Characterization of proteins secreted from a type III secretion system of Edwardsiella tarda and their roles in macrophage infection.

The Type III secretion system is essential for intracellular replication of Edwardsiella tarda in phagocytes of fish and mammals. We identified the secreted proteins of the Type III secretion system by comparing the wild-type strain and the Type III mutant mET1229. The wild-type strain secreted 55, 25, and 22 kDa proteins into the culture supernatant, whereas the Type III mutant did not. These proteins were identified as EseB, EseC, and EseD and are similar in sequence to Salmonella SseB, SseC, and SseD that function as a translocon. The EseB, EseC, and EseD knockout mutants did not replicate in murine macrophages, suggesting that these proteins are essential for intracellular replication of E. tarda. Highest secretion of EseBCD proteins was observed when bacterial cells were cultured in neutral and alkaline pHs but not in acidic pH. When the pH of the phagosomes was examined using an acidotropic probe, the phagosomes containing the wild-type strain showed neutral pH, whereas those containing the Type III mutant exhibited acidic pH. These results suggest that the Type III-dependent interference with formation of the acidic environment in phagosomes is essential for intracellular replication of bacteria in murine macrophages.