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1 Zoonotic and Animal Pathogens Research Laboratory, Centre for Infectious Disease, The Chancellor's Building, 49 Little France Crescent, Edinburgh EH16 4SB, UK
2 Moredun Research Institute, Pentlands Science Park, Bush Loan, Penicuik, Midlothian EH26 0PZ, UK
3 School of Biosciences, University of Birmingham, Birmingham B15 2TT, UK
4 Roslin Institute, Roslin BioCentre, Midlothian EH25 9PS, UK
5 Institute for Comparative Medicine, University of Glasgow, Glasgow G61 1QH, UK
Correspondence
David L. Gally
d.gally{at}ed.ac.uk
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ABSTRACT |
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These authors contributed equally to this work.
The array data discussed in this publication have been deposited in the NCBI Gene Expression Omnibus (GEO; http://www.ncbi.nlm.nih.gov/geo/) and are accessible through GEO series accession number GSE6296.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Coombes & Finlay, 2005; Cornelis, 2002; Galan, 2001; Journet et al., 2005; Stebbins & Galan, 2003; Troisfontaines & Cornelis, 2005). The effectors have numerous functions, including inhibition of phagocytosis, invasion, cytotoxicity and bacterial attachment, mostly through effects on signal-transduction pathways (Bruckner et al., 2005; Crane et al., 2001; Garmendia et al., 2005; Hapfelmeier et al., 2005; Kenny et al., 1997; Kim et al., 2005; Knodler et al., 2005; Nataro & Kaper, 1998; Patel & Galan, 2005; Yuk et al., 2000; Zhang & Bliska, 2005). Our understanding of EPEC and EHEC pathogenesis has benefited from the study of the mouse pathogen Citrobacter rodentium, which also expresses a T3SS encoded from a homologous locus of enterocyte effacement (LEE), and this pathogen forms characteristic attaching and effacing lesions in vivo and in eukaryotic HeLa cells (Deng et al., 2004; Gruenheid et al., 2004; Mundy et al., 2004b). Through an understanding of the regulation of LEE expression in C. rodentium, a number of putative type III (T3)-secreted effector proteins have been identified (Deng et al., 2005). As the genes for these proteins are not encoded on the LEE, they are termed non-LEE-encoded effector (Nle) proteins. Seven putative effector proteins have been identified from C. rodentium and designated NleA–G. Subsequent research has established that NleA is conserved in many pathogens containing an LEE, including EHEC O157 : H7 from which it is expressed, exported and localized to the Golgi apparatus (Gruenheid et al., 2004; Mundy et al., 2004a, b). Less is known about the other putative effector proteins, although the T3-dependent export of NleC and NleD can be induced from EPEC, and deletion of these genes in E. coli O157 : H7, has no obvious impact on pathology in lambs (Marches et al., 2005). More recently, an nleB mutant has been shown to be important for the colonization of mice by C. rodentium, and induced export of an NleB : : Bla fusion from C. rodentium and EPEC has been shown to be T3-dependent (Kelly et al., 2006). A recent study of proteins exported from an sepL mutant of E. coli O157 : H7 has identified 39 potential effectors in the bacterial supernatant, and induced plasmid-based expression of these effectors from EPEC results in their delivery into host cells (Tobe et al., 2006). Therefore, apart from NleA, the expression and secretion of the different Nle proteins from wild-type E. coli O157 : H7 are unknown.
