transcriptional regulation of the esp genes of enterohemorrhagic

JOURNAL OF BACTERIOLOGY,0021-9193/99/$04.0010

June 1999, p. 3409–3418 Vol. 181, No. 11

Copyright © 1999, American Society for Microbiology. All Rights Reserved.

Transcriptional Regulation of the esp Genes ofEnterohemorrhagic Escherichia coli

FABRIZIO BELTRAMETTI, ANDREAS U. KRESSE, AND CARLOS A. GUZMAN*

Department of Microbial Pathogenicity and Vaccine Research, Division of Microbiology,GBF-National Research Centre for Biotechnology, D-38124 Braunschweig, Germany

Received 21 December 1998/Accepted 31 March 1999

We have determined that the genes encoding the secreted proteins EspA, EspD, and EspB of enterohemor-rhagic Escherichia coli (EHEC) are organized in a single operon. The esp operon is controlled by a promoterlocated 94 bp upstream from the ATG start codon of the espA gene. The promoter is activated in the earlylogarithmic growth phase, upon bacterial contact with eukaryotic cells and in response to Ca21, Mn21, andHEPES. Transcription of the esp operon seems to be switched off in tightly attached bacteria. The activationprocess is regulated by osmolarity (induction at high osmolarities), modulated by temperature, and influencedby the degree of DNA supercoiling. Transcription is sS dependent, and the H-NS protein contributes to its finetuning. Identification of the factors involved in activation of the esp operon and the signals responsible formodulation may facilitate understanding of the underlying molecular events leading to sequential expressionof virulence factors during natural infections caused by EHEC.

Enterohemorrhagic Escherichia coli (EHEC) is the mostcommon cause of hemorrhagic colitis, a bloody diarrhea whichcan lead to the life-threatening hemolitic-uremic syndrome (4).This pathogen can cause large food-borne epidemic outbreaksand belongs to the group of Shiga toxin-producing E. coli(STEC) (34). Infections caused by EHEC and the closely re-lated enteropathogenic E. coli (EPEC) are associated withhistopathological changes called attaching and effacing (A/E)lesions (33, 42). These changes consist of effacement of theintestinal microvilli followed by intimate association of bacte-ria with host cells and reorganization of cytoskeletal compo-nents beneath adherent bacteria (8). Most of the factors re-quired to produce A/E lesions are encoded by a largechromosomal locus called LEE (for “locus of enterocyte ef-facement”) (31). LEE codes for a type III secretion system(30); an outer membrane protein called intimin (EaeA), whichis required for intimate attachment to host cells (22, 46); thesecreted proteins EspA, EspD, and EspB, which are requiredin EPEC for signal transduction events leading to formation ofA/E lesions; the Tir (EPEC), or EspE (STEC), protein, which,after translocation within the host cell, phosphorylation, andsurface display, constitutes the intimin receptor (6, 26); and thePas (EHEC), or EscD (EPEC), protein, which seems to beinvolved in the secretion process (28). Other genes that appearto be involved in the pathogenesis process are located onplasmids (14, 22, 24).

The EspA protein plays a key role during the infectionprocesses of both EHEC and EPEC (11, 25). It has recentlybeen shown that EspA is involved in the formation of a noveltype of pilus-like structure, which is essential for early bacterialattachment to epithelial cells and seems to be involved in EspBtranslocation within host cells (11, 27). Formation of thesesurface structures is transient, disappearing once the attach-ment is strengthened (11, 27). Thus, major synthesis and se-cretion of the EspA and EspB proteins presumably occur dur-

ing early infection and are enhanced when bacteria are grownat 37°C in tissue culture medium and by the presence of mi-cronutrients or signals produced by eukaryotic cells (10, 21).However, neither the transcriptional regulation of Esp pro-teins nor the real signals required for gene activation areknown.

