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 ...

The type III secretion system (T3SS) plays a crucial role in the pathogenesis of many Gram-negative bacteria, including , an important fish pathogen. Within the T3SS, there are three proteins (EsaB/EsaL/EsaM) that are homologous to proteins present in many other bacteria, including SpiC/SsaL/SsaM in , SepD/SepL/CesL in enteropathogenic (EPEC) and enterohemorrhagic (EHEC), and YscB/YopN/SycN in EsaL was found to interact with both EsaB and EsaM within the bacterial cell, as revealed by a coimmunoprecipitation assay. Moreover, EsaM is required for EsaB stability, and the two proteins interact with each other. EsaB, EsaL, and EsaM are all indispensable for the secretion of the T3SS translocon protein EseC into supernatants under pH 5.5 and pH 7.2 conditions. Unlike EseC, EseG is a T3SS effector whose secretion is suppressed by EsaL at pH 7.2 while it is promoted at pH 5.5 condition. Despite this finding, mutant strains lacking EsaB, EsaL, or EsaM (i.e., the Δ, Δ, or Δ strain, respectively) were all outcompeted by wild-type during a coinfection model. These results demonstrate that EsaB/EsaL/EsaM form a ternary complex controlling the secretion of T3SS translocon and effector proteins and contributing to pathogenesis.