There is considerable variation in the levels of T3-associated proteins secreted from EHEC O157 : H7 strains. This has been shown to be due, in part, to heterogeneous expression of the EspADB translocon apparatus (Roe et al., 2003, 2004). The expression of the translocated intimin receptor (Tir) is co-ordinated with production of EspA filaments at the level of the single cell (Roe et al., 2004). The molecular basis of this heterogeneity and whether it governs expression of effector proteins located outside the LEE are not known. The aim of the current study was to analyse NleA–F expression and secretion from E. coli O157 : H7. To address this, we screened for Nle proteins in the supernatant of an E. coli O157 : H7 strain capable of high-level secretion. This was carried out by tandem MS of bacterial supernatants, as well as the construction of full-length translational fusions of the Nle proteins to 
-lactamase to allow detection by Western blotting. Through the construction of promoter fusions to both green fluorescent protein (GFP) and red fluorescent protein (RFP), and an analysis of global transcription, expression was analysed, as was co-ordination with EspA filament production. Expression of nleA–F in different genetic backgrounds and on contact with eukaryotic cells was also determined.
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METHODS |
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Western analysis.
Bacteria were cultured at 37 °C in MEM-HEPES medium to OD600 0.8. Secreted proteins were extracted by TCA precipitation as described previously (Roe et al., 2003). For protein-localization experiments, whole-cell fractions were prepared by centrifugation (20 min, 4000 g), washing twice in 20 ml PBS, and final suspension in 400 µl protein A buffer (Roe et al., 2003). Proteins were analysed by SDS-PAGE, and Western blotting for -lactamase was carried out as described by Karavolos et al. (2005).
MS analysis of secreted proteins.
E. coli strain TUV93-0 was grown statically to mid-exponential phase (OD600 0.4) in MEM-HEPES supplemented with 2 mM L-glutamine at 37 °C in a 5 % CO2 atmosphere. Bacteria were pelleted by centrifugation in a swing-out rotor at 4000 g for 40 min at 4 °C. The clarified supernatants were then filter-sterilized by passing through 0.2 µm low-protein-binding filters (Millipore) under a vacuum. Secreted proteins were then precipitated from the supernatant by the addition of TCA to 10 % (v/v) final volume, and incubated at 4 °C overnight. Precipitates were pelleted via centrifugation in a swing-out rotor at 4000 g for 40 min at 4 °C. Pellets were then washed twice in 20 ml ice-cold acetone and pelleted before being allowed to briefly air dry.
Protein pellets were resuspended in Laemmli sample buffer before separation on a 12 % SDS-PAGE Mini-PROTEAN III (Bio-Rad) gel. Resolved proteins were visualized using colloidal Coomassie blue stain (Genomics Solutions). The lane containing resolved secreted protein was excised from the gel and sliced horizontally into 25 equal slices of 
2.5 mm. These slices were then processed for MS, essentially as described by Batycka et al. (2006).
Liquid chromatography was performed using an Ultimate 3000 nano-HPLC system (Dionex; LC-Packings), comprising a WPS-3000 well-plate micro autosampler, an FLM-3000 flow manager and column compartment, a UVD-3000 UV detector, an LPG-3600 dual-gradient micropump, and an SRD-3600 solvent rack controlled by Chromeleon software. The monolithic column (200 µm internal diameter x5 cm; LC-Packings) was maintained at a constant 50 °C and was run at a final flow rate of 3 µl min–1. Samples of 4 µl were applied to the column by direct injection. Peptides were eluted by the application of a 15 min linear gradient from 8 to 45 % solvent B (80 % acetonitrile, 0.1 % formic acid) and directed through a 3 nl UV detector flow cell. The LC system was interfaced directly with a 3D high-capacity ion trap mass spectrometer (Esquire HCTplus; Bruker Daltonics) utilizing a low-volume (50 µl min–1 maximum) stainless steel nebulizer (Agilent), and electrospray ionization (ESI)-MS/MS analysis was initiated on a contact closure signal triggered by the Chromeleon software. m/z data were processed, and MASCOT-compatible files were created using DataAnalysis 3.2 software (Bruker Daltonics) with the following parameters: compounds (autoMS) threshold, 1000; number of compounds, 500; retention time window, 0.8 min. Searches were performed using MASCOT software (Perkins et al., 1999; Matrix Science) and an in-house E. coli O157 : H7 EDL933 database. The interpretation and presentation of MS/MS data were performed according to published guidelines (Taylor, 2005). The peptide and fragment mass tolerances were 2.5 and 0.8, respectively. Individual MS/MS spectra for peptides with a Mascot MOWSE (molecular weight search) score lower than 40 were inspected manually, and only included in the statistics if a series of at least four continuous y or b ions were observed.
