Genome-wide Screening For Edwardsiella Tarda Effectors And Primary Characterization For One Of The Effectors, EseG

Effector is a class of bacterial protein transferred into host cells during infection. Bacteria could utilize effectors to manipulate host cells’physiological process like membrane transport, MAPK signaling pathway and ubiquitylation. However, as an important marine pathogen, there is limited report about Edwardsiella tarda effectors. In our previously work, we have proved that intra-macrophage infection induced significantly changed transcriptional profile in E. tarda, indicating that many genes are involved in the process of infection as virulence effectors.In this work, we aim to screen and characterize some effectors from them.Firstly, we used TEM-1 report system to screen and get a series of candidate effectors. Then, we used immunofluorescence and western blot assays to verify 9 novel effectors, ETAE₀247, ETAE₀275, ETAE₀490, ETAE₁303, ETAE₁640, ETAE₂080,ETAE₂136, ETAE₂188, and ETAE₂399. Next, we explored the extracellular secretion of these effectors and revealed the diversity of effectors’ secretion mechanism, suggesting that translocation is uncoupled with secretion. Further bioinformatics analysis predicted that some effectors contain signaling peptide and transmembrane domains, which might be important for effectors’localization and function. Finally, we focoused on one of the identified effectors, EseG, and investigated its intracellular translocation and localization features. We demonstrated that EseG could be transported into host cells only after E. tarda was internalized, indicated that extracellular E. tarda couldn’t inject EseG into host cells.Further subcellular fractionation analysis and immunofluorescence detection suggested that EseG specifically targeted the E. tarda-containing vacuoles （ECVs） in host cells. These unique features might be important to interpret the interaction of EseG with host cells upon infection.Taken together, our work identified 9 novel effectors of E. tarda and illuminated the internalization-depending translocation and ECV-targeting localization of EseG, which is important for resolving effectors’function and further interpreting the pathogensis of E. tarda.