Operon structure and gene expression of the espJ–tccP locus of enterohaemorrhagic Escherichia coli O157:H7

FEMS Microbiology Letters 247 (2005) 137–145 www.fems-microbiology.org

Operon structure and gene expression of the espJ–tccP locus of enterohaemorrhagic Escherichia coli O157:H7 Junkal Garmendia, Gad Frankel

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Centre for Molecular Microbiology and Infection, Department of Biological Sciences, Imperial College London, London SW7 2AZ, UK Received 16 February 2005; received in revised form 25 April 2005; accepted 29 April 2005 First published online 17 May 2005 Edited by P.H. Williams

Abstract In this study, we investigated the genetic organisation and expression of the espJ–tccP locus of enterohaemorrhagic Escherichia coli (EHEC O157:H7), which encodes the type III secretion system effector proteins EspJ and TccP. Using flow cytometry and fluorescence microscopy, we found that espJ and tccP constitute an operon, whose expression in vitro is affected by the composition of the medium and environmental signals such as temperature, pH, osmolarity and O2 pressure. Moreover, expression of the espJ–tccP operon is not regulated by Ler, a LEE-encoded transcriptional regulator, but by an as yet unidentified regulatory system(s).
2005 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved. Keywords: EHEC; Effectors; Type III secretion system; Environmental signals; Gene expression

1. Introduction Infections with enterohaemorrhagic Escherichia coli (EHEC) O157:H7 are associated with a wide spectrum of illnesses, from mild diarrhoea to severe diseases such as hemorrhagic colitis and haemolytic-uremic syndrome [1], while enteropathogenic E. coli (EPEC) is a diarrhoeal pathogen prevalent in developing countries [2]. During colonisation of the gut mucosa, EHEC and EPEC produce
attaching & effacing (A/E) lesions, which are characterised by effacement of the brush border microvilli and intimate attachment of the bacterium to the enterocyte plasma membrane [3]. Additionally, infected cells produce elongated actin-rich pedestal-like structures at the site of intimate bacterial adhesion [4]. The locus of enterocyte effacement (LEE) pathogenicity is-

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Corresponding author. Tel./fax: +44 20 7595 5253. E-mail address: [email protected] (G. Frankel).

land [5] is necessary for A/E lesion formation by EHEC and EPEC. It contains genes encoding a filamentous type III secretion system apparatus (FTTSS) [6], effector proteins, chaperones, the outer membrane adhesion molecule intimin [4] and the transcriptional regulatory proteins locus of enterocyte effacement (LEE)-encoded regulator (Ler) [7], global regulator of LEE activation (GrlA) and global regulator of LEE repressor (GrlR) [8]. Ler negatively regulates its own gene expression [9] and activates the expression of both LEE- and nonLEE virulence genes [10]. Moreover, Ler is the molecular channel used by several global regulatory systems (e.g. Fis, BipA, H-NS, PerC1, IHF and Hha) to influence LEE gene expression [11–16]. EtrA and EivF, encoded by genes located within the ETT2 pathogenicity island have recently been identified as negative regulators of LEE-carried genes [17]. In addition, quorum sensing influences LEE gene expression via the LuxS/ autoinducer AI-3 system, which cross-talks with the mammalian hormone epinephrine [18–20].

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2005 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.femsle.2005.04.035

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Within the LEE, tir, map, espF, espG, espH and sepZ encode effector proteins whose expression is Ler-dependent (reviewed in [21]). Additionally, recent studies identified a number of prophage-carried effectors that are also translocated by the LEE-encoded FTTSS (reviewed in [21]. Among them are Tir-cytoskeleton coupling protein (TccP) [22] (also know as EspFU [23]) and EspJ [24], which are located contiguously on prophage CP-933U. Like the LEE-genes, expression of tccP and espJ is down regulated in bacteria attached to eukaryotic plasma membranes [25]. While not required for A/E lesion formation, EspJ influences colonisation and clearance dynamics of in vivo [24]. TccP binds and activates NWASP leading to recruitment of the Arp2/3 complex and actin polymerisation at the site of intimate bacterial attachment [22,23]. The aim of this study was to define the promoter region(s) responsible for expression of espJ and tccP and to characterise the environmental cues and regulatory elements responsible for the control of espJ and tccP expression.