Plasmid-based promoter fusion construction.
Promoters for nleA–E were amplified from strain ZAP193 and cloned into pAJR70 to create pTD-1A-pTD7El (Table 1). Fig. 1 shows the putative promoter regions that were amplified and cloned to create the constructs. For nleC and nleE, only small intergenic regions are present 5' of the ATG codons. The close proximity to the upstream gene presented the possibility of these genes being co-transcribed as part of an operon. To address this, two constructs were created that utilized primers spanning both putative promoter locations (Fig. 1). As an additional reporter, the enhanced RFP gene (rfp) was cloned into pACYC184. Primer pair RFP5'/RFP3' (Table 2) was used to amplify the DsRed T3_S4T gene (Sorensen et al., 2003). The resultant plasmid (pDW16) had single BamHI and KpnI sites 5' of the rfp+ gene, allowing promoters of interest to be cloned in-frame with the reporter gene. The promoters from nleA and nleB were cloned into pDW16 to create pTD-15A and pTD-16B (Table 1).
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). The slides were then examined by fluorescence microscopy using appropriate filter sets, and the images were captured as above.
Expression on contact with EBL cell lines.
Embryonic bovine cells (German Collection of Microorganisms and Cell Cultures, no. ACC192) were prepared and cultured as described previously (Roe et al., 2004). The ZAP193 strain transformed with the appropriate RFP reporter plasmids was cultured in MEM-HEPES to OD600 0.6 at 37 °C, added to the multichamber slide, and centrifuged onto the EBL cells (1000 g) for 5 min. The cells were fixed at intervals by removal of the culture and addition of 4 % paraformaldehyde. Time points analysed were 0, 5, 30 and 180 min after addition. Fluorescence analysis using Openlab and Leica software was then performed as described above and previously (Roe et al., 2004).
Plasmid-based translational fusions to -lactamase.
To allow the export of the Nle proteins to be assayed, translational fusions were created to -lactamase. The region amplified for these fusions consisted of the promoter regions described in Fig. 1
, but also covered the entire coding sequence. The PCR products were cloned into pAJR104 (Karavolos et al., 2005) to create pTD-8AT–pTD-14ELT (Table 1).
Whole-genome array analysis.
Preparation of mRNA, labelling, hybridization and analysis of array data were carried out as described by Zhang et al. (2004), with the following amendments. To provide the biological replicates, triplicate cultures of strain TUV93-0 were grown with shaking at 37 °C in MEM-HEPES or DMEM to OD600 0.6. Culture (15 ml) was mixed with 30 ml RNAprotect bacterial reagent (Qiagen), and an RNeasy minikit was used to prepare total RNA according to the manufacturer's instructions (Qiagen). Any contaminating DNA was removed using a DNase column kit (Qiagen). Total RNA was transcribed to Cy3- and Cy5-labelled cDNA, hybridized onto Corning GAPSII glass slides with the 6112 70-mer oligonucleotides of the Operon Array Ready E. coli set 1.0, as described by Zhang et al. (2004). The slides were washed and scanned, and data were analysed using Genepix and Genespring software as described by Zhang et al. (2004).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). Table 3 summarizes the highest MOWSE scores obtained for each of 10 recognized secreted proteins of E. coli O157 : H7. Proteins detected in supernatants included seven proteins known to be secreted via T3SS (EspA, EspB, EspD, Tir, Map, EspF and NleA), as well as proteins secreted independently of T3SS (EspP, StcE and EhxA). For all proteins except NleA, multiple tryptic peptides (at least three) were detected, giving confidence in the identification from the tandem MS data (Table 3). Although the MOWSE score for NleA was below the significance threshold, closer examination of the ion data from tandem MS analysis indicated a continuous stretch of 7 aa represented by y or b ions. This heptapeptide (GETPLTP) was searched against the entire NCBI database using BLAST with default settings. Despite the high probability of a random match, NleA/EspI represented top hits along with putative and hypothetical proteins from other bacterial genera, although the latter could be disregarded, since the sample material was a pure culture of E. coli O157 : H7.
