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1 Department of Biological Sciences, Faculty of Science, National University of Singapore, 117543, Singapore
2 Institute of Oceanology, Chinese Academy of Sciences, Qingdao 266071, China
Correspondence
Ka Yin Leung
dbslky{at}nus.edu.sg
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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escC restored the export of EseB and EseD into the culture supernatant. Further studies showed that EscC is not a secreted protein and is located on the membrane and in the cytoplasm. Mutation of escC did not affect the transcription of eseB but reduced the amount of EseB as measured by using an EseB–LacZ fusion protein in Ed. tarda. Co-purification studies demonstrated that EscC formed complexes with EseB and EseD. The results suggest that EscC functions as a T3SS chaperone for the putative translocon components EseB and EseD in Ed. tarda.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). In humans, Ed. tarda can cause gastro- and extra-intestinal infections, including myonecrosis (Slaven et al., 2001), septic arthritis (Osiri et al., 1997), bacteraemia (Yang & Wang, 1999), wound infections (Banks, 1992) and peritonitis (Clarridge et al., 1980). Ed. tarda also causes haemorrhagic septicaemia in many farmed and feral fish and other aquatic animals, and leads to great losses in aquaculture industries (Thune et al., 1993). Therefore, it is important to study the pathogenesis of Ed. tarda and find suitable strategies to prevent these diseases.
Many factors are reported to contribute to the pathogenesis of Ed. tarda. These include the abilities to adhere to, invade and replicate within epithelial cells (Janda et al., 1991; Ling et al., 2000) and fish tissues (Ling et al., 2001) and to resist serum- (Janda et al., 1991; Ling et al., 2000) as well as phagocyte-mediated killing (Srinivasa Rao et al., 2001). Ed. tarda also produces toxins and exoenzymes such as haemolysins (Hirono et al., 1997), catalases (Srinivasa Rao et al., 2003b) and dermatotoxins (Ullah & Arai, 1983) for disseminating infection. Recently, we have identified two important virulence mechanisms that are related to secretion systems, namely, a type III secretion system (T3SS) (Tan et al., 2005) and a novel secretion system, EVP (Ed. tarda virulence protein) (Srinivasa Rao et al., 2004). These two secretion systems cross-talk through a two-component system EsrA–EsrB and a regulator EsrC (Zheng et al., 2005). Disruption of the T3SS or EVP gene cluster resulted in 
1–2 log increase in LD50 in the blue gourami (Trichogaster trichopterus Pallas) host.
The T3SS is a contact-dependent protein secretion system and it is generally composed of about 20 proteins, including apparatus, effectors, regulators and chaperones (Hueck, 1998). The translocon proteins of the apparatus form a needle structure 2.8 nm in diameter to inject effector proteins directly into eukaryotic cells (Marlovits et al., 2004), where the normal cellular functions are subverted for the benefit of the bacteria. In Ed. tarda, a T3SS was identified by using a combination of genomics and proteomics approaches (Tan et al., 2002; Srinivasa Rao et al., 2003a; Tan et al., 2005). The T3SS of Ed. tarda PPD130/91 contains 35 open reading frames, which include Ed. tarda secretion system apparatus (esa), chaperones (esc), effectors (ese), regulators (esr) and some unknown genes (orf) (Tan et al., 2005). By using two-dimensional electrophoresis (2-DE), EseB, EseC and EseD were identified as the major components of the extracellular proteins (ECPs) and their secretion was shown to be T3SS-dependent. These three Ese proteins contribute to Ed. tarda pathogenesis as mutation of eseB, eseC or eseD led to a 10-fold decrease in virulence compared to the wild-type (Tan et al., 2005). Sequence analysis showed that EseB, EseC and EseD are homologous to EspA, EspD and EspB, respectively, of enteropathogenic Escherichia coli (EPEC) (Tan et al., 2005). EspA forms a sheath-like structure, and EspB and EspD form a translocon pore in EPEC. EspA, EspB and EspD together constitute a molecular syringe and channel effector proteins into the host cells (Ide et al., 2001). In addition, the homologues of EseB, EseC and EseD in Salmonella (SseB, SseC and SseD, respectively) have also been shown to function as translocon components, and to be essential for translocation of the effectors (Nikolaus et al., 2001). The sequence similarities of EseB, EseC and EseD to their homologues in EPEC and Salmonella suggest that these three Ese proteins may have similar functions, forming a translocon complex to facilitate the translocation of effectors.