Coordinated regulation of gene activation according to en-vironmental stimuli is a common feature among microorgan-isms to optimize performance, avoiding the energetic cost ofsynthesizing unnecessary products. This becomes a more com-pelling requirement for those infectious agents that duringtheir biological cycle transit across different niches. In fact,untimely expression of virulence factors may have a devastat-ing effect on pathogenic bacteria (1). Thus, genes encodingproteins involved in the pathogenesis process are expressedonly when required in response to environmental regulatorysignals. The control process is usually very complex and or-chestrated by a cascade of regulatory factors (12, 32). How-ever, the underlying mechanism and true nature of the signalsinvolved in triggering and fine tuning this response remainelusive. In enteropathogenic bacteria, expression of virulencegenes is mainly required within the intestinal tract. Therefore,control circuits which respond to a range of local signals haveevolved. In Salmonella spp., different regulators and stimulat-ing factors have been identified (9, 13); however, there is verylimited information concerning EHEC. Thus, we have investi-gated the regulation of expression of the esp genes, which areessential during the first steps of the infection process.

MATERIALS AND METHODS

Bacterial strains, plasmids, media, and growth conditions. The bacterialstrains and plasmids used in this study are listed in Table 1. Strains were grownin Luria-Bertani medium, M9 minimal medium supplemented with 0.2% glucoseas a carbon source (39), or Dulbecco modified Eagle medium (DMEM)(GIBCO, Karlsruhe, Germany). Where required, media were supplemented withampicillin (100 mg/ml), nalidixic acid (20 mg/ml), or novobiocin (5, 20, or 50mg/ml). For b-galactosidase assays, bacteria were grown until they reached theexponential phase, and cultures were reinoculated to an optical density at 600 nm(OD600) of 0.1 into the appropriate medium. To test the influence of oligoele-ments on gene expression, bacteria were grown in M9-glucose medium supple-mented with either MgSO4 (1, 7, or 30 mM), MnSO4 (0.0033, 0.33, or 3.3 mM),CaCl2 (0.01, 0.1, or 1 mM), FeSO4 (0.25, 25, or 250 mM), or Fe(NO3)3. NH4Clwas added to nitrogen-free M9 medium at a concentration of 0.5, 2, or 10 mM.

* Corresponding author. Mailing address: Department of MicrobialPathogenicity and Vaccine Research, Division of Microbiology, GBF-National Research Centre for Biotechnology, Mascheroder Weg 1,D-38124 Braunschweig, Germany. Phone: 49-531-6181558. Fax: 49-531-6181411. E-mail: [email protected].

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Osmotic regulation was tested in M9-glucose minimal medium by the addition ofNaCl or sucrose to a final concentration ranging from 10 to 600 mM.

Tissue culture and cell infections. HeLa cells (ATCC CCL2) were cultured insix-well Nunclon Delta tissue culture plates (Inter Med Nunc, Roskilde, Den-mark) in DMEM supplemented with 10% fetal calf serum and glutamine (2 mM)at 37°C. Semiconfluent monolayers were infected for 4 h at 37°C with a bacte-rium/cell ratio of 100:1. For immunofluorescence studies, cells were seeded onto12-mm-diameter glass coverslips in 24-well tissue culture plates (Inter MedNunc), infected with overnight-grown bacteria resuspended in DMEM for 3 h,fixed with 3.7% paraformaldehyde in phosphate-buffered saline (PBS), and per-meabilized with 0.2% Triton X-100 in PBS. Bacteria were stained with a rabbitpolyclonal antiserum against O157 K2 (Behring, Marburg, Germany) as primaryantibody and tetramethylrhodamine isothiocyanate (TRITC)-conjugated goatanti-rabbit as secondary antibody (Dianova, Hamburg, Germany), whereas F-actin was stained with fluorescein isothiocyanate (FITC)-labelled phalloidin (Sig-ma, Deisenhofen, Germany). Then, coverslips were washed and mounted, andcells were examined by epifluorescence with a Zeiss axiophot microscope (CarlZeiss, Jena, Germany).

