Secretion Of Translocon And Effector Proteins In Type Ⅲ Secretion System(T3SS)is Regulated By The EsaB/EsaL/EsaM Complex In Edwardsiella Tarda

Edwardsiella tarda,the causative pathogen of Edwardsiellosis,is a Gram-negative intracellular bacterium which infects a wide range of hosts,including fish and mammals.Importantly,E.tarda infection often results in huge economic losses in farmed flatfish,such as turbot(Scophthalmus maximus)and Japanese flounder(Paralichthys olivaceus).Many Gram-negative bacteria utilize type Ⅲ secretion system(T3SS)to translocate virulent proteins(or called effectors)into host cells to intervene normal cellular processes,and to promote the replication of bacteria in host cells.This project was focussed on a protein complex,EsaB/EsaL/EsaM encoded in the E.tarda T3 SS gene cluster in order to understand how EsaL controls the secretion of T3 SS translocon/effector proteins.The results obtained in the present study provide supportive evidence that therapeutic inhibitors targeting at the EsaB/EsaL/EsaM complex and their homologues may be used to combat infections caused by E.tarda and other T3SS-containing pathogens.Firstly,it was found that EsaL is not secreted into culture supernatants,but was detected in membrane fractionation of E.tarda PPD130/91.EsaL is required for the efficient secretion of translocon proteins.The homologues of EsaL from other bacteria,such as SsaL,SepL and YopN,have been reported also to form a complex with two other proteins i.e.SpiC/SsaL/SsaM,SepD/SepL/CesL,and YscB/YopN/SycN,to control the T3SS activity of their bacteria.To identify these proteins in E.tarda,we further analyzed the sequences of E.tarda T3 SS gene cluster.Indeed,EsaB and EsaM were found to be the possible homologues which may interact with EsaL to form a complex.Like in esa L mutant,the secretion of EseB/EseC/EseD decreased dramatically in either esaB or esaM mutants as compared with wild-type strain.This result suggests that,similar to EsaL,both EsaB and EsaM promote the secretion of translocon proteins.Further study identified that EsaL interacts with EsaB and EsaM as revealed by yeast two hybrid and co-immunoprecipitation assays.EsaB and EsaM can interact separately with EsaL within bacterial cells.In addition,EsaB is able to interact with EsaM even in the absence of EsaL.Deletion of esaB and esaM could alter the steady-state protein level of each other.The intracellular EsaB protein level decreased sharply in the ΔesaM strain(compared with WT strain)starting as early as 30 min post chloramphenicol treatment.Similarly,the intracellular EsaM protein level decreased over time in the ΔesaB strain.These data suggest that the interaction between EsaB and EsaM is important for maintaining their mutual stability within bacterial cells.SpiC/SsaL/SsaM in Salmonella can form a heterotrimeric complex at acidic pH,promoting the secretion of translocon proteins but suppressing the secretion of effectors from the Salmonella-containing vacuole.It is hypothesized that pH shift might also trigger the secretion of effectors by the T3 SS in E.tarda.When bacteria were cultured in acidic condition,EsaB,EsaL and EsaM promote the secretion of EseG.However,after pH was shifted to neutral,a higher proportion of EseG was secreted into the supernatants in the ΔesaL strain,but the secretion of EseG in the ΔesaB and ΔesaM strains decreased drastically.These results indicated that EsaL suppresses the secretion of effector,while EsaB and EsaM promote effector secretion in neutral condition.It was found that deletion of esa L has no influence on effector translocation,whereas translocation of EseG decreased markedly in ΔesaB and ΔesaM.In vivo infection experiments of blue gourami fish showed that the median lethal dose,LD50 of the ΔesaL mutant strain is 3.39 times higher than that of the wild-type.The survival rate of the fish infected with Δesa L,ΔesaB and ΔesaM is 97.22%,97.22% and 100%,respectively,during a 7-days infection period;however,the survival rate infected with wild-type is only 30.55%.The CIs from spleens for ΔesaL,ΔesaB,or ΔesaM strains were 0.17±0.05,0.12±0.03,and 0.05±0.03,respectively,demonstrating that each of the mutant strains is far out-competed by wild-type strain.These in vivo results indicate that the deletion of esaL,esaB and esaM can respectively attenuate the virulence of E.tarda in blue gourami infection model.Furthermore,strong interactions between EsaK and EsaL,and EsaE and EsaL as well,were detected by yeast two hybrid.Deletion of esaK or esaE leads to a sharp decrease of intracellular protein level of EsaL.Whether or not EsaK/EsaL or EsaE/EsaL complex is involved in T3 SS protein secretion control awaits further study.