Many of the T3SS secreted proteins are dependent on specific chaperones for secretion as well as stabilization in the bacterial cytosol prior to secretion. The binding of a chaperone with the secreted protein can prevent premature interaction of a secreted protein with other secreted protein(s) and/or with parts of the secretion and translocation machinery (Wattiau et al., 1996; Bennett & Hughes, 2000). In addition, chaperones were shown to be required for the regulation of genes in T3SS (Darwin & Miller, 2000, 2001) and conferred secretion pathway specificity to their cognate secreted proteins (Lee & Galan, 2004). In Ed. tarda, EscA and EscB have been proposed to be the chaperones for T3SS proteins (Tan et al., 2005). However, the chaperones for the secreted proteins EseB, EseC and EseD have not been characterized. In the present study, we first determined that EseB, EseC and EseD formed a complex after their secretion, which is consistent with their possible roles as translocon components. We also found that Orf2 is a chaperone of EseB and EseD, and thus it was renamed EscC.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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. Ed. tarda stains were grown in tryptic soy broth (TSB) or Dulbecco's modified Eagle medium (DMEM) at 25 °C without shaking, while E. coli strains were cultured in Luria broth (LB) at 37 °C. Cultivation of bacteria in DMEM was carried out in a 5 % (v/v) CO2 atmosphere. The appropriate antibiotics were added when necessary at the following concentrations: ampicillin (Amp, 100 µg ml–1); kanamycin (Km, 50 µg ml–1); colistin (Col, 12.5 µg ml–1); chloramphenicol (Cm, 30 µg ml–1).
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) was used to generate in-frame deletion of orf2 (escC) and eseE on the Ed. tarda wild-type PPD130/91 chromosome as described previously (Zheng et al., 2005). For the construction of escC, two PCR fragments were generated from PPD130/91 genomic DNA with the primer pairs escC-for (5'-AGGTACCGTTAGTCCTCGGCTGGTGCT-3') plus escC-int-rev (5'-ATGGATGGCGTGTTGGCT-3') and escC-int-for (5'-GCCAACACGCCATCCATTTCATCTAACTCCATCCCTC-3') plus escC-rev (5'-TGGTACCGGCGTAGTCCTCTGCGTCG-3'). The resulting products were a 767 bp fragment, containing the upstream region of escC, and a 791 bp fragment, containing the downstream region of escC, respectively. A 17 bp overlap in the sequences (underlined) permitted amplification of a 1558 bp product during a second PCR with primers escC-for and escC-rev, both of which introduced a KpnI restriction site (bold). The resulting PCR product, containing a deletion from nucleotides 22 to 339 of escC, was ligated into suicide vector pRE112 (Cmr) (Edwards et al., 1998) and transformed into SM10 
pir. The single crossover mutants were obtained by conjugal transfer of the resulting plasmid into Ed. tarda PPD130/91. Deletion mutants were screened on 10 % sucrose TSA plates. The construction of the eseE deletion mutant followed the same protocol. The primers used for eseE were eseE-for (5'-TGGTACCGCAGTTCATGCTGGAGGA-3' plus eseE-int-rev (5'-GTCATCCATCGTGCCTCC-3') and eseE-int-for (5'-GAGGCACGATGGATGACCTGTGGCTGCGCTATTGCTGC-3') plus eseE-rev (5'-AGGTACCAGGCTCCTTAGGGTTGATT-3').
