscicida was released into the supernatant.The release of E.piscicida at the late phase of infection depends on caspase-1-dependent cell pyroptosis and exhibited a T3SS/T6SS-responsive transcription profile.In this work,the identified responsive genes were systematically analyzed,and the top 200 regulated genes were selected and tested for intracellular translocation using the TEM1 ?-lactamase protein translocation reporter assay.We found 25 intracellular translocation-positive effector candidates which are probably the E.piscicida effectors including five previously reported EseG,EseJ,EseH,EseK,and EvpP.A subsequent secretion analysis revealed divergent secretion patterns of the 25 effector candidates,suggesting that multiple transport pathways were involved in the internalization of these effectors.We also verified two new T6SS-dependent effectors,EseL and EseM,which are encoded by ETAE₁303 and ETAE₂136,and three outer membrane vesicle(OMV)-dependent effectors,EseN,EseO and EseP,which are encoded by ETAE₀247,ETAE₀490 and ETAE₂080,respectively.Further analysis of their role in bacterial virulence showed that these new effectors collectively contributed to the virulence of E.piscicida.As an intracellular pathogen,E.piscicida is able to survive and replicate in the host cells,yet the underlying mechanisms remain unclear.Here we characterized its intracellular lifestyle in non-phagocytic cells and showed that the intracellular replication of E.piscicida is dependent on its T3SS.Following internalization,E.piscicida,which was contained in vacuoles that transiently interact with early endosomes,suppressed the classical endosome pathway and prevented their fusion with lysosome in a T3SS dependent manner.Quickly escaped from the degradative pathway,E.piscicida was found in a special replication-permissive niche that characterized by endoplasmic reticulum(ER)markers like Calnexin.Meanwhile,we found that the T3SS effector EseJ is responsible for intracellular replication of E.piscicida by preventing endosome/lysosome fusion.Furthermore,in vivo experiments confirmed that EseJ is necessary for bacterial colonization in both mice and zebrafish.This work delineates the strategy used by E.piscicida to survive and proliferate within the host cell.The natural immune system is the first line of host defense against invading pathogenic microorganisms.Inflammasome is a multiprotein complex assembled by intracellular pattern-recognition receptors and an important part of the natural immune system.A growing number of studies showed that inflammasome activation is critical for host defense against pathogenic infections.NLRP3 inflammasome is one of the most widely studied but its activation mechanism is not fully interpreted.We further investigated the activation mechanism of NLRP3 and found that Rack1 is a critical regulator in activating NLRP3.Knock-down of Rack1 inhibits NLRP3 activation but not NLRC4 and AIM2.By binding to NLRP3,Rack1 acts at the downstream of potassium efflux to regulate ASC oligomerization and NLRP3 activation.Meanwhile,in vivo knock-down of Rack1 showed that Rack1 is required for NLRP3 inflammasome activation.