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Table 4 shows a selection of genes on the E. coli chromosome that are differentially regulated when cultured in MEM-HEPES compared with DMEM. The majority of genes are unaffected in their level of transcription, with 88 % showing less than a twofold increase or decrease in transcription. Under the conditions used, 166 genes (3 % of the genome) showed significant (P
0.05) upregulation, with the O157 LEE pathogenicity island showing marked changes. For the LEE, genes encoding basal apparatus (escJ/escN), translocon (espA/espD/espB) and effector proteins (tir) showed significant changes in transcription when cultured in MEM-HEPES (Table 4). Analysis of nle genes showed that nleA–E gave a hybridization signal when cultured in DMEM or MEM-HEPES (Table 4). nleF gave no hybridization signal and was not investigated further in this study. The data showed that nleA displayed the most marked (ninefold) and significant increase (P=0.001) in transcript level when cultured in MEM-HEPES. Slight increases were detected for nleB (twofold), nleD (1.8-fold) and nleE (2.6-fold) (Table 4).
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). These fusion constructs were used to analyse the expression profiles of the nle genes in liquid culture and during contact with bovine epithelial cells. E. coli O157 : H7 strain ZAP193 was used for this work, as our previous research has characterized the expression of LEE-encoded factors in this background. When cultured in MEM-HEPES, both nleA : : gfp (pTD-1A) and nleB : : gfp (pTD-2B) fusions gave a high level of expression in exponential phase and into stationary phase (Fig. 2a). In comparison, nleC : : gfp (pTD-4l) and nleD : : gfp (pTD-5D) were expressed at a 20-fold lower level (determined at OD600 0.6) than nleA : : gfp (Fig. 2b). The reporter pTD-3Cs, which consisted of a 484 bp putative promoter region amplified immediately 5' of the nleC ATG was measured at <50 relative fluorescence units (RFU) throughout the growth curve. In addition, expression of either nleE : : gfp construct (pTD-6Es and pTD-7El) was <50 RFU through the growth curve in this medium (Fig. 2b). To validate the microarray results for MEM-HEPES versus DMEM expression of nleA and nleB, the expression of the nleA : : gfp and nleB : : gfp fusions was measured in the two media. At OD600 0.6 (the same optical density used for the microarray experiments), there was a 5.1-fold increase in nleA expression in MEM-HEPES compared to that in DMEM, and a 2.5-fold increase for nleB. These data are similar to the nine- and twofold differences, respectively, measured by microarray analysis (Table 4).
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). This production of EspA filaments is also co-ordinated with the expression of the LEE5 genes tir and eae (intimin). As both nleA and nleB showed a high level of expression when cultured in MEM-HEPES by both microarray and reporter gene assays, the possibility of co-ordinate regulation with the T3SS was addressed. Single-cell imaging of the fusions showed that nleB : : rfp+ was expressed in all the cells in the population (Fig. 3a), with no correlation to the expression of heterogeneous EspA filaments (data not shown). In contrast, nleA : : rfp+ expression was clearly heterogeneous, with only a subset of cells expressing rfp+ when cultured in MEM-HEPES (Fig. 3b). Co-staining for EspA filaments showed that the subset of cells actively transcribing nleA : : rfp+ was also producing the T3SS filament required for protein export (Fig. 3c). These data indicate that nleA is co-ordinately regulated with the translocon of E. coli O157 at the single-cell level.