For the construction of LacZ transcriptional or translational strains, the eseB internal fragment was amplified with the forward primer eseB-int-for (5'-GAATTCCATGACGCTATTCACCGAT-3') and the reverse primer eseB-int-rev (5'-TTCTAGACGGATATTCTGGGCGATGG-3') from PPD130/91 genomic DNA. The PCR product was digested with XbaI and EcoRI and ligated into the same sites within pVIK112 or pVIK111. The resulting plasmids were then transferred from S17-1pir into Ed. tarda by conjugation and the LacZ transcriptional or translational strains were selected for kanamycin and colistin resistance.
To generate GST hybrid proteins, eseB, eseC and eseD were amplified from PPD130/91 chromosomal DNA and ligated into the pGEX-4T-1 vector. The plasmid expressing the His-tagged EseB protein was constructed by ligating the eseB gene into the pET32a vector.
For the construction of pSAescC-FLAG, the complete escC gene was amplified from PPD130/91 genomic DNA. The PCR product was digested with SalI and EcoRI and ligated into the same sites within the pSA10 plasmid (Schlosser-Silverman et al., 2000).
To generate co-expression plasmids pETB-D and pETD-D, eseB and eseD, respectively, were amplified from the Ed. tarda PPD130/91 chromosome and cloned into BamHI and HindIII (eseB) or BamHI and EcoRI (eseD) sites in pETDuet-1 (Novagen). The resulting plasmids were then digested with NdeI and XhoI, and escC PCR product amplified from the Ed. tarda wild-type chromosome and digested with the same enzymes was introduced into the constructs.
Production of recombinant EseB, EseC and EseD and generation of polyclonal antibodies.
The recombinant proteins GST-EseB, GST-EseC and GST-EseD were expressed overnight at 25 °C and purified by affinity chromatography with Sepharose 4B-glutathione resin under conditions recommended by the manufacturer (Amersham Biosciences). The purified GST-EseB, GST-EseC and GST-EseD proteins were used to immunize New Zealand White rabbits. For co-immunoprecipitation, the anti-EseB antibody in the immune serum was purified by affinity adsorption onto a nitrocellulose membrane blotted with the His-tagged EseB fusion protein. Briefly, His-tagged EseB protein was expressed from the pET32a expression vector and purified with Ni-NTA resin. The purified His-tagged EseB was then blotted onto the nitrocellulose membrane and blocked with 5 % milk. The membrane was then incubated with the anti-EseB serum. The anti-EseB antibody adsorbed on the membrane was eluted with 100 mM glycine (pH 2.0). The eluate was centrifuged briefly and neutralized with 0.1 vol. 1.5 M Tris (pH 8.0). The Tris buffer was replaced with PBS by spinning through an Amicon column (Millipore).
SDS-PAGE, 2-DE and Western analysis of proteins.
Protein samples were prepared as described previously (Srinivasa Rao et al., 2004). Proteins were separated on 12 % SDS-polyacrylamide gels. For Western analysis, proteins were transferred to PVDF membrane with a semi-dry system and examined by using the SuperSignal WestPico Chemiluminescent substrate under conditions recommended by the manufacturer (Pierce). EseB, EseC and EseD were detected by the addition of diluted anti-EseB (1 : 10 000), anti-EseC (1 : 10 000) and anti-EseD (1 : 5000) polyclonal antisera, respectively, followed by a 1 : 5000 dilution of mouse anti-rabbit IgG HRP (Santa Cruz Biotechnology). For detection of EscC-FLAG, the anti-FLAG M2 monoclonal antibody (Sigma) was used at 1 : 2500. Protein isolation and 2-DE were performed as previously described (Srinivasa Rao et al., 2004). Proteins were identified by MALDI-TOF MS as described elsewhere (Zheng et al., 2005). Anti-DnaK monoclonal antibody (Stressgen) was used at 1 : 2000, and anti-EvpA1 (J. Zheng & K. Y. Leung, unpublished data) was used at 1 : 5000.
