Edwardsiella Piscicida T4SS Effector Thioredoxin-like-Protein(Trxlp)Restrains ASK1-MAPK Pathway To Promote Infection Process

The relationship between pathogen infection and host immune defence is a dynamic combat.Conversely,microbial pathogens have evolved their own strategies to evade,inhibit,or otherwise manipulate the innate immune response,including MAPK pathway,NF-kB pathway,inflammasome activation and so on.The secretion system,as a crucial bacterial virulence system,is tightly correlated with the survival and pathogenesis of pathogens during infection.So far,at least six secretion systems?types I-VI?have been elucidated in bacteria,which are macromolecule sophisticated nanomachines to export proteins,DNA or other substrates to the the extracellular or into the host cells.The T4SS is a major determinant of bacterial virulence,which is responsible for DNA exchange and effectors transport.However,little is known about T4SS effectors and their functions during infection.Edwardsiella piscicida is an important intracellular-replicated gram-negative pathogen,which manipulate the genes expression to adapt complicated environment during infection.We recently discovered that E.piscicida EIB202 replicates and induces cell death in murine macrophages.The macrophage-released E.piscicida population displays a reprogrammed transcriptional profile characterized by the upregulation of T3SS/T6SS-related genes as well as some uncharacterized genes.In this study,we demonstrated that a putative ETAE₂186-expressed gene was greatly induced during macrophage infection.Crystal structure analysis revealed that it is a Trx family protein,which shares the signature WCXXC motif and a ?1-?l-w2-?2-?3-?3-?4-?5-?4 topology.Thus,we named the ETAE₂186 as Trxlp?Thioredoxin-like protein?in our study.However,through enzymatic function analysis,we found Trxlp was much less active than the classical reducing Trxs in E.piscicida Furthermore,Trxlp can be secreted and translocated into the cytosol of infected cells in a T4SS-dependent manner.Collectively,we first identified a T4SS effector protein,Trxlp,which is a thioredoxin protein in E.piscicida,hile its role during infection remains unknown.Trxlp shares a conserved structure and critical WCXXC-motif site not only with bacterial Trxs,but also with mammalian Trxs.Thus,we hypothesize that the T4SS effector Trxlp can mimic endogenous human Trx1?hTrx1?when it is translocated into the cytosol of host cells during infection.According to the CO-IP and Pull-down analysis,Trxlp could mimic endogenous thioredoxin to bind with the ASK1-TBD domain and abrogate ASK1 homophilic interaction and phosphorylation.Furthermore,Trxlp significantly suppresses the phosphorylation of Erk1/2-MAPK and p38-MAPK downstream of ASK1 signaling,and the catalytic active site of WCXXC motif in Trxlp plays a critical role in this process.Finally,to evaluate the function of the E.piscicida T4SS effector Trxlp in regulating the ASK1 and MAPK signaling cascades in vivo,we firstly established the ZF4 infection model,and confirmed Trxlp-mediated inhibition of ASK1-Erk/p38-MAPK axis and regulation of TNF-? and IL-10 cytokines,given the similarity between fish MAPK pathways and those of mammals.Secondly,we stepped forward to establish the 3 days post-fertilization?dpf?zebrafish larvae infection model,and used the wild type and ask1 morpholinos zebrafish larvae to reveal that ASK1 is a target of the Trxlp,which promotes the bacterial survival and pathogenicity in vivo.