Edwardsiella tarda is a Gram-negative enteric pathogen that causes hemorrhagic septicemia in fish and both gastrointestinal and extraintestinal infections in humans. A type III secretion system (T3SS) was recently shown to contribute to pathogenesis, since deletions of various T3SS genes increased the 50% lethal dose (LD 50 ) by about 1 log unit in the blue gourami infection model. In this study, we report EseG as the first identified effector protein of T3SS. EseG shares partial homology with two Salmonella T3SS effectors (SseG and SseF) over a conserved domain (amino acid residues 142 to 192). The secretion of EseG is dependent on a functional T3SS and, in particular, requires the chaperone EscB. Experiments using TEM-1 ␤-lactamase as a fluorescence-based reporter showed that EseG was translocated into HeLa cells at 35°C. (EseG 142-192 ), since truncated versions of EseG devoid of this motif lose their ability to cause microtubule destabilization. By demonstrating the function of EseG, our study contributes to the understanding of E. tarda pathogenesis. Moreover, the approach established in this study to identify type III effectors can be used to identify and characterize more type III and possible type VI effectors in Edwardsiella. Fractionation of infected HeLa cells demonstrated that EseG was localized to the host membrane fraction after translocation. EseG is able to disassemble microtubule structures when overexpressed in mammalian cells. This phenotype may require a conserved motif of EseG

The type III secretion system (T3SS) of Edwardsiella tarda plays an important role in infection by translocating effector proteins into host cells. EseB, a component required for effector translocation, is reported to mediate autoaggregation of E. tarda. In this study, we demonstrate that EseB forms filamentous appendages on the surface of E. tarda and is required for biofilm formation by E. tarda in Dulbecco's modified Eagle's medium (DMEM). Biofilm formation by E. tarda in DMEM does not require FlhB, an essential component for assembling flagella. Dynamic analysis of EseB filament formation, autoaggregation, and biofilm formation shows that the formation of EseB filaments occurs prior to autoaggregation and biofilm formation. The addition of an EseB antibody to E. tarda cultures before bacterial autoaggregation prevents autoaggregation and biofilm formation in a dose-dependent manner, whereas the addition of the EseB antibody to E. tarda cultures in which biofilm is already formed does not destroy the biofilm. Therefore, EseB filament-mediated bacterial cell-cell interaction is a prerequisite for autoaggregation and biofilm formation. Edwardsiella tarda is a Gram-negative bacterium with a wide range of hosts, including fish and humans. E. tarda causes hemorrhagic septicemia in fish and gastrointestinal and extraintestinal infections in humans (1-3). The type III secretion system (T3SS) of E. tarda plays a pivotal role in infection and enables the bacteria to survive and replicate in phagocytes and epithelial cells (4-7).The bacterial T3SS nanomachine, delivering effector proteins directly from the bacterial cytosol to host cells (8, 9), consists of three parts: the basal body, needle, and translocation pore (10). The gene cluster of the T3SS in E. tarda contains 34 genes, which encode secretion apparatus, chaperones, translocators, effectors, and regulators (5, 11). The esrA-esrB (5) and esrC (12) genes in the T3SS gene cluster together with phoP-phoQ (13) and phoB-phoR (14) outside the T3SS gene cluster control the virulence of E. tarda. Deletion of esrB abolished the secretion of the translocon proteins EseB, EseC, and EseD (5), which can form a protein complex after secretion (5, 15, 16). Mutation of eseB led to an E. tarda replication defect in host cells (5). EseB is required not only for translocating effectors into host cells (11) but also for bacterial autoaggregation in a T3SS-inducing medium, Dulbecco's modified Eagle's medium (DMEM) (5).EseB is homologous to EspA of enteropathogenic Escherichia coli (EPEC) or enterohemorrhagic Escherichia coli (EHEC), and it has been reported that EspA forms a sheath-like structure on the bacterial surface, as revealed by immunofluorescent staining and immunogold labeling, and is required for effector translocation (17-19). EspA of EPEC or EHEC also functions as an adhesin in microcolony formation on epithelial cells and is involved in bacterial aggregation during biofilm formation on abiotic surfaces or salad leaves (19,20). The contribution of the T3SS to biofilm ...