Dissection On The Mechanism Of Orf1B Regulating Protein Secretion Of Type Ⅲ Secretion System In Edwardsiella Piscicida

Edwardsiella piscicida is a gram-negative intracellular pathogen that infects a wide range of hosts and can cause hemorrhagic septicemia in fish.Type III secretion system(T3SS)is one of its two most important virulence factors in E.piscicida.T3SS effectors can be delivered into the host cell through this nanomachine,to cause dieases.In this study,we characterized an functional unknown protein-Orf1B,encoded by the T3SS gene cluster of E.piscicida.The protein consists of 122 amino acids and shares structural similarity with YscO of Yersinia spp.By this project,the mechanism on how Orf1B regulates T3SS protein was revealed,below are the main findings.1.Sequence analysis on Orf1BThe orf1B gene encodes a 14.20 k Da protein with p I of 8.04.Similarity analysis on Orf1B revealed that Orf1B protein shares similarity with EscO of enteropathogenic Escherichia coli(EPEC),Spa13 of Shigella flexneri,Spa M/Inv I of Salmonella SPI-1,and YscO of Yersinia pseudotuberculosis by 26.4%,24.1%,20.8%,and 32.5%.Moreover,Orf1B shows a DNA binding probability of 99.86%,indicating that Orf1B might be a regulator.2.Orf1B plays a pivotal role in T3SS protein secretion in E.piscicidaDisruption of Orf1B abolishes autoaggregation of E.piscicida.Autoaggregation of E.piscicida in DMEM is mediated by its T3SS translocon protein EseB.Neither T3SS translocon proteins EseB,EseC,EseD nor T3SS effector EseJ were secreted fromΔorf1B strain,and complementation ofΔorf1B strain with the pACYC184-HA-orf1B restored their secretion to the level of wild-type strain.Increased protein levels of T3SS translocon EseB/EseC/EseD and effector EseJ were observed in TBPs ofΔorf1B strain orΔesa N strain than that in WT strain.These data indicate that,like EsaN,Orf1B is necessary for the activity of E.piscicida T3SS,and disruption of Orf1B or EsaN blocks the secretion of T3SS translocon and effectors,resulting in their accumulation inside E.piscicida.3.Orf1B interacts with EsaN as revealed by co-immunoprecipitationConsidering that several homologues of Orf1B interact with ATPase,the interaction between Orf1B and the T3SS ATPase EsaN was investigated through immunoprecipitation.WT esa N::3×FLAG/orf1B and WT ese E::3×FLAG/orf1B strains cultured in DMEM were harvested and sonicated to produce cell lysates.These cell lysates were immunoprecipitated(IP)with an anti-HA antibody.It was observed that EsaN-3×FLAG was precipitated by HA-Orf1B.Taken together,Orf1B specifically interacts with EsaN in E.piscicida.4.Orf1B contributes to E.piscicida pathogenesis in vivoTo determine the contribution of Orf1B to E.piscicida pathogenesis,the survival rates of blue gourami fish were assessed following bacterial infections.To do this,blue gourami fish were injected with 4.5×10~5 CFU of E.piscicida per fish.Ten days post infection,10%of the fish infected with the wild type survived,whereas 85%of the fish infected with E.piscicidaΔorf1B strain survived.This indicates that deletion of orf1B significantly attenuates the virulence of E.piscicida PPD130/91 in vivo.To sum up,it was demonstrated that Orf1B controls secretion of T3SS translocon and effectors by interacting with ATPase.This helps to better understand the mechanism of pathogenesis of E.piscicida.