2. Materials and methods 2.1. Bacterial strains and growth conditions The bacterial strains used in this study are listed in Table 1. Bacteria were grown in Luria Bertani (LB) medium, Dulbeccos Modified Eagles Medium (DMEM) or Vogel–Bonner minimal medium (0.8 mM MgSO4 Æ 7H2O, 9.5 mM citric acid Æ H2O, 57 mM K2HPO4, 16 mM NaNH5PO4 Æ 4H2O, 25 mM dextrose) pH 7.5 or 4.5, supplemented with ampicillin (50 lg/ml) when necessary. Bacteria were grown at 37 and 30 C, with aeration, in the presence of 5% CO2 or in an anaerobic chamber (10% CO2, 10% H2, 80% N2), as appropriate.

2.2. Plasmids The plasmids used in this study are listed in Table 1. Plasmid pFVP25.1, carrying gfpmut3A under the control of a constitutive promoter, was introduced into bacterial strains for fluorescence visualisation where indicated [26]. pICC310 is a derivative of pFVP25, a vector carrying a promoterless gfpmut3A [26]. pICC310 contains a transcriptional fusion of the putative promoter region 1 (P1) to gfp. A 1200 bp fragment containing the 5 0 -end of the prophage CP-933U and the sequence encoding 5 0 -25 bp of espJ was amplified by PCR from EDL933 genomic DNA using the primers 5 0 -CCGGAATTCACGAATGATGCCTCGCCGC and 5 0 -CGCGGATCCATAAGCAGTTTTTTATAATTGACAT. The 1218 bp PCR product, containing terminal EcoRI and BamHI sites was digested and ligated into pFVP25, generating pICC310. pICC311 is a derivative of pFVP25, containing a transcriptional fusion of the putative promoter region 2 (P2) to gfp. A 660 bp fragment containing the 5 0 -end of the prophage CP-933U without including the region upstream Z3069 coding sequence and the sequence encoding 5 0 -25 bp of espJ was amplified by PCR from EDL933 genomic DNA using the primers 5 0 -CCGGAATTCGATTGTTCTGTTTAGGAAAAG and 5 0 -CGCGGATCCATAAGCAGTTTTTTATAATTGACAT. The 696 bp PCR product, containing terminal EcoRI and BamHI sites was digested and ligated into pFVP25, generating pICC311. pICC312 is a derivative of pFVP25, containing a transcriptional fusion of the putative promoter region 3 (P3) to gfp. A 572 bp fragment containing P3 and the sequence encoding 5 0 -25 bp of Z3069 was amplified by PCR from EDL933 genomic DNA using the primers 5 0 -CCGGAATTCCCGCAAATAAGTCGATCCTCT and 5 0 -CGCGGATCCATCCCCCTCTCTCCGCCACTTTATC. The 590 bp PCR product, containing termi-

Table 1 Strains and plasmids used in this study Name

Description

Source of Reference

Strains EDL933 DF1291 DF1292 LZ2 LZ3

Wild type EHEC O157:H7 stx
EDL933 ler::Km EDL933 ihf::Km etrA deletion mutant of EHEC Sakai 813 eivF deletion mutant of EHEC Sakai 813

ATCC [35] [35] [17] [17]

Plasmids pFPV25.1 pFPV25 pICC310 pICC311 pICC312 pICC313 pICC314 pICC315

Derivative Derivative Derivative Derivative Derivative Derivative Derivative Derivative

[26] [26] This This This This This This

of of of of of of of of

pBR322, gfpmut3A under constitutive promoter pBR322, promoterless gfpmut3A pFPV25, transcriptional fusion P1:gfp pFPV25, transcriptional fusion P2:gfp pFPV25, transcriptional fusion P3:gfp pFPV25, transcriptional fusion P4:gfp pFPV25, transcriptional fusion P5:gfp pFPV25, transcriptional fusion Pler:gfp