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Expression of nleA–E on contact with EBL cell lines
The expression of the nleC, nleD and nleE reporter constructs in MEM-HEPES gave rise to the hypothesis that the culture conditions being tested did not contain the environmental signals required for the activation of their respective promoters. In order to test this hypothesis, the interaction of the promoter fusions was analysed during contact with eukaryotic cells. E. coli O157 : H7 ZAP193 was transformed with the nle : : gfp fusion plasmids (pTD1A-7El, Table 1), cultured in MEM-HEPES, and added to EBL cells. At different time points 0, 30 and 180 min, the supernatant was removed, the samples were fixed, and bacteria were stained with fluorescently labelled anti-O157 antibodies to allow identification by fluorescence microscopy. The expression of the six nle : : gfp fusions was quantified over the time course as described in Methods. During the initial culture to OD600 0.6, nleA : : gfp and nleB : : gfp fusions gave the highest level of expression, with lower but detectable levels from nleC and nleD fusions. As described above, nleA : : gfp gave a heterogeneous expression pattern (Fig. 4a, b). On contact with EBL cells, the expression of nleA : : gfp was markedly reduced, even within 30 min of contact with host cells (Fig. 4a). At 180 min, expression had fallen to <30 % of the initial level before cell contact. The nleB : : gfp fusion showed a reduction in expression to 18 % of the starting value over the same time course. A control plasmid consisting of the rpsM transcriptional fusion to GFP+ (Roe et al., 2004) gave consistent levels of expression throughout the course of the experiment (Fig. 4a). Constructs for nleC, nleD and nleE showed no stimulation on contact with EBL cells, indicating that the environmental signals from the EBL cells, or physical contact with eukaryotic cell surfaces, were not stimuli for their expression (data not shown).
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-lactamase fusion analysis-lactamase fusions to the whole proteins were constructed. These fusion proteins allowed the localization of the proteins to be investigated, including whether any export was dependent on T3 secretion. When cultured in MEM-HEPES, Western blotting for -lactamase detected NleA : : Bla, NleB : : Bla, NleC : : Bla, NleD : : Bla and NleE : : Bla in the bacterial whole-cell preparations (Fig. 5a). NleA : : Bla and NleD : : Bla were also detected in the supernatant fraction (Fig. 5b). Using an escN (T3SS ATPase) mutant, the export of NleA : : Bla and NleD : : Bla was shown to be dependent on the presence of a functional T3SS (Fig. 5c, e).
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), the production of NleA : : Bla and NleD : : Bla in the whole-cell fractions was also reduced in the ler mutant when compared with that in the wild-type strain (Fig. 5d). |
DISCUSSION |
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sepL mutant, with six clear homologues in E. coli O157 (Deng et al., 2004). Using a sepL mutant of E. coli O157, Tobe et al. (2006) have recently identified some 39 proteins, falling into over 20 separate families. These advances in identification of novel effector proteins raise the question of how the Nle family of effector proteins is regulated. If the effectors are secreted through the T3SS, the expectation would be that the expression of the effectors would be co-ordinated with that of the Esp translocation complex, as this is the physical apparatus required for injection of the proteins into eukaryotic cells. In this work, we characterized the secretome of an sepL wild-type strain to see which Nle effectors (from the originally described NleA–F) were secreted in co-ordination with the complete T3SS. Using tandem MS, we observed many of the established proteins that have been shown to be secreted by EHEC O157. These included the Esp family of translocon proteins (EspA, B, D and F) (Abe et al., 1998; Viswanathan et al., 2004), the translocated intimin receptor, Tir (DeVinney et al., 1999), the mitochondrion-associated protein, Map (Kenny & Jepson, 2000), secreted proteases EspP (Brunder et al., 1997) and StcE (Lathem et al., 2002) (both encoded on pO157), and enterohaemolysin (Schmidt et al., 1995) (also on pO157). Using