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

Abstract

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...

ORF13 in the Type III secretion system gene cluster of Edwardsiella tarda binds to the mammalian factor Cugbp2

“…We previously reported that the highest expression of the EseBCD translocon proteins, secreted through the TTSS, occurs when the wild-type strain was incubated at 25 to 30°C in neutral or alkaline pH (Okuda et al 2009). In this study, we determined whether ORF13 can be secreted into the culture supernatant under the same conditions as for the EseBCD translocon proteins.…”

“…The highest expression of the Ese-BCD translocon proteins, secreted through the TTSS, is induced when the wild-type strain is incubated at 25 to 30°C in neutral or alkaline pH (Okuda et al 2009). According to this observation, in a time course experiment, the wild-type strain and the orf13 mutant were incubated for 0, 2,5,8,12,18 and 24 h at 30°C in Tripto-soy broth (TSB, Nissui) medium (pH7.3).…”

“…The wild-type strain, mET1229, and the orf13 mutant were incubated for 18 h at 30°C in TSB (pH7.3). Secreted proteins in the culture supernatant were isolated using trichloroacetic acid precipitation as de scribed previously (Okuda et al 2009). Western blot analysis was performed using anti-ORF13 polyclonal antibody, which was prepared by immunizing a rabbit with a synthetic oligopeptide corresponding to amino acids 61 to 74 (VRVIDTLWTLHQGTC) of ORF13.…”

Transcriptomic dissection of the horizontally acquired response regulator EsrB reveals its global regulatory roles in the physiological adaptation and activation of T3SS and the cognate effector repertoire inEdwardsiella piscicidaduring infection toward turbot

“…22,26 These 2 secretion systems enable the bacterium to enter, survive and replicate inside epithelial cells and phagocytes and to spread and invade deeper tissues. 18,23,27 At present, only 3 T3SS effectors have been documented in E. piscicida. EseG (ETAE_0866) is a T3SS effector that localizes the host membrane fraction after translocation; it destabilizes microtubules when overexpressed in mammalian cells through a conserved domain that includes amino acid residues 142-192.…”

“…18,19,20,21 E. piscicida invades and colonizes various cells, including Hep-2, HeLa, epithelioma papillosum of carp (EPC), macrophage-like cells J774A.1 and fish primary phagocytes. 22,23,24,25 E. piscicida attaches to host cells and translocates effector proteins into these cells via type III and type VI secretion systems (T3SS and T6SS, respectively), which are necessary for bacterial survival, replication and pathogenesis. 22,26 These 2 secretion systems enable the bacterium to enter, survive and replicate inside epithelial cells and phagocytes and to spread and invade deeper tissues.…”