| Edwardsiella tarda is a major pathogen threatening aquaculture industry. Its pathogenesis results from a series of pathogenic genes expressed in temporal and spatial order. Two-component system EsrA-EsrB is a global regulator in Edwardsiella tarda. It mainly implements invasion and virulence on the host by regulating type III secretion system, type VI secretion system and hemolysin EthA. Clarifing their different regulation contributes to a more thorough understanding of the pathogenesis network of Edwardsiella tarda, as well as to the development of hyperinvasive and low virulent novel vaccines.Random mutation was introduced by error-prone PCR, resulting in hundreds of variant strains containing substituted esrB segment featured randomness. Combining hemolytic activity test and ELISA for T3SS and T6SS secretion, two strains, i.e. No.15and No.47, with stronger ability of invasion but weaker production capacity of secreted proteins were selected. At the same time, AT6SS was constructed by unmarked in-frame deletion mutation. AT3SS/AT6SS mutant strain was also obtained by deletion of AT6SS in the context of AT3SS.Phenotypes including pathogenicity, infection kenetics, extracellular protein profiles, transcription level of some pathogenic genes, and survival and replication activities in macrophages were tested in EIB202, AesrB and those strains mentioned above. The results indicated that either T3SS or T6SS is essential to Edwardsiella tarda during pathogenesis, while T3SS is more important than T6SS. Differences between AT3SS/AT6SS and AesrB on survival and replication activities in macrophages indicated that esrB works more than regulating T3SS and T6SS. Phenotypes of No.15and No.47mutants were quite different from those of both wild type EIB202and AesrB. Taking together the results of homology modeling, the phenotypical differences between No.15and No.47mutants might result from the mutation in both promoter and ORF region. |
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