Co-immunoprecipitation of EseB, EseC and EseD.
ECPs from Ed. tarda PPD130/91 were prepared and quantified as described previously to obtain high concentrations (Tan et al., 2002). The Seize Primary Immunoprecipitation kit (Pierce) was used for the co-immunoprecipitation experiment. Generally, 200 µl of slurry and 100 µg of purified anti-EseB antibody were mixed and incubated at room temperature for 4 h with gentle shaking. After washing the mixture according to the product manual, a 50 µg freshly prepared ECP sample was loaded. The mixtures were incubated overnight at 4 °C with shaking. The beads were eluted with elution buffer and the eluted sample was subjected to SDS-PAGE, followed by Western analysis with the anti-EseB, anti-EseC or anti-EseD antibody.
Fractionation of bacterial cells.
Bacteria were fractionated as described by Neyt & Cornelis (1999). Ed. tarda cultured in DMEM was harvested by 10 min centrifugation at 5000 g at 4 °C. The ECPs were prepared by precipitation of the supernatant with TCA as described previously (Srinivasa Rao et al., 2004). The bacterial pellet was resuspended in cold 10 mM Tris/HCl (pH 8.0), 5 mM MgCl2, then sonicated. The intact cells were pelleted by centrifugation at 10 000 g for 5 min. The supernatant was then centrifuged for 30 min at 100 000 g to separate the membrane fraction (insoluble fraction, pellet) from the cytosolic fraction (soluble fraction). The membrane fraction (pellet) was washed once with cold 10 mM Tris/HCl (pH 8.0), 5 mM MgCl2 and dissolved in an appropriate volume of ReadyPrep reagent 3 (Bio-Rad) for SDS-PAGE.
β-Galactosidase assays.
Ed. tarda strains were grown in DMEM for 24 h at 25 °C with 5 % (v/v) CO2. β-Galactosidase activities were determined with cells permeabilized with SDS and chloroform as described by Miller (1972).
Co-purification assay.
The ability of a Ni2+-NTA column (Qiagen) to bind 6His-EseB-EscC and 6His-EseD-EscC complexes was determined according to the product manual with slight modifications. Briefly, 6His-EseB and EscC, and 6His-EseD and EscC were expressed from plasmids pETB-D and pETD-D, respectively, by induction with 0.5 mM IPTG. The bacterial pellets were resuspended in buffer containing 300 mM NaCl, 50 mM Tris (pH 8.0) and 10 mM β-mercaptoethanol supplemented with 20 mM imidazole, and lysed by sonication. The lysates were cleared by centrifugation and incubated at 4 °C for 1 h with 50 µl Ni2+-NTA resin (Qiagen) with gentle mixing. The suspensions were then transferred into a column, followed by five washes with buffer containing 300 mM NaCl, 50 mM Tris (pH 8.0) and 10 mM β-mercaptoethanol supplemented with 30 mM imidazole. The bound proteins from the column were eluted with 50 µl buffer containing 300 mM NaCl, 50 mM Tris (pH 8.0) and 10 mM β-mercaptoethanol supplemented with 200 mM imidazole.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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), suggesting that they may have similar functions in Ed. tarda. In several Gram-negative pathogens, translocon components of T3SS have been shown to form homo- and hetero-oligomers after their secretion (Hartland et al., 2000; Büttner & Bonas, 2002). Bioinformatics analysis with COILS (http://www.ch.embnet.org/software/COILS_form.html) revealed the coiled-coil regions in EseB, EseC and EseD, which are implicated in the mediation of homo- or hetero-oligomeric protein–protein interactions (Lupas, 1996; Pallen et al., 1997). To test whether these three proteins are interacting with one another, co-immunoprecipitations were performed. The ECPs from Ed. tarda PPD130/91 were harvested and the anti-EseB antibody was used to co-immunoprecipitate the protein complex. The eseB mutant was used as a negative control. The flow-throughs and the immunoprecipitates were then subjected to Western blot analysis. As shown in Fig. 1, EseB, EseC and EseD, and EseC and EseD were detected in the flow-throughs of the wild-type ECPs and the eseB ECPs, respectively (lanes 1 and 2). However, the three Ese proteins (EseB, EseC and EseD) were only detected in the immunoprecipitate of the wild-type ECPs and not in the immunoprecipitate of the eseB ECPs (lanes 3 and 4). These results indicated that EseC and EseD specifically interact with EseB, and these three Ese proteins form a protein complex. Furthermore, a control using anti-GFP rabbit antibody did not co-immunoprecipitate any EseB, EseC or EseD proteins, suggesting that the immunoprecipitation was specific (data not shown). Therefore, our bioinformatics analysis and co-immunoprecipitation results suggested that EseB, EseC and EseD form a protein complex after secretion, which is consistent with their putative functions as translocon components.