study study study study study study

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nal EcoRI and BamHI sites was digested and ligated into pFVP25, generating pICC312. pICC313 contains a transcriptional fusion of the putative promoter region 4 (P4) to gfp. A 208 bp fragment containing P4 and the sequence encoding 5 0 -25 bp of espJ was amplified by PCR from EHEC EDL933 genomic DNA using the primers 5 0 -CCGGAATTCTCTCGTCACTGAGCTCAATCA and 5 0 -CGCGGATCCATAAGCAGTTTTTTATAATTGACAT. The 226 bp PCR product, containing terminal EcoRI and BamHI sites was digested and ligated into pFVP25, generating pICC313. pICC314 contains a transcriptional fusion of the putative promoter region 5 (P5) to gfp. A 325 bp fragment containing P5 and the sequence encoding 5 0 -25 bp of tccP was amplified by PCR from EDL933 genomic DNA using the primers 5 0 -GGGGTACCCTCAAAAAATAATCTTAAAGG and 5 0 -CGCGGATCCAAAGTGAAGAAACATTGTTAATCAT. The 343 bp PCR product, containing terminal KpnI and BamHI sites was digested and ligated into pFVP25, generating pICC314. pICC315 contains a transcriptional fusion of ler promoter (Pler) to gfp. A 1071 bp fragment containing Pler and the sequence encoding 5 0 -25 bp of ler was amplified by PCR from EDL933 genomic DNA using the primers 5 0 -CCGGAATTCCGGTTCACAGATGCCCCCGGCGGT and 5 0 -CGCGGATCCTATTCATAATAAATAATCTCCTCA. The 1089 bp PCR product, containing terminal EcoRI and BamHI sites was digested and ligated into pFVP25, generating pICC315. 2.3. Cell culture and antibodies HeLa cells (clone HtTA1) were grown in Dulbeccos modified Eagle medium (DMEM) supplemented with 10% fetal calf serum (FCS) and 2 mM glutamine at 37 C in 5% CO2. Cells were seeded onto glass coverslips (12 mm-diameter) in 24-well plates at a density of 5 · 104 cells per well, 24 h before infection. Bacterial cultures, incubated in LB with the appropriate antibiotics for 16 h at 37 C with aeration, were added to HeLa cells at a multiplicity of infection of 100:1. Infected cells were incubated for 6 h. For immunofluorescence, cell monolayers were fixed in 3.7% paraformaldehyde in phosphate buffered saline (PBS) pH 7.4, for 15 min at RT and washed three times with PBS (phosphate buffered saline). Antibodies were diluted in 10% horse serum, 0.1% saponin in PBS. Coverslips were washed twice in PBS containing 0.1% saponin, incubated for 30 min with primary antibody, washed twice with 0.1% saponin in PBS and incubated for 30 min with secondary antibody. Coverslips were washed twice in 0.1% saponin in PBS, once in PBS and once in H2O, and mounted on Aqua Poly/Mount (Polysciences). Anti-E. coli O157:H7 goat polyclonal (Fitzgerald Industries International) was diluted 1:500. Texas Red sulfonyl chloride (TRSC)-conjugated donkey anti-goat antibody

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(Jackson Immunoresearch Laboratories) was diluted 1:200. Samples were analysed using a Zeiss LSM510 confocal laser scanning microscope. 2.4. Preparation of bacteria for flow cytometric analysis EHEC EDL933 wild-type and mutant strains carrying the appropriate plasmids were centrifugated at 4000g, and pellets were resuspended in PBS. In each experiment, strains EDL933 and EDL933 (pFPV25.1) were used as negative and positive controls for fluorescence, respectively. For each sample, 105 cells were analysed on a FACS Calibur cytometer (Becton Dickinson). GFP was detected at 525 nm in the FL1 channel. Data were analysed as follows. The geometric mean of the fluorescence of each strain in three independent experiments was calculated. The fold increase in fluorescence of the different growth conditions was calculated dividing the geometric mean fluorescence of the condition 1 by the geometric mean of the condition 2.

