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. KEY WORDS: Edwardsiella tarda · EseBCD proteins · Translocon · Phagosomal pH · Macrophage Resale or republication not permitted without written consent of the publisherDis Aquat Org 84: [115][116][117][118][119][120][121] 2009 macrophages is important for efficient intracellular growth of bacteria in the macrophages (Okuda et al. 2006). However, only scant information is available on the pathogenicity of E. tarda.Many gram-negative pathogenic bacteria use a conserved protein secretion machinery termed 'Type III secretion system' (TTSS) to transport virulence factors and cause disease. The TTSS apparatus consists of approximately 20 to 25 proteins. The unique feature of the TTSS is a needle-like structure through which particular proteins (termed 'effectors') are injected into host cells. Effectors play an important role in the pathogenic relationship between host and bacterium. Injection of effectors into host cells occurs through pores (termed 'translocons)' formed in the host cell membrane by the TTSS using Type III-secreted proteins (Ghosh 2004). The TTSS encoded within Salmonella pathogenicity island 2 (SPI2) is essential for its intracellular accumulation in macrophages. SseB, SseC, and SseD proteins secreted from the TTSS encoded within SPI2 are reported to function as translocons (Nikolaus et al. 2001). Using TnphoA transposon tagging and the proteomics approach, a TTSS was found in Edwardsiella tarda that was similar to that encoded within SPI2 of Salmonella (Srinivasa Rao et al. 2003, Tan et al. 2005. Three TTSS proteins of E. tarda, homologous to SseBCD were identified and these proteins were designated as EseB, EseC, and EseD (Srinivasa Rao et al. 2004, Tan et al. 2005. We reported that, similar to Salmonella, the TTSS encoded within E. tarda is required for its intra...
Edwardsiella tarda is a pathogen with a broad host range infecting animals and humans. We have reported recently that the type III secretion system (TTSS) is essential for intracellular replication of the bacterium in murine macrophages. The present study shows that the TTSS is also needed for intracellular growth of the bacterium in human epithelial cells (HEp-2). However, different from the previous microarray analyses on murine macrophages, upregulation of the mRNA expression level of NF-kappaB target genes was not detected in the infected HEp-2 cells. The wild-type E. tarda, but not its TTSS mutant, actually repressed the tumor necrosis factor alpha-dependent NF-kappaB activation in an NF-kappaB reporter gene assay. These results suggest TTSS-dependent repression of the NF-kappaB activation in HEp-2 cells infected with E. tarda.
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