Recombinant DNA techniques. All DNA manipulations were performed bystandard methods (39). Amplification by PCR of the chromosomal region en-compassing the espA, espD, and espB genes from strain EDL933 was performedas previously described (28); all reported DNA positions refer to the EMBLdatabase (accession no. Y13068) (28). Plasmid DNA was isolated with the QIA-prep Spin Miniprep kit (Qiagen, Chatsworth, Calif.) and sequenced with a Taqdyedeoxy terminator cycle sequencing kit and an automatic DNA sequencer,model 373A (Applied Biosystems, Foster City, Calif.), according to the manu-facturer’s instructions.

esp promoter fragments were generated by PCR (see Fig. 3) with primerswhich incorporated restriction sites (underlined) to facilitate construction oftranslational fusions with the lacZ gene present in the promoter probe vectorpUJ9TT (23). Plasmid pUJ3 contains a 653-bp BamHI fragment generated withthe oligonucleotides EspA-lac1 (2164 59-CCGGATCCGGTATCCAGAAGATCAAGAAGC-39 2185) and EspA-lac2 (2817 59-GCGGATCCTTACCTAAGTCATAGATCGTCGAT-39 2794). Plasmid pUJ3-285 was constructed by subclon-ing the 371-bp EcoRV/BamHI fragment from plasmid pUJ3 into the SmaI/BamHI-digested pUJ9TT. Finally, pUJ3-56 contains a BamHI fragmentgenerated with the primers EspA-lac1 (see above) and FAB56 (2742 59-GGGGATCCATCTATATACCTCTTGATAATTTTTC-39 2728).

The espA, espD, espB, and sepL (region upstream of espA) probes used forNorthern blot analysis were generated by PCR with the primer pairs A293 (261159-GATAGTGAGCAGAGAGAATGC-39 2633) and EspAP1 (3161 59-CCGCCTTCACTGTTTGCAGATC-39 3139), 9189 (3618 59-GCTATCCCTATCTCTCTCAGGT-39 3640) and 9530 (4113 59-CCAATTTTGTTAGCAACATTAC-394091), 6556 (4477 59-ATGAATACTATTGATAATACTC-39 4499) and 7191(4739 59-GCTTTATTCTGGCTCTCAAAAA-39 4717), and A291 (1616 59-GTGAGTTTCCAATGGCTAATGG-39 1638) and A292 (1880 59-AGCAGCTTCTCGATTGTCGAGC-39 1858), respectively. [a-32P]dATP (Amersham Life Sci-ence, Braunschweig, Germany) was incorporated into the probes with theRandom Primed DNA labelling kit (Boehringer, Mannheim, Germany), accord-ing to the manufacturer’s instructions.

Generation of a nonpolar mutation of the espA gene. Overlap extension PCR(18) was used to generate an in-frame deletion of the espA gene. Two PCR

fragments were generated with the primer pair 9188 (2524 59-CGGGTATCGATTGTCGAAG-39 2542) and 9187 (2803 59-GATCGTCGATGTCGAAGAACTCG-39 2780) and the primer pair 9186 (59-CTTCGACATCGACGATC-3254-AGTGCACGTTCTGATGTGCAATC-39 3277) and 9185 (3523 59-CGTCACTAATGAGTGACCTGCC-39 3501). The resulting products contained the first 63bp and the last 66 bp of the espA open reading frame (ORF), respectively. A17-bp overlap in their sequences (underlined) permitted amplification of a548-bp fragment during a second PCR performed with primers 9188 and 9185.The resulting product was cloned into plasmid pCR2.1 (Invitrogen), digestedwith KpnI and XbaI, and subcloned into the pMAK700oriT (43) derivativepANK1, thereby generating plasmid pANK111. Transfer of the suicide vector byconjugation, cointegration, and excision was performed as previously described(28). The in-frame deletion was confirmed by PCR with the primers ANK25(2164 59-GGTATCCAGAAGATCAAGAAGC-39 2185) and A289 (3549 59-CAACCCGGGCTAAGGACATCCTCAGCAGC-39 3578), which hybridize withadjacent external sequences.