3. Results and discussion 3.1. espJ and tccP constitute an operon In order to identify the promoter region(s) responsible for espJ and tccP expression, the 1.2 kb DNA region upstream of espJ, including the 5 0 -end of prophage CP933U, (indicated as filled block P1 in Fig. 1) was amplified by PCR and ligated upstream of a promoterless gfp in the promoter trap vector pFPV25 [26]. Since growth in tissue culture medium (DMEM) is known to stimulate secretion of EHEC proteins involved in infection, such as LEE-encoded proteins [27], and logarithmicphase bacteria grown at 37 C elicit strong, EPEC-induced, A/E activity on HeLa cells [28], wild-type EHEC, strain EDL933, carrying the resultant plasmid pICC310 was grown overnight in LB, diluted 1:100 into DMEM medium and grown to logarithmic-phase at 37 C. Expression from the transcriptional fusion P1:gfp was detected by flow cytometry, reaching levels comparable to those of EDL933 carrying pICC315, a plasmid containing gfp fused to the promoter region of ler (Pler), which is known to be expressed in the conditions tested [10,15] (Fig. 2(a)). In addition, using fluorescence microscopy to visualise HeLa cells infected with EDL933(pICC310), P1 was shown to trigger gfp expression during infection. However, the contact with eukaryotic cells by itself did not modify gfp expression, as the intensity of GFP generated by EDL933(pICC310) was comparable between bacteria attached to the epithelial cells or to the glass substrate (Fig. 2(b)). The infections were performed during 6 h in DMEM; therefore, it is likely that bacteria attached to the glass coverslip rather

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Fig. 1. Schematic representation of EHEC espJ and tccP genomic organisation at the 5 0 -end of the prophage CP-399U. Filled blocks P1, P2, P3, P4 and P5 were independently fused to gfp in pFPV25. Localization of putative
35/
10 boxes and RBS sequences in P4, the intergenic Z3069-espJ region, is shown.

Fig. 2. (a) Flow cytometric analysis of wild-type EHEC EDL933 strain carrying different intergenic region-gfp transcriptional fusions, grown to logarithmic phase at 37 C in DMEM. Lines depict GFP expression in EDL933 carrying P1:gfp (pICC310) (red dashed), P2:gfp (pICC311) (black dashed), P3:gfp (pICC312) (black continuous), P4:gfp (pICC313) (red continuous), P5:gfp (pICC314) (blue continuous) or Pler:gfp (pICC315) (blue dashed). The black thin line shows a non-fluorescence negative control (EDL933(pFPV25)). In each individual experiment 105 bacterial-sized particles were analysed. In the upper panel, the fluorescence intensity of each particle is reported on the x axis and the number of bacterial-sized particles is shown on the y axis. Lower panel shows the geometric mean of the fluorescence detected by flow cytometry for every transcriptional fusion analysed. (b) The expression of P1:gfp, P2:gfp, P3:gfp, P4:gfp and P5:gfp in EDL933, 6 h after infection of HeLa cells, was analysed by fluorescence microscopy. EHEC was detected with goat anti-EHEC and TRSC-conjugated donkey anti-goat antibodies (red).

than to the eukaryotic cells would have reached a growth phase in which P1:gfp is active. Considering the genomic organization of the espJ– tccP locus (Fig. 1), Z3069, a gene located at the 5 0 -end of the prophage CP-933U and encoding a protein of unknown function, espJ and tccP could constitute a single

transcriptional unit. In order to elucidate the transcriptional organisation of these genes, four additional gfp transcriptional fusions were generated: P2, which contains the region located upstream of espJ excluding the region upstream of Z3069 (pICC311); P3, which contains the region located upstream of Z3069 (pICC312);