this wild-type strain, only NleA of the Nle proteins was identified in the supernatants of E. coli O157 cultured in MEM-HEPES. Shotgun proteomics methods do not provide quantification of proteins, although levels may be inferred from data parameters such as the number of peptides detected for proteins; on that basis, NleA was secreted at very low levels compared to the LEE-encoded translocon (EspA, B, D) or effector (Tir, Map, EspF) proteins. These data suggested that either secretion of the other Nle proteins was at a very low level, or the culture conditions did not support expression or secretion of the effectors. We therefore analysed expression of the nleA–F genes using a combination of arrays and fusions in liquid media, and (for the fusions) during interaction with a bovine-derived eukaryotic cell line. To analyse the expression of the nleA–F genes on a DNA microarray, expression conditions were used that were highly selective for upregulation of the LEE. The hypothesis was that non-LEE-encoded effector proteins would be upregulated in a co-ordinated manner with the LEE operon, as the T3SS is the mechanism required for effector protein delivery. The E. coli O157 : H7 transcriptome of bacteria cultured in either MEM-HEPES or DMEM was compared. Transcript levels of LEE-encoded genes increased in MEM-HEPES, e.g. escJ (6.4-fold) and espB (16.7-fold). For the nle genes examined, nleA was markedly upregulated (ninefold), which correlated well with its detection in the bacterial supernatant and likely cross-regulation with the LEE. Expression of nleA was then examined using an nleA : : rfp promoter fusion that demonstrated co-ordinate expression with EspA filaments at the single-cell level. This result showed that a gene encoding an effector protein located outside the LEE locus is regulated co-ordinately with the physical apparatus for its delivery. In addition, we showed that NleA production and expression are dependent on the LEE-encoded regulator (ler). Therefore, NleA expression appears to be regulated by a two-stage process involving both LEE-encoded and non-LEE-encoded regulators.
We have demonstrated heterogeneous expression of a number of EHEC virulence factors, including EspA (Roe et al., 2003), intimin, tir and map (Roe et al., 2004), and now nleA, using fluorescent gene fusions. The molecular mechanism that controls this single-cell ‘cross-talk’ between multiple T3SS effectors and the translocon is known to be controlled by factors not encoded by the LEE (Roe et al., 2003, 2004). The likely biological function of heterogeneous expression is to restrict expression of antigenic factors such as type 1 fimbriae or T3SS needle EspA filaments, and also to prevent co-expression and therefore physical interference between surface factors on individual cells. Furthermore, co-ordination of EspA expression with effector proteins including NleA and Tir is logical, as this is the physical delivery system into eukaryotic cells. Indeed, a previous study has demonstrated the co-ordinated down-regulation of non-LEE-encoded factors on contact with eukaryotic membranes (Dahan et al., 2004).
In contrast to nleA, the expression and export of nleB–E are less clear. The gfp promoter fusion to nleB showed high-level expression throughout the growth curve, but no exported protein was detected by MS. The -lactamase fusion to NleB confirmed that no secretion was detectable and indicated that very little full-length protein was being produced inside the bacterial cell. Therefore, expression of NleB appears to be tightly controlled by a post-transcriptional mechanism under the conditions tested. While NleB has been shown to be required for the colonization of mice by C. rodentium (Kelly et al., 2006), it has yet to be identified as a colonization factor for EHEC.
The expression and regulation of nleC and nleE can be summarized relatively easily; very-low-level expression was observed by the use of gfp and bla fusions, and no protein was detectable by MS. As gene expression was low, coupled with no detectable protein, we can conclude that under the conditions tested, these factors were produced either at a low level or not at all. It is interesting to note that a deletion of nleC in EHEC O157 has shown no difference in the colonization of a lamb animal model (Marches et al., 2005).