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). Three deletion mutants were constructed and autoaggregation of overnight Ed. tarda cultures in DMEM was used as a quick assay to confirm the secretion of EseB. Our previous results suggested that extracellular EseB is required for the autoaggregation phenotype (Tan et al., 2005). Autoaggregation was found for mutants escA and escB (Z. L. Mo & K. Y. Leung, unpublished data) as well as eseE, which suggests that EseB is secreted in these mutants. We also analysed Orf2, which is located immediately upstream of eseB (Fig. 2a). A homology search of Orf2 against the NCBI database did not retrieve any characterized homologues. However, the close proximity of orf2 and eseB suggests that Orf2 may be the chaperone for EseB. To investigate the role of orf2, an in-frame deletion mutant orf2 was constructed. The resulting mutant lacked an internal fragment of 318 bp, and contained the first 21 bp and the last 6 bp of the orf2 gene. The orf2 mutant did not show an autoaggregation phenotype after 24 h of culture in DMEM. The results of 2-DE revealed that EseB and EseD but not EseC were absent in the ECPs of the orf2 mutant (Fig. 2b, d). Complementation of the orf2 mutant in trans with pSAorf2-FLAG restored the secretion of EseB and EseD in the supernatant and the autoaggregation phenotype. These results suggested that Orf2 is essential for the secretion of EseB and EseD. In contrast, EseC but not EseB and EseD was absent from the ECPs in the deletion mutant eseE (Fig. 2c), indicating that EseE is essential for the secretion of EseC.
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-helices (Bennett & Hughes, 2000; Cornelis & Van Gijsegem, 2000), and these characteristics were observed in Orf2. The predicted molecular mass of Orf2 is 13 kDa, which is within the 10–15 kDa range of most known T3SS chaperones. It has an -helical structure, and a stretch of N-terminal sequence with strong characteristics of coiled-coils motifs. Thus, we renamed orf2 as escC (Ed. tarda secretion system chaperone C). However, the predicted pI of EscC is 7.3, which is near neutral and differs from the predicted acidic pI of most identified type III cytosolic chaperones as well as the basic pI of chaperone SseA from Salmonella SPI-2 T3SS (Zurawski & Stein, 2003).
To localize EscC in Ed. tarda, the escC mutant was complemented with pSAescC-FLAG and the secreted proteins were collected. After sonication, the soluble and insoluble fractions of Ed. tarda were separated by centrifugation. The three fractions were then analysed by Western blotting with an anti-FLAG antibody. As shown in Fig. 3, EscC-FLAG was found in both the soluble (bacterial cytoplasm) and the insoluble fractions (bacterial membranes) but not in the ECPs. In contrast, no EscC-FLAG was detected in the escC background (Fig. 3). DnaK, a cytosolic protein, was found only in the soluble fractions and not in the ECPs or in the insoluble fractions (Fig. 3), indicating that the membrane fraction was not contaminated by the bacterial cytoplasm. Taken together, these results suggest that EscC is not a secreted protein but a protein located in the cytoplasm or on the bacterial membrane. However, we could not eliminate the possibility that EscC formed small insoluble aggregates and thus was detected in the insoluble fractions.
