EseC,A Type Ⅲ Secretion System Translocon Protein,Promote Edwardsiella Tarda Intracellular Survival

Edwardsiella tarda(E.tarda)is an important gram-negative pathogen affecting both animals and humans.Enabled to survive and reproduce in macrophages is the key to its pathogenesis after phagocytosis by macrophages.A type III secretion system(T3SS)has been found to be an important virulence factor of E.tarda.EseC,together with EseB and EseD has been suggested to form a putative T3SS translocon complex,EseC and EseD inserted into the host cell membrane,whereas EseB is free protein content in T3SS translocon complex.When E.tarda contacted with the host cell,EseB,EseC and EseD quickly formed complexes inserted into the host cell membrane,drilling that bacteria invade the host cell,It was confirmed that EseC affect the intracellular survival of E.tarda in mircophage,the but the exact mechanisms hasn’t been understood.A suicide plasmid pHM5 was used to construct the recombination suicide plasmid,according to the principle of homologous recombination,eseC gene was deleted from Et.CD strain,△eseC mutant strain was got and identified.The eseC gene was cloned into pACYC 184 vector to construct a recombination plasmid,which entered into the△eseC strain by electric shock,a complementary strain eseComp was got and identified.To explore whether EseC affect the bacterial toxicity and cell apoptosis,CCK-8 kit was used to test the survival rates of macrophages after the wild type,△eseC and eseComp strains of E.tarda infected RAW264.7 macrophages at different MOI,respectively.The results showed that the AeseC mutant exhibited lower bacterial toxicity than the wild type.After the cells were infected with E.coil,the wild type,△eseC and eseComp strains of E.tarda at MOI of 10:1 to prolong 2h or 6h culture time,respectively,flow cytometry was adopted to detect the percentage of apoptosis cells after internalized 2 h and 6 h.The results showed that EseC affect the expres-sion of the T3SS,inhibit early the cell apoptosis and induced lately the cell pyroptosis,thus influenced the intracellular survival of E.tarda.To explore whether EseC was able to affect the intracellular survival,numbers of Et.CD,the wild type,AeseC and eseComp strains infected the macrophages at MOI 100:1 to compare their intracellular survival numbers in cultural time prolonged by the method of counting colony on plate.The results showed that the wild type and AeseC strain were able to survive and reproduce in the macrophages,but the surviving rate of AeseC strain was lower than the one of the wild type and eseComp strain,there was no difference between the surviving rate of the wild type and eseComp strain.These results suggested that EseC played an important role in sustaining speed of intracell-ular survival of E.tarda.To explore the mechanism of EseC affected intracellular survival of E.tarda,flow cytometry was adopted to compare the product of NO and ROS in RAW264.7 after the wild,AeseC and eseComp internalized 2 h and 6 h.It was founded that the product of NO and ROS showed a significant increase after AeseC internalized 2 h,however,the product of NO and ROS after AeseC internalized 6 h were similar to the ones in the wild group,and the product of NO and ROS in the eseComp group approached to the ones in the wild type group at 2h and 6h time prolonged.These results indicated that EseC played an important role in the production of ROS and NO after microph-ages infected in the early phase of infection.These data of stress experiment in vitro showed that the mutant was more sensitive to H2O2 than the wild type,flow cytometry was adopted to establish the standard relationship between intracellular pH and the rate of the cells tagged by acid probe.After the wild type,AeseC and eseComp strains infected the macrophages to prolong different cultural times,intracellular pH has been got from the standard curve.These results suggested that T3SS of the wild type can interference with formation of the acidic environment in the macrophages,whereas the AeseC mutant failed in resisting the acidic environment in macrophages.RT-PCR was used to detect the expression levels of eseE and eseJ genes encoding the effector proteins of T3SS,with 16S rRNA as control.The results showed that the transcriptive levels of eseE and eseJ genes in the mutant were lower than those in the wild type,and there were no differences between the gene levels in the eseComp and wild type.These results suggested that These results suggest that the deletion of eseC have an influence on EseE and EseJ exerting biological effects in the host cell.In order to explore whether EseC affect E.tarda virulence in mouse in vivo,the wild type and AeseC strains infected mice to compare their virulence of half lethal dose(LD50).The results showed that the virulence of the mutant was 4.05 times lower than that of the wild type,which indicated that the virulence of the mutant decreased obviously.Compare with the survival rate of the wild type infected mice in several days,the CFUs of the AeseC mutant in the liver,spleen,lung and kidney of mice decreased obviously.The differences between appearance and weight was compared in the liver,spleen,lung and kidney of mice after the wild and AeseC infected mice in several days,except of the kidneys,the appearance and weight of other tissues infected by the wild increased more significantly than those by the AeseC.Optical microscopy was applied to obsever the difference between tissue damage of wild and that of AeseC after H-E straining,show that the AeseC reduce the inflammatory reaction and decrease the degree of tissue damage.ELISA method was used to compare the expression of IL-1β and TNF-α of wild and that of the AeseC,After 1～11d of infection,the expression of IL-1β and TNF-α in the wild type group were obviously higher than those in the AeseC group.Therefore,the deletion of eseC has affected the generation of cytokines in mice activated by E.tarda.On the whole,The results showed EseC can inhibit the cellular apoptosis and toxi-city,induce the cellular pyroptosis,inhibit microphages to produce ROS and RNS,and interfer the cellular acid environment in favour of E.tarda intracellular survival,survive in mice in vivo,and cause acute tissue damage and acute inflammation through regulating the translocation of EseE and EseJ.This research provided an important clue to further investigate the pathogenic mechanism of T3SS of E.tarda.