Northern blot and primer extension analyses. Bacterial strains were grown onDMEM-HEPES (pH 7) to an OD600 of 0.8, and total RNA was extracted withthe RNeasy Midi Kit (Qiagen), according to the supplier’s instructions. Aliquotsof 10 mg of RNA were denatured at 100°C in the presence of formaldehyde (2 M)and 50% formamide, separated on a 1% agarose–10% formaldehyde gel, blottedon a Byodine B transfer membrane (0.45 mm) (Pall, Dreieich, Germany), andthen hybridized as described by Sambrook et al. (39) at 50°C with the probesdescribed above. For primer extension analysis, strains were grown to an OD600of 0.8 on M9 minimal medium with or without NaCl (430 mM), and total RNAwas extracted as described above. Primer FAB56 (2743 59-CATCTATATACCTCTTGATAATTT-39 2720) was end labelled with [g-32P]dATP at 37°C for 40min. The labelled primer was hybridized with 25 mg of RNA at 50°C for 20 minand extended with 1 U of avian myeloblastosis virus reverse transcriptase (Pro-mega, Madison, Wis.) at 42°C for 40 min. Sequencing ladders were generated byusing the same primer with the Deaza G/A T7Sequencing Mixes kit (PharmaciaBiotech, Piscataway, N.J.), according to the supplier’s instructions. Primer ex-tension products were analyzed on a sequencing gel with the sequence ladder asa reference.

Detection of secreted proteins. Bacteria were grown in DMEM-HEPES (pH 7)until they reached an OD600 of 0.6. Then, the proteins present in the supernatantfluids were precipitated by the addition of 10% (vol/vol) trichloroacetic acid,overnight incubation at 4°C, and subsequent centrifugation at 4,000 3 g for 30min. The dry pellet was resuspended in 1.5 M Tris (pH 8), and proteins (20mg/lane) were fractionated by discontinuous sodium dodecyl sulfate-polyacryl-amide gel electrophoresis (39) with a 12.5% separating gel. They were thentransferred to a positively charged Biodyne B nylon membrane (Pall) with asemidry device (Bio-Rad Laboratories, Richmond, Calif.), and proteins weredetected with monoclonal antibodies against EspA, EspB, and EspD (10, 11) andhorseradish peroxidase-conjugated rabbit anti-mouse immunoglobulin G andimmunoglobulin M as second antibodies (Bio-Rad Laboratories). Antigen-anti-body complexes were visualized by chemiluminescence with the ECL system(Amersham Life Science).

b-Galactosidase assays. Samples were taken at different time intervals, theOD600 was determined, and aliquots were removed and centrifuged at 8,000 3g to recover bacterial pellets, which were immediately processed to determineb-galactosidase activity or were stored at 280°C. To study activation of the esp

TABLE 1. Bacterial strains and plasmids

Strain or plasmid Genotype or description Reference or source

StrainsE. coli EDL933 Prototypic O157:H7 EHEC strain 35E. coli MC4100 F2 D(argF-lac)U169 araD139 rpsL150 ptsF25 flbB5301 rbsR deoC relA1 29E. coli RH90 MC4100 rpoS359::Tn10 29E. coli GM37 Wild type 19E. coli GM230 GM37 hns 19

PlasmidspCR2.1 Apr Kmr; high-copy-number vector for cloning of PCR products InvitrogenpMAK700oriT Cmr; mobilizable suicide vector bearing the oriT region of pJFF350 43pANK1 Cmr Kmr; pMAK700oriT derivative containing additional cloning sites and the Kmr gene

(aphA3) inserted in the HindIII restriction siteThis work

pANK111 Cmr Kmr; pANK1 derivative containing the 548-bp PCR fragment encompassing the espAgene containing an in-frame deletion, generated with primers 9188 and 9185