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escC mutant and its complemented strain were separated by SDS-PAGE and immunoblotted with either anti-EseB or anti-EseD antibody. Samples were loaded to represent the same number of bacteria for each strain. As shown in Fig. 4(a), the amounts of EseB and EseD were reduced in the escC background. After pSAescC-FLAG was introduced into the escC mutant, EseB and EseD were restored to nearly the same levels as in the wild-type (Fig. 4a). However, the amount of EvpA1, a non-T3SS secreted protein used as a control, was nearly the same in the different strains (Fig. 4a). Taken together, these results suggest that EscC is required for the accumulation of EseB and EseD. We also used 2-DE to analyse the total bacterial proteins of the wild-type and the escC mutant, and the results further confirmed the reduction of EseB and EseD in escC (Fig. 4b). Thus, EscC is required for the accumulation and stabilization of EseB and EseD in Ed. tarda.
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, 2001). The effect of escC on EseB and EseD accumulation could also be explained if escC affected their transcription. To address whether or not the decrease of EseB and EseD accumulation was due to decreased transcription, we examined the effect of a deletion mutation in escC on EseB at the transcription and translation levels. pVIK112 is a transcriptional fusion suicide plasmid carrying lacZY and has been used to study the regulation of genes at the transcriptional level (Kalogeraki & Winans, 1997). We cloned a 462 bp fragment of eseB and generated the plasmid pVIK212, in which eseB was in transcriptional fusion with lacZY. The plasmid was integrated by conjugation into the chromosomes of Ed. tarda wild-type PPD130/91, escC and esrC mutants, creating the strains eseB : : pVIK212, escC eseB : : pVIK212 and esrC eseB : : pVIK212, respectively. The β-galactosidase activity was then examined. Strong β-galactosidase activity was detected in eseB : : pVIK212 (172±9.8 Miller units; n=3), and nearly the same β-galactosidase activity was found in escC eseB : : pVIK212 (185±11 Miller units; n=3). However, the β-galactosidase activity decreased significantly (P<0.01) in esrC eseB : : pVIK212 (24±1.0 Miller units; n=3). These results further confirm the role of esrC as a transcriptional regulator (Zheng et al., 2005) and suggest that the deletion of escC does not affect the transcription of eseB. Thus, EscC is not a transcriptional regulator like EsrC. In order to further address the role of escC in the stabilization of EseB and EseD, pVIK111, which is a translational fusion suicide plasmid carrying lacZY (Kalogeraki & Winans, 1997), was used to construct the translational fusion of eseB with lacZY. The internal fragment eseB was cloned into pVIK111 and generated the plasmid pVIK211. The resulting plasmid was integrated by conjugation into the chromosome of Ed. tarda wild-type PPD130/91, escC and esrC mutants, creating the strains eseB : : pVIK211, escC eseB : : pVIK211 and esrC eseB : : pVIK211. The β-galactosidase activity in these strains was examined. As expected, mutation of the transcriptional regulator esrC decreased the level of EseB–LacZ fusion proteins in the bacteria and the β-galactosidase activity in esrC eseB : : pVIK211 (437±10 Miller units; n=3) was reduced more than sixfold compared to that in eseB : : pVIK211 (3075±124 Miller units; n=3). A decreased level of β-galactosidase activity in escC eseB : : pVIK211 (2183±300 Miller units; n=3) was also observed, demonstrating that mutation of escC affected the EseB–LacZ level in the bacteria. These results suggest that deletion of escC affected the stability of EseB in the cytoplasm, as escC did not affect the transcription of eseB. Taken together, these results suggest that EscC is not required for the transcription of eseB but is important for the accumulation of EseB in the cytoplasm. However, we still can not rule out the possibility that EscC could have a role in the translation of EseB.
