Identification And Preliminary Functional Evaluation Of Edwardsiella Tarda Effectors

Edwardsiella tarda is an important pathogenic bacterium in the aquaculture of economically important fish species. Its infection usually leads to huge losses due to the outbreaks of the infectious diseases, such as ascites and sepsis. In our previous work, a strong virulent strain, EIB202 was separated from infected turbot and 25 candidate effectors were screened from EIB202 genome by comparative transcriptomics and other strategies in our lab. In this work, the secretion and translocation of 4 candidate effectors, including ETAE₀866, ETAE₁757, ETAE₂186 and ETAE₂428, were investigated by multiple identification methods, and their roles in bacterial infection also preliminarily evaluated both in vitro and in vivo.Firstly, extracellular protein profile was analyzed by Western blot and the results showed that ETAE₀866 and ETAE₁757 are secreted depending on T3SS, while ETAE₂428 on T6SS. Furthermore, FRET was used to confirm the translocation of the four candidate effectors, and the intracellular localization of the effectors was investigated by immunofluorescence technique and Western blot for cell fractions, and the results show that ETAE₀866 and ETAE₂428 were located in the cell membrane fraction, while ETAE₂186 was located in the cytoplasm fraction, ETAE₁757 was likely to locate in cell nucleus fraction.Next, the gene deletion mutants were constructed for each effectors, and their capability of adherence, internalization in non-phagocytic cells （HeLa） and macrophage cells （J774A.1） and replication in macrophage cells （J774A.1） compared with that of wild type strain. Furthermore, their virulences were evaluated in zebrafish. Among the four mutants, A2428 showed the most significant attenuation in zebrafish, implying that ETAE₂428 is the major virulent effectors in EIB202.Finally, all the four effectors were expressed in E. coli, and the purified protein of ETAE₂428 was preparated, which could be used for further study on protein structure and function.Above work laid a solid foundation to further explore the function of Edwardsiella tarda effectors and its mechanism on interaction with host cells.