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P4, which contains the intergenic Z3069-espJ region (pICC313); and P5, which contains the intergenic espJ–tccP region (pICC314). Each plasmid was transformed independently into EHEC EDL933 wild-type and the generated strains were grown as described above. Expression from the transcriptional fusions P2:gfp, P3:gfp, P4:gfp and P5:gfp was detected in vitro by flow cytometry, and during infection by using fluorescence microscopy in order to visualise HeLa cells infected during 6 h with EDL933 (pICC311, pICC312, pICC313, pICC314) (Fig. 2(a) and (b)). P2::gfp and P4::gfp generated levels of GFP intensity equivalent to those of P1::gfp, both in vitro and during infection of HeLa cells (Fig. 2(a) and (b)). However, P3, located upstream of Z3069, and P5, corresponding to the intergenic espJ–tccP region, did not trigger gfp expression in the conditions tested (Fig. 2(a) and (b)). These results suggest that espJ and tccP constitute a two-gene operon; the intergenic espJ–tccP region does not contain internal promoter sequences, and the promoter region responsible for the expression of the espJ–tccP transcriptional unit is located in the intergenic Z3069-espJ region (P4). Analysis of the features present in the intergenic Z3069-espJ region revealed the presence of putative
10/
35 and RBS sequences, consistent with the results obtained with the transcriptional fusions (Fig. 1). The polycistron espJ–tccP cluster is expressed in growth conditions similar to those required for LEE-encoded gene expression [10,28–30]. Accordingly, gfp expression was found to be threefold higher in logarithmic cultures of EDL933(pICC313) grown in DMEM than grown in LB (Fig. 3(a)). Although logarithmicphase EPEC has been reported to elicit strong A/E activity on epithelial cells [28], no significant differences in gfp expression were detected when EHEC EDL933(pICC313) was grown in DMEM to logarithmic or to stationary phase (Fig. 3(c) and (f)). 3.2. Contribution of different environmental signals to espJ–tccP expression in vitro The success of a pathogen such as EHEC is fully dependent on its complex interactions within the milieu and on a coordinated regulation of gene expression according to environmental cues. EHEC faces a sudden increase in temperature when it first infects its host. Accordingly, the effect of changes in growth temperature on activity of the espJ–tccP promoter was analysed. EDL933(pICC313) was grown to logarithmic phase in DMEM at 30 or 37 C and gfp expression was measured by flow cytometry, being found to be fourfold higher in cultures grown at 37 C than at 30 C (Fig. 3(b)). In addition, during the infection process EHEC finds initially a highly acidic environment in the stomach, then it transits across the alkaline duodenum, due to the biliary content, and even-

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tually it reaches the ileum, caecum and colon, where the pH is neutral or slightly alkaline. The effect of changes in pH of the medium on espJ–tccP promoter activity was analysed. EDL933(pICC313) was grown at 37 C to logarithmic phase in Vogel–Bonner minimal medium pH 7.5 or 4.5 and gfp expression was measured by flow cytometry. This revealed an approximately twofold increase in gfp expression in bacteria grown at pH 7.5 compared to pH 4.5 (Fig. 3(b) and (f)). The external niches where E. coli is commonly present have generally a low osmolarity. Considering that the high osmolarity of the gut lumen could affect EHEC virulence gene expression, experiments were performed to analyse the effect of osmolarity changes on the activity of the espJ–tccP promoter. EDL933(pICC313) was grown at 37 C to logarithmic phase in Vogel–Bonner minimal medium pH 7.5, in the absence or presence of 0.3 M NaCl and gfp expression was measured by flow cytometry. Unexpectedly, a threefold increase in gfp expression was observed in bacteria grown in low osmolarity conditions, when compared to bacteria grown in the presence of 0.3 M NaCl (Fig. 3(e) and (f)). Given that the anaerobic environment of the large intestine is the target for EHEC, the effect of changes in O2 partial pressure levels on the activity of the espJ–tccP promoter was analysed. EDL933(pICC313) bacteria were grown to logarithmic phase in DMEM at 37 C in the absence or presence of 5% CO2. gfp expression was measured by flow cytometry and found to be twofold higher in cultures grown without 5% CO2 (Fig. 3(a)). Additionally, levels of gfp expression of EDL933(pICC313) grown to logarithmic phase in DMEM at 37 C were compared to those detected when the same bacteria were grown in an anaerobic chamber (10% CO2, 10% H2, 80% N2). The levels of GFP were measured by flow cytometry and found to be eightfold higher in cultures growing aerobically than in cultures grown in anaerobic conditions (Fig. 3(d) and (f)). Studies performed in EPEC have previously shown that partial O2 pressure does not affect its ability to form A/E lesions [28], and that the increase in the secretion of EPEC LEE-encoded proteins observed in the presence of 5% CO2 is independent of CO2 itself because this molecule acts only as a component of the medium buffering system [29,31]. Therefore, the results obtained for both EPEC and EHEC suggest that the partial or total lack of O2 in the growth medium in vitro is not a significant parameter that influences positively the expression of LEE-encoded and non-LEE encoded virulence factors. The influence of the anaerobicity of the intestinal tract in espJ–tccP expression is a question that remains unanswered with the experimental conditions used in this study. We can speculate that when EHEC, exposed to the anaerobic environment of the colon, intimately attaches to the epithelial cells, the FTTSS physically