This work

pUJ9TT Apr; multicopy promoter probe vector to generate fusions with the lacZ gene 23pUJ3 Apr; pUJ9TT derivative containing a 653-bp BamHI PCR fragment generated with primers

EspA-lac1 and EspA-lac2This work

pUJ3-285 Apr; pUJ9TT derivative containing a 371-bp EcoRV/BamHI fragment from pUJ3 insertedinto the SmaI/BamHI sites

This work

pUJ3-56 Apr; pUJ9TT derivative containing a 580-bp BamHI PCR fragment generated with primersEspA-lac1 and FAB 56

This work

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promoter during bacterial infection of HeLa cells, monolayers were infected; atdifferent time intervals, supernatants fluids were removed and unattached bac-teria were collected by centrifugation. Then, the monolayers were gently washedand lysed with 1% Triton X-100 in PBS to collect attached bacteria. Thesesamples were processed to determine the number of viable microorganisms andb-galactosidase activity. The b-galactosidase assay was performed with theb-GAL Reporter Gene Assay Chemiluminescent Kit (Boehringer) according tothe supplier’s instructions, except that lysis was performed by resuspendingbacteria in 500 ml of the lysis solution from the kit supplemented with chloroform(20 ml) and 0.1% SDS (20 ml) for 30 min at room temperature. The samples weremeasured with a Victor 1420 Multilabel Counter fluorometer (EG&G Wallac,Turku, Finland), and the results were normalized for the number of bacterialcells.

RESULTS

A functional espA gene is necessary for production of A/Elesions after infection with the EHEC strain EDL933. Al-though the relevance of the EspA protein has been establishedfor EPEC, limited information is available for EHEC. There-fore, we analyzed the role played by EspA in the initial inter-action between the prototypic EHEC strain EDL933 (O157:H7) and eukaryotic cells. To assess whether the product

encoded by the espA gene was also necessary for formation ofthe A/E lesion in EDL933, a mutant which contains an in-frame deletion in the espA gene was generated (see Materialsand Methods). Immunofluorescence studies revealed a markedreduction in the numbers of attaching bacteria and actin accu-mulation when EDL933 DespA was compared with the paren-tal strain (Fig. 1). To confirm that the observed effect was dueto production of a truncated (i.e., nonfunctional) EspA proteinand not to an affected transcription or translation of geneslocated downstream, production of the EspA, EspB, and EspDproteins was analyzed by Western blotting. As expected, EspAwas not present in concentrated culture supernatants, whereasbands reacting with EspD- and EspB-specific antibodies weredetected (not shown). This demonstrated that the EspA pro-tein plays similar roles in the interactions between eukaryoticcells and EPEC, STEC, or EHEC.

The espADB genes of EHEC are transcribed as a singleoperon. sepL and the genes encoding the secreted proteinsEspA, EspD, and EspB are positioned in tandem on LEE,suggesting that they are cotranscribed as a polycistronic

FIG. 1. Infection of HeLa cells with the EHEC strain EDL933. Cells were infected with EDL933 (a and c) or its DespA derivative (b and d) for 3 h. Then, monolayerswere fixed, bacteria were labelled with TRITC-conjugated antibodies (a and b) and F-actin was stained with FITC-labelled phalloidin (c and d), and coverslips wereexamined by immunofluorescence microscopy. While the wild-type strain forms microcolonies with consistent actin accumulation (a and c), the DespA mutant has lostthe ability to attach to HeLa cells (b) and to induce actin accumulation (d). Scales are in micrometers.

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mRNA. The available information about secreted proteins inEPEC and STEC indicates that both the temperature and thecomposition of the culture medium are critical factors forexpression (10, 21, 26). Therefore, to identify the transcript ofthe esp genes, Northern blot analysis was performed with RNAextracted from bacteria grown in DMEM supplemented withHEPES (100 mM) and PCR-generated fragments encompass-ing internal sequences from the three esp ORFs as probes. Allprobes hybridized with a unique band of approximately 2.8 kb.The length of the transcript corresponds to that of the espA,espD, and espB genes, suggesting that the promoter is locatedimmediately upstream of espA. Probes specific for sepL, whichis located upstream of espA, did not give any signal, ruling outthe possibility that the observed band resulted from 59 process-ing of a major transcript. This suggests that the esp genes, butnot sepL, are transcribed as a single operon (subsequentlydesignated the esp operon) (Fig. 2a).