EscC interacts with both EseB and EseD
To determine the interaction between EscC and EseB, we co-expressed EseB and EscC in plasmid pETDuet-1. The resulting plasmid produced EseB with a 6His tag at the N-terminus (6His–EseB) and EscC in E. coli. Lysates of E. coli expressing 6His–EseB and EscC were incubated with a Ni2+-NTA resin column. The binding substrates were eluted and examined on a Coomassie blue stained SDS-PAGE gel. As shown in Fig. 5(a), co-expression of 6His-EseB with EscC in E. coli led to two proteins being retained on the column and they could be eluted together by using a high concentration (200 mM) of imidazole. The molecular masses of these two proteins on the SDS-PAGE corresponded to those of 6His-EseB and EscC, respectively, and the identity of the protein corresponding to EscC was further confirmed by MALDI-TOF MS. However, when lysate of E. coli expressing 6His–EseB alone was applied to the column, the band corresponding to EscC was not observed from the eluted sample (Fig. 5a). In another control experiment, EscC was not retained on a Ni2+-NTA column when lysates of E. coli expressing EscC only were used (Fig. 5a). Thus, our results suggest that EscC specifically interacts with EseB.
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, EscC co-purified with 6His–EseD when lysates of E. coli expressing 6His–EseD together with EscC were incubated in a Ni2+-NTA resin column (Fig. 5b). As a control, EscC alone could not bind to Ni-NTA resin and flowed through the column (Fig. 5b), suggesting that EseD specifically interacts with EscC. The co-purification of EseB or EseD with EscC strongly supports the direct association of EseB or EseD with EscC, which is consistent with the chaperone function of EseBD.
EscC is required for virulence
The contribution of EscC to the pathogenesis of Ed. tarda was investigated by examining the LD50 values in blue gourami fish. The deletion of escC increased the LD50 value by about 1 log (105.9±0.3; n=3) as compared to the wild-type (105.0±0.2, n=3). This increase is comparable to that of eseB or eseD mutants (LD50 of eseB=106.0, LD50 of eseD=106.1) (Tan et al., 2005), but is less than that of the regulator mutant esrC (LD50=107.6) (Zheng et al., 2005). These results suggest that EscC is required for virulence and contributes to the pathogenesis of Ed. tarda.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). EseB, EseC and EseD are the major ECPs in Ed. tarda and their secretion is T3SS-dependent. The amino acid sequences of these three Ese proteins are homologous to those of the translocon components in other bacteria (Tan et al., 2005). EseB was predicted to be a major component of the Ed. tarda T3SS translocon, based on its similarity to EspA, and to form a translocon complex together with EseC and EseD on the bacterial cell surface (Tan et al., 2005). In this study, we showed that EseB, EseC and EseD formed a complex after secretion, which is consistent with their roles as translocon components. The interaction of EseB, EseC and EseD is reminiscent of that of EspA and EspB from EPEC. EspA interacted with EspB and this interaction was necessary for EPEC type III effector translocation. However, an interaction between EspA (EseB) and EspD (EseC) was not shown in EPEC as co-immunoprecipitation of EspA filaments with EspD failed to yield positive results (Hartland et al., 2000). In contrast to EPEC, EseB (EspA) interacted not only with EseD (EspB) but also with EseC (EspD). The reason for this difference is not clear, but it may be due to the organisms' respective infection mechanisms. The interaction of EseB, EseC and EseD is probably essential for the translocation of Ed. tarda T3SS effector(s) although no effector proteins have been reported in Ed. tarda thus far. Future study will investigate the actual roles of EseB, EseC and EseD by evaluating their abilities to form pores in biological membranes.