NleD expression and export were the most complex, as MS analysis failed to detect this protein in the bacterial supernatant, even though a full-length NleD : : Bla fusion was detected in the whole-cell and supernatant fractions. The difference is likely to be a result of the high sensitivity of Western blot analysis. The secretion of the -lactamase fusion was shown to be dependent on a functional T3SS, and expression in the cell was reduced (twofold) in a ler mutant. One explanation for the low-level expression of nleC, nleD and nleE is a lack of appropriate signals in the liquid media used in this study. However, the finding that no additional activation of nleA : : gfp was observed after contact with eukaryotic cells suggests that neither T3-permissive liquid media nor contact with EBL eukaryotic cells over a 3 h time period provides signals for activation. The same applies to nleB–E, as no further expression of the promoters was measured following contact of the bacteria with eukaryotic cells, compared to culture in MEM-HEPES. This lack of activation must be considered in the context of previous research that has demonstrated down-regulation of LEE-encoded factors on contact with eukaryotic cells (Dahan et al., 2004; Roe et al., 2004). In our study, expression of both espJ and tccP was shown to be increased by culturing E. coli O157 : H7 in secretion-permissive conditions [espJ expression was increased threefold in MEM-HEPES (P=0.16) and tccP 2.4-fold (P=0.007)]. This co-ordinate expression agrees with earlier published data (Dahan et al., 2004). It is not possible to rule out the activation of promoters by additional signals in vivo.
Work to date has focused on the localization of the NleA–D proteins and their effects on eukaryotic cells. These questions have been addressed in studies that have relied on overexpression of the proteins by exogenous promoters, coupled with creation of constructs to reporters such as -lactamase and haemagglutinin. Such systems ensure high-level expression of the protein coupled with sensitive detection by Western analysis, and have proved very valuable: for example, the subcellular localization of NleA in eukaryotic cells has been demonstrated using such technology (Gruenheid et al., 2004). More recently, the repertoire of T3-secreted proteins has been expanded in EHEC O157 by analysis of an sepL deletion strain. SepL, along with SepD, is considered to act as a gating switch between translocon and effector-protein secretion. Mutation of sepL results in higher levels of effector-protein secretion, possibly by reducing competition for the export apparatus of EspA, EspB and EspD. In theory, this mutation then allows easier detection in the bacterial supernatant of effector proteins otherwise secreted at lower levels. A caveat with the sepL mutation approach is that the mechanism of SepL/SepD control of translocon secretion is unknown, and it is not possible to know whether the secretion profile of this mutant reflects that of the wild-type strain. Many of the targets identified have been overexpressed and detected in the bacterial supernatant, and furthermore can be exported into host cells using induced expression from EPEC as the delivery vehicle. Therefore, these proteins all have the potential to be effectors of E. coli O157 infecting host cells, but for the majority of these proteins, whether they are in fact expressed or secreted by wild-type E. coli O157 : H7 remains to be determined.
In conclusion, we have confirmed that NleA is exported from EHEC O157, and that this is dependent on a functional T3SS. Furthermore, we have demonstrated that expression of nleA is heterogeneous under the conditions tested. Only a proportion of bacteria expressed nleA, as determined by a gfp fusion, and this subpopulation correlated with bacteria that produced EspA filaments. The evidence from this work implies that NleD is produced at a low level, and that this protein is exported in a T3-dependent manner. No evidence for production of NleB, NleC and NleE was obtained, but specific environmental stimuli may be required for their expression.
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ACKNOWLEDGEMENTS |
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Edited by: M. P. Stevens
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Received 26 October 2006;
revised 18 December 2006;
accepted 9 January 2007.
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) and nleB : : gfp (pTD-2B, 
); (b) expression of nleC : : gfp (pTD-3C, 
and pTD-4C, 
), nleD : : gfp (pTD-5D, 
), and nleE : : gfp (pTD-6Es, 
and pTD-7El, 
). All values were corrected for background by deducting the fluorescence value for ZAP193 transformed with pAJR70 (promoterless gfp) measured at the same optical density. Experiments were repeated at least three times with the data plotted being representative of the results obtained.