Primer extension analysis was performed to identify the startof transcription of the esp promoter. RNA was extracted from

cells grown on M9-glucose medium supplemented with either10 or 430 mM NaCl. The major start site was mapped to 94 bpupstream from the ATG start codon of the espA gene (position2646 of the published sequence) (Fig. 2b). The intensity of thesignal was increased when bacteria were grown at high osmo-larity, confirming the data obtained in studies on esp promoterregulation (see below). Analysis of the region upstream fromthe start of transcription led to identification of putative 210and 235 sequences (Fig. 3). The 210 sequence exhibits a highdegree of homology both to the 210 sequences of the bfpAgene of EPEC (37), which seems to be s70 dependent, and tothe osmE promoter, which is sS dependent (5).

Transcription of the esp operon is activated upon contactwith HeLa cells. To study regulation of the esp operon, a DNAfragment spanning nucleotides 2577 to 176 (with respect tothe espA ATG start codon) was amplified by PCR and used togenerate a translational fusion with the lacZ gene present inplasmid pUJ9TT, thereby generating plasmid pUJ3 (Fig. 3).This fragment was considered sufficiently long both to includethe promoter and upstream regions containing potential bind-ing sites for regulatory factors and to retain intact the transla-tion initiation region to avoid potential artifacts resulting fromaltered translational efficiency (40).

EspA seems to be produced in the early phase of infection,and it disappears when bacteria are stably attached to eukary-otic cells (11, 27). However, EspA was also detected in super-natant fluids and attached to bacterial surfaces, and it is un-clear whether its transcription or translation results frominteraction with eukaryotic cells. To elucidate this point, HeLacells were infected with EDL933 harboring pUJ3 and the ki-netics of espA activation was analyzed by determining the levelof b-galactosidase produced by bacteria present in supernatantfluids or attached to HeLa cells. As shown in Fig. 4, rapidtranscriptional activation was observed when bacteria came incontact with the eukaryotic cells, whereas almost no incrementin b-galactosidase activity over the basal level was observed inbacteria present in supernatant fluids. Thus, the esp promoterappears to be induced upon contact with HeLa cells. Thedetected enzymatic activity began to decrease 1 to 2 h afterinfection, suggesting that a repression of the esp promotertakes place after the initial attachment. During the course ofinfection, the ratio between tightly and loosely attached bac-teria increases; thus, transcription of the esp operon is probablyswitched off in tightly attached bacteria. These results demon-strate that transcription of the esp operon is induced by directbacterial contact with HeLa cells rather than by componentspresent in tissue culture medium or by soluble factors releasedby eukaryotic cells.

esp operon induction by growth in different media. Sincegrowth in tissue culture medium is known to stimulate secre-tion of EHEC proteins involved in the infection process (21),activation of the esp promoter in DMEM was analyzed. StrainEDL933(pUJ3) was grown in DMEM and expression of b-ga-lactosidase was determined at different time intervals. An in-crement in b-galactosidase activity was observed in the expo-nential phase; however, this activation was blocked whenbacteria were grown in DMEM without HEPES (Fig. 5a).