The secretion of T3SS translocon components is generally chaperone-dependent. Up to now, various chaperones involved in the translocon components have been reported in EPEC, Salmonella and Shigella species. However, their roles in the secretion of EseB and EseD in Ed. tarda are still unclear. We report here the identification and characterization of EscC, a specific chaperone for EseB and EseD. EscC shares most of the characteristics of typical T3SS chaperones, such as -helical propensity, small size, and a carboxy-terminal amphipathic -helical segment (Wattiau et al., 1996; Bennett & Hughes, 2000). However, the predicted pI of EscC is near neutral, which is different from most characterized acidic or basic type III chaperones (Wattiau et al., 1996; Bennett & Hughes, 2000; Page & Parsot, 2002). Chaperones with near neutral pI are not unprecedented. CesD in EPEC has a predicted pI of 7.4 (Wainwright & Kaper, 1998). Furthermore, the association of neutral pI chaperones with their cognate proteins remains uncharacterized. Crystallographic study of the EscC complex is needed to provide insights into the mechanism. Another atypical feature of EscC is its location both on the membrane and in the cytoplasm. Most of the characterized type III chaperones are found in the cytosol, and only a few chaperones have membrane associations. For example, CesD from EPEC and SseA from Salmonella were localized to both the cytoplasmic membrane and cytoplasm (Wainwright & Kaper, 1998; Zurawski & Stein, 2003). The mechanism of membrane localization of chaperones is unclear. Cycles of chaperone membrane insertion and deinsertion may be a possible explanation for the dual location. The cytoplasmic chaperone CesT has been demonstrated to recruit Tir and co-localize to the EPEC inner membrane (Elliott et al., 1999; Thomas et al., 2005). The ATPase of T3SS plays critical roles in chaperone release from and unfolding of the cognate secreted protein in an ATP-dependent manner (Akeda & Galan, 2005). Thus, membrane insertion may be the initial step in the reorganization of the secreted protein–chaperone complex with the T3SS ATPase.
T3SS chaperones have been divided into three classes: class I, chaperones which associate with one or several effectors, typified by the Yersinia SycE chaperone, which serves a single substrate (YopE); class II, chaperones which associate with translocators, typified by the Yersinia SycD protein, which is required for the proper secretion of two proteins (YopB and YopD); and class III, chaperones of the flagellar export system, which are believed to be the ancestors of the T3SS (Neyt & Cornelis, 1999; Page & Parsot, 2002; Parsot et al., 2003). Chaperones from the flagellar system participate in flagellar assembly and recognize the C-terminal domain of their substrates, and these C-terminal domains are thought to form amphipathic helices that mediate interactions between the subunits of the flagellum (Fraser et al., 1999; Auvray et al., 2001). Despite the evidence for EscC as a chaperone, this protein also possesses features different from most other chaperones. For example the EscC substrate of EseB is neither an effector nor a translocon pore, and EscC could also be detected in the bacterial membrane. The characteristics of EscC are reminiscent of SseA of Salmonella SPI-2. SseA interacts with the C-terminal coiled-coil domain of SseB and is classified as a class III flagellar chaperone (Zurawski & Stein, 2004). Similarly, coiled-coil domains in the C-terminal regions of EseB and EseD were predicted with COILS. EscC may interact with EseB and EseD at their coiled-coil domains. Thus, EscC seems functionally more similar to SseA. However, EscC does not share sequence similarity with SseA. The predicted coiled-coil domain of EscC is in the N-terminal region while that of SseA is in the C-terminal region. Another chaperone functionally similar to EscC is CesAB of EPEC, which is required for the secretion and stability of EspA (the homologue of EseB) and EspB (the homologue of EseD) (Creasey et al., 2003). CesAB has been shown to interact with EspA with the N-terminal coiled-coil domain (Yip et al., 2005). Although there is a lack of sequence similarity between EscC and CesAB, they may have similar three-dimensional structures, and the coiled-coil domain of EscC may be important for its binding with EseB or EseD. Further studies to delineate how EscC interacts with EseB and EseD will allow better comparisons with well defined chaperone families.
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ACKNOWLEDGEMENTS |
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Edited by: P. van der Ley
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Received 6 December 2006;
revised 14 February 2007;
accepted 19 February 2007.
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