Localization And Pathogenicity Of EseH,Eseland Escd Encoded In Native Plasmids Of Edwardsiella

Edwardsiella ictaluri, belonging to the family Enterobacteriaceae, is a Gram-negative, facultative intracellular bacterium, and is known as the etiological agent of enteric septicaemia of channel catfish (Ictalurus punctayus)(ESC). Recently, this bacterium has been found from several species of siluriform fish in China, such as yellow catfish (Pelteobagrus fulvidraco), with the typical symptom being a hole in the head, thus often called as red-head disease. In this study, two native plasmids pEI1and pEI2, which were present in Edwardsiella ictaluri HSN-1isolated from diseased yellow catfish, were sequenced, and some open reading frames (ORFs) in the plasmids are found homologous to genes in type Ⅲ secretion system (T3SS) of Gram-negative bacteria. The present study was therefore designed to understand pathogenic effects of three proteins, EseH, Esel and EscD, which are encoded in plasmids of the bacterium and share homology with T3SS proteinsThe sequenced plasmid, pEIl in strain HSN-1lacks the transposition fragment, which was reported in pEIlin strain93-146isolated from channel catfish in the US. EseH encoded by pEIl and EscD by pEI2contain the conserved domains in PRK15372and PRK15336superfamilies, respectively. EseI in pEI2shares a low degree of similarity with T3SS effector OspB at the C-terminus in Shigella. It is hypothesized that the three proteins may be involved in pathogenesis of E. ictaluri.In consideration of the multi-copy presence of pEIl and pEI2in E. ictaluri, each of the two plasmids was cured completely in E. ictaluri based on plasmid incompatibility for the construction of mutants. The mutants with deletion of eseH, eseI and escD, were used to determine the median lethal dose (LD50) and competitive index (CI) in yellow catfish. The LD50value of HSN-1was105.4775(R2=0.9945), and the value of△eseHand△eseI was higher than the wild-type, being105.6567(R2=0.9808) and105.8758(R2=0.9874), respectively. The LD50value of△escD was quite similar to the wild-type, being105.5497(R2=0.9642). All mutants as revealed by CI values were related to the virulence of the bacterium. The eseH and eseI mutants had mean CI values of0.7246and0.7044, and0.6373and0.6158for liver and spleen respectively, showing lower virulence than strain HSN-1(P<0.01). However, the CI of AescD was significantly higher than that of HSN-1(P<0.01), being above1.5. When all mutants used in the infection of epithelioma papillosum cyprinid (EPC) cell,△eseH,△eseI and pEIl-pEI2-showed decreased adherence to EPC cells, and EseI and pEIl-pEI2-increased abilities in invasion when compared with wild-type HSN-1strain. Moreover, all mutants were analysed in respects with their replication in macrophages derived from head kidney of yellow catfish.△eseH showed a decrease in replication, and Aesel a defected colonization in macrophages.△escD showed almost similar multiplication as the wild-type HSN-1(P>0.05).As T3SS in E. ictaluri is similar to that in E. tarda in gene organization, the T3SS in E. tarda was used to analyse the secretion and translocation of EseH, EseI and EscD. Both EseH and Esel were secreted and translocated with the secretion of EseH depending on T3SS. Once translocated, EseH was localized in membrane fraction of ZF4cells, and EseI in cytosol fraction. But, EscD was not secreted. Using yeast two-hybrid system, the ’bait’ protein EscD was not detected interactively against ’prey’ protein EseH or EseI, indicating that EscD cannot be a chaperone of EseH or EseI.In summary, two native plasmids, pEIl and pEI2, were sequenced and successfully cured from the strain HSN-1of E. ictaluri, and three genes eseH, eseI and escD encoded in the plasmids were deleted respectively with the construction of mutants. EseH and Esel were found secretory, with the secretion of EseH being dependent on T3SS. Mutants, AeseH and Aesel were attenuated, while AescD had increased virulence, indicating that these two plasmids are involved in virulence and pathogenesis of E. ictaluri. Furthermore, the method developed in this study may be applicable for investigating other plasmid-encoded proteins in E. ictaluri.