Many micronutrients are known to induce expression ofvirulence genes in a wide range of pathogenic microorganisms(reviewed in reference 32). The influence of some micronutri-ents on protein secretion by EPEC and STEC has been ana-lyzed previously but only in connection with other inducingconditions (e.g., HEPES and tissue culture medium). There-fore, their individual contributions to activation of the esppromoter were analyzed in the present study. The virulence ofEPEC appears to be inhibited by the consistent amount of

FIG. 2. (a) Northern blot analysis of the mRNA transcript encompassing theespA, espD, and espB genes. Total RNA extracted from EDL933 grown onDMEM-HEPES was fractionated on a 1% agarose gel, transferred to Byodine Bmembranes, and hybridized with probes specific for espA, espD, and espB. As acontrol, a probe that hybridizes within regions located upstream of espA (sepL)was used. The main RNA transcript is indicated by an arrow (approximately 2.8kb). (b) Identification of the transcriptional start site from the esp operon byprimer extension analysis. Total RNA was extracted from EDL933(pUJ3) grownexponentially at 37°C in medium supplemented with either 10 (lane 1) or 430(lane 2) mM NaCl. A 24-bp oligonucleotide (FAB56), which hybridizes withpositions 13 to 221 of the espA region, was used to perform primer extensionand to generate a sequence ladder. The position of the first base in the mainRNA message relative to the adenosine (base 11) of the ATG start codon isindicated.



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NH41 present in the colon (26, 32). Supplementation of nitro-

gen-deficient M9 medium with different concentrations ofNH4Cl did not affect the basal activity of the esp promoter,indicating that promoter activation is independent of the pres-ence of either NH4

1 or chloride (data not shown). Kenny et al.(26) reported that addition of calcium and iron to the culturemedium resulted in improved export of secreted proteins inEPEC. Supplementation of M9-glucose medium with CaCl2resulted in increased b-galactosidase activity from the early tomiddle exponential growth phases (Fig. 5b). In contrast, nosignificant changes in transcription were observed when theminimal medium was supplemented with FeSO4 or Fe(NO3)3,suggesting that iron, nitrate, and sulfate contribute very little,

if at all, to activation of the esp operon (data not shown).Interestingly, the addition of MnSO4 resulted in an increasedtranscription, similar to that observed with CaCl2 (Fig. 5b andc). However, activity of the esp promoter was not affected inthe presence of Mg21, indicating that divalent ions per se werenot responsible for the observed effect.

Effects of temperature, pH, and osmolarity on activation ofthe esp promoter. The first sudden change that enteropatho-genic bacteria face when they infect their hosts is the incrementin temperature. Previous studies have suggested that secretedproteins are upregulated at 37°C (10, 26); however, the indi-vidual contribution of temperature was buried among otherpotential stimuli (e.g., pH and culture medium, etc.) due to the

FIG. 3. (a) Sequence of the esp promoter region. The start of transcription (11), the putative 210 and 235 consensus sequences (underlined), the Shine-Dalgarnosequence (SD), the consensus binding sequence for H-NS (59-TNTNAN-39 [in boldface italic type]), and several inverted and direct repeats (underlined) are indicated.(b) Schematic representation of the constructs employed to study transcriptional regulation of the esp operon. Abbreviations: EV, EcoRV; lacZ, b-galactosidase-encoding gene.



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poor sensitivity of the reading system. Therefore, the effects ofchanges in growth temperature on induction of the esp pro-moter were analyzed. Interestingly, no significant differenceswere observed in the activity of the promoter when strainEDL933(pUJ3) was incubated in standard M9 medium (10mM NaCl) at 25, 37, or 42°C (Fig. 6). EHEC is also confrontedduring the first phase of infection with a very acidic environ-ment in the stomach. It then transits across the duodenum,which receives the alkaline biliary content. Finally, it reachesthe ileum, cecum, and colon, which constitute its primary tar-gets and in which the pH is neutral or slightly alkaline. How-ever, no significant differences in promoter activity were ob-served when bacteria were grown at pH 6, 7, or 8 (data notshown).

The external niches in which E. coli is present are in generalcharacterized by low osmolarity. Therefore, it might be ex-pected that the high osmolarity of the gut lumen can be ex-ploited by EHEC as an expression signal. In fact, osmolarityplays an important role in activation of virulence genes fromother enteropathogenic microorganisms (26, 32, 36). It