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Fig. 3. Flow cytometry analysis of P4:gfp expression by EDL933(pICC313) grown in: DMEM (DMEM1) (red)/LB (blue)/DMEM 5%CO2 (DMEM2) (black) at 37 C to logarithmic phase (a); DMEM at 37 C (black continuous)/30 C (black dashed) to logarithmic phase (b); DMEM at 37 C to logarithmic (black continuous)/stationary (black dashed) phase (c); DMEM at 37 C to logarithmic phase (black continuous)/DMEM anaerobic chamber (black dashed) (d); Vogel–Bonner minimal medium pH 7.5 (black)/pH 4.5 (blue)/pH 7.5, 0.3 M NaCl (red) (e). The black thin line shows a non-fluorescence negative control (EDL933 (pFPV25)). In each individual experiment 105 bacterial-sized particles were analysed, the fluorescence intensity of each particle is reported on the x axis and the number of bacterial-sized particles is shown on the y axis. (f) Fold increase in expression detected by flow cytometry, calculated as the geometric mean (n = 3) of the fluorescence of EDL933(pICC313) in condition 1 divided by the fluorescence of the same strain in condition 2.

bridges the bacterial cytoplasm with the eukaryotic cell cytosol. O2 levels existent in the epithelial cell could then be at least partially available to the bacterium, which would be then in a microaerophilic environment. These changes in the partial O2 pressure could contribute to changes in the pattern of bacterial gene expression, such as activation of the espJ–tccP operon expression. The results presented in Fig. 3 were obtained with a plasmid-encoded transcriptional fusion; this could contribute to the observation that gfp levels were not reduced to undetectable levels when bacteria were grown in the less favourable conditions tested. Together, these results indicate that changes in temperature, pH, osmolarity and partial O2 pressure have significant effects on espJ–tccP expression during logarithmic growth in DMEM.

3.3. Regulation of espJ–tccP expression Regulation of EHEC virulence factors expression in response to environmental signals is a complex issue governed by a number of regulatory elements. Ler has been characterised as a negative autoregulator for LEE1 [9] and as a positive regulator for LEE2, LEE3, LEE4, LEE5 and non-LEE-encoded tagA [10,13,15,32]. P4:gfp expression was analysed in an EDL933 ler mutant strain (DF1291) grown to logarithmic phase in DMEM at 37 C. gfp expression in this strain was found to be comparable to the levels measured in the wild-type EDL933 strain (Figs. 1(a) and 4(a)). Additionally, P4 was shown to trigger gfp expression during infection, by using fluorescence microscopy to visualise HeLa cells infected with EDL933 ler (pICC313). As shown before, the levels of GFP ob-

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Fig. 4. (a) Flow cytometric analysis of EDL933 ler, EDL933 ihf, Sakai etrA, Sakai eivF mutant strains carrying pICC313 grown to logarithmic phase at 37 C in DMEM. Lines depict GFP expression in EDL933 ler mutant (pICC313) (red continuous), EDL933 ihf mutant (pICC313) (blue continuous), Sakai etrA mutant (pICC313) (black continuous), Sakai eivF mutant (pICC313) (red dashed) strains. The black thin line shows a nonfluorescence negative control (EDL933(pFPV25)). In each individual experiment 105 bacterial-sized particles were analysed. In the upper panel, the fluorescence intensity of each particle is reported on the x axis and the number of bacterial-sized particles is shown on the y axis. Lower panel shows the geometric mean of the fluorescence detected by flow cytometry for P4:gfp in each mutant strain analysed. (b) Expression of P4:gfp from EDL933 ler, EDL933 ihf, Sakai etrA, Sakai eivF mutant strains 6 h after infection of HeLa cells, analysed by fluorescence microscopy. EHEC was detected with goat anti-EHEC and TSRC-conjugated donkey anti-goat antibodies (red).

