| The innate immune system,as a natural,universal,and non-specific defense mechanism in animals,is the first line of host defense against invading pathogens.Programmed cell death is a fundamental biological and pathological phenomenon for the development of multicellular organisms and especially a critical event during innate immune response to invading pathogens.Upon pathogen infection,the programmed suicide of infected cells eliminates the damaged cells as well as the intracellular pathogen and promotes the exposure of pathogen antigen to dendritic cells further activating the adaptive immune response,benefitting for pathogen clearance and host defense.Conversely,pathogen has also evolved multiple strategies to manipulate the host cell death for successful infection.For example,pathogen could suppress cell death signaling pathway to elude host recognition and successfully invade and proliferate in cells during early period of infection while pathogen could promote cell death for bacterial release and further infection during late period of infection.Thus,the question of whether and how pathogen modulating cell death during infection is a key to understand pathogen-host interaction and bacterial pathogenesis,and develop therapeutic approaches to control infection.As an important maricultured fish pathogen,although Edwardsiella piscicida has been studied for many years of its pathogenesis,very little is known about the regulation of host cell death in E.piscicida infection.In a previous work of our laboratory,a transposon insertion mutant library of E.piscicida has been utilized to identify mutants inducing cell death based on cellular morphology changes and release of cellular content such as lactate dehydrogenase.Two novel E.piscicida mutants,namely 0909I and 1906I,were found to induce cell morphological changes and LDH release,which attracted our attention and raised our curiosity.Thus,the aim of this work is to reveal the specific mechanism of both 0909I and 1906I in modulating cell death and gain a deep insight into the role of regulation of cell death during E.piscicida infection.Firstly,we found that the transposon insertion in 0909I resulted in an increase of transcriptional level of hemolysin-encoding gene ethA,and hemolytic activity assays further validated that 0909I mutant is a hemolysin-overexpressing strain.The upregulation of hemolysin expression in E.piscicida promoted a robust caspase 5-like-activity-dependent pyroptotic-like cell death in diverse fish nonphagocyte cells.Furthermore,E.piscicida hemolysin was found to mainly associate with bacterial outer membrane vesicles(OMVs)and secreted out of the bacteria together with OMVs via examining the localization of hemolysin.More interestingly,the purified hemolysin-associated OMVs were found to internalize into cytosol and induce the pyroptotic cell death when incubated with fish nonphagyocyte cells.Likewise,in E.piscicida the hemolysin-associated OMVs could enter cells via a dynaminmediated endocytosis to induce pyroptotic-like cell death during infection.To mimic the natural pathogen infection in fish,we established the intestinal mucosal infection model in both larvae and adult zebrafish.Bacterial immersion infection of both larvae and adult zebrafish suggested that dysregulated expression of hemolysin alerts the innate immune system and induces intestinal inflammation to restrict bacterial colonization in vivo.Additionally,we found that the transposon insertion in 1906I caused up-regulation of diguanylate cyclase expression and resulted in elevated bis-(3’,5’)-cyclic dimeric GMP(c-diGMP)which facilitated E.piscicida biofilm formation,exopolysaccharides production and adhesion to HeLa cells.The up-regulation of c-di-GMP synthesis in E.piscicida triggered a robust membrane-ruptured cell death in HeLa cells and limited the bacterial colonialization as well as attenuated bacterial virulence in zebrafish.Moreover,the cell death induced by E.piscicida c-di-GMP was neither dependent on caspases or gasdermin D/E,nor mediated by the RIP1/3-MLKL axis,but an iron-dependent,oxidative,non-canonical ferroptosis.It was observed that dysregulation of c-di-GMP in E.piscicida promoted iron accumulation in both cytosol and mitochondrial,which could be blocked by iron chelator.In addition,the cellular iron accumulation leaded to a decrease of mitochondrial potential and ATP production and an increase of ROS generation both in mitochondria and cytosol,facilitating cellular oxidative stress and triggering cell death.More importantly,unlike classical ferroptosis executed via excess lipid peroxidation,no lipid peroxidation was detected in the infected cells.Furthermore,lipoxygenase inhibitors and lipophilic antioxidants were not able to suppress morphological changes and cell death induced by 1906I,validating that c-di-GMPdysregulation induced a non-canonical ferroptosis independent of lipid peroxidation.Our work on specific mechanism of E.piscicida 0909I and 1906I triggering cell death reveals the importance of tight regulation of hemolysin and c-di-GMP in modulating cell death during E.piscicida infection,which deepens our understanding of pathogen-host interaction and provides a new perspective for controlling infection.In addition,the hemolysin-mediated pyroptotic-like cell death in fish nonphagyocyte cells and modulation of zebrafish innate immunity suggests the existence of non-canonical inflammasome signaling in lower vertebrates.Moreover,the finding of E.piscicida c-di-GMP promoting a novel,lipidindependent and non-canonical ferroptosis during infection provides new evidence for the pathological role of ferroptosis in regulation of pathogen infection and more importantly reveals a novel non-canonical ferroptotic pathway independent of lipid peroxidation. |
