Identification And Function Of Edwardsiella Piscicida Type ? Secretion System Effector EseK And Its Chaperones

Edwardsiella piscicida is an invasive enteric bacterium,which infects a wide range of hosts including fish,amphibians,reptiles,birds,mammals,and even humans.It can infect the epithelial cells and cause important diseases in animals.Edwardsiellosis caused by E.piscicida is more apparent in various fish,including blue gourami fish,turbot and zebrafish.E.piscicida has two sets of important secretion system,namely T3SS?Type III secretion system?and T6SS?Type VI secretion system?.During the infection,E,piscicida usually utilizes T3SS or T6SS to deliver effectors directly into host cells.These effectors can affect various signaling pathways to help the strain to survive and proliferate in the host cells.Type ? secretion system is very important for most pathogenic bacteria,such as Salmonella enterica,Yersinia spp.and Shigella spp.In the processes of infection,the pathogen uses T3SS to inject its effector proteins into the host cells and regulate the function of the host cells so as to be beneficial for the survival of the bacteria.T3SS is one of the most important virulence factors for E.piscicida,which can help E.piscicida to survive and proliferate in host cells.Hence,it is very important to identify E.piscicida T3SS effectors for better understanding of host-pathogen interactions.In this study,animal cells were used as a model for study.It was found that EseK,which encoded by the gene ETAE 1586,is a new T3SS effector protein and is translocated by E.piscicida.EseK was secreted into the bacterial extracellular supernatants dependent on the T3SS pathway and injected into the host cells,and then localized in the membrane fraction of the host cell.For EseK secretion,20 amino acids at the N-terminus of EseK served as secrete signal peptides.EseK could inhibit not only the phosphorylation of p38,JNK and ERK1/2 in MAPKs signaling pathway directly,but also the expression of TNF-a in host cells.T3SS-translocated effectors in many pathogens generally have an associated chaperone that is not secreted.The expression,secretion and transcription of T3SS effector proteins are usually accompanied by chaperones.Here,we showed that T3SS effector EseK has two class I chaperones named EscH and EscS,which are encoded by ETAE₁587 and ETAE 1588 in E.piscicida.Bioinformatic analysis indicated that EscH and EscS have the features of class I chaperones,and both of them can enhance the secretion and translocation of EseK,but not promote EseK expression or prevent EseK degradation.Chaperones usually bind effectors directly.Therefore,understanding the dynamic interactions between T3SS chaperones and their binding partners is necessary in elucidating the T3SS secretion and translocation process.In vitro pull-down experiments showed that both of EscH and EscS could bind EseK directly,and EseK might act as an adaptor protein to bind the two chaperones EscH and EscS and form a ternary complex.Further investigation indicated that EscH directly binds EseK through undetermined binding domains,whereas EscS binds EseK via its N-terminal 10-21 amino acids.We also found that EseK has an N-terminal chaperone-binding domain?CBD?,and the CBD is important to binding EscH or EscS.Finally,in vivo zebrafish experiments showed that EseK as well as its two chaperones EscH and EscS could reduce the bacterial burden of E.piscicida in the livers and kidneys of zebrafish,all of which played a critical role in the virulence of bacteria.