served were similar in bacteria attached to either the cells or the glass substrate, at 6 h after infection (Fig. 4(b)). These results were in agreement with those obtained for the Pler:gfp transcriptional fusion (pICC315) which, considering that Ler autoregulates negatively its own expression, was active in the EDL933 ler mutant strain in the conditions tested (data not shown). A number of additional positive regulatory elements have been associated with the regulation of LEE gene expression; the majority of them, including LEE-encoded GlrA [8], global regulators Fis [11], BipA [12], PerC1 [14] and IHF [15], positively influence LEE expression through Ler. Considering that Ler does not seem to regulate espJ–tccP expression, it is unlikely that any of these elements would contribute to the regulation of this transcriptional unit. Consistently, an EDL933 ihf mutant strain (DF1292) transformed with pICC313 (encoding P4:gfp) and grown to logarithmic phase in DMEM at 37 C, was found to be similar to the levels measured in the wild-type EDL933 strain (Figs. 1(a) and 4(a)); moreover, P4 triggered gfp expression during infection of HeLa cells with EDL933 ihf (pICC313) (Fig. 4(b)). Several negative regulators have been identified as responsible for repressing LEE-encoded gene expression, such as the LEE-encoded GlrR [8], ETT2-encoded EtrA and EivF [17], and the general regulatory systems

YhiE, YhiF [33,34] and Hha [16]. Considering the high gfp levels obtained in the wild-type strain-based experiments (Figs. 2 and 3), the sensitivity of the methods used in this study could make difficult the measure of the derepressing effect of the absence of a potential negative regulator. The putative roles of EtrA and EivF in the control of espJ–tccP expression were analysed in EHEC Sakai etrA and eivF mutant strains (LZ2 and LZ3), independently transformed with pICC313, and grown to logarithmic phase in DMEM at 37 C. P4:gfp expression was analysed as described above and GFP levels in the etrA and eivF mutant strains were found to be similar to that measured in the wild-type strain (Figs. 1(a) and 4(a)). Equally, P4 triggered gfp expression during infection of HeLa cells with EHEC Sakai etrA (pICC313) or eivF (pICC313) (Fig. 4(b)). These results indicate that EspJ and TccP constitute the first example of EHEC effector proteins translocated through the LEE-encoded FTTSS whose expression is not regulated by Ler. Moreover, the regulatory systems known to control LEE-encoded gene expression are unlikely to play a significant role in the control of espJ and tccP transcription. As mentioned before, regulation of the expression of EHEC virulence factors is complex, and the elucidation of the regulatory elements governing espJ–tccP expression will be the subject of future research.

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Fig. 5. Model for espJ–tccP gene expression; these two genes constitute an operon located at the 5 0 -end of the prophage CP-933U, which encodes EspJ and TccP, two effector proteins that are translocated into the host cell cytosol via the LEE-encoded FTTSS. Expression of the espJ–tccP operon in vitro is affected by media composition, changes in temperature, pH, osmolarity and partial O2 pressure, and it is independent of bacterial growth phase. EspJ and TccP constitute the first example of EHEC FTTSS-dependent effectors whose expression is not regulated by the transcriptional regulator Ler, but by an as yet unidentified regulatory system(s).

4. Conclusion EspJ and TccP, two EHEC non-LEE encoded effectors translocated through the FTTSS, involved in the dynamics of clearance from the hosts intestinal tract and in cytoskeletal reorganisation beneath adherent bacteria respectively, are encoded in a two-loci containing operon located at the 5 0 -end of the prophage CP933U. As shown in the model proposed in Fig. 5, their in vitro expression is affected by the composition of the medium, by changes in temperature, pH, osmolarity and partial O2 pressure, and appears to be independent of the bacterial growth phase. Moreover, EspJ and TccP constitute the first example of EHEC FTTSS-dependent effectors whose expression is not regulated by the transcriptional regulator Ler, but by an as yet unidentified regulatory system(s).

Acknowledgements We wish to acknowledge A. Rae and D. Goulding for technical advice, Dr. Ilan Rosenshine and Dr. Mark Pallen for EHEC mutant strains and Dr. S. Wiles for helpful discussion. This project was supported by the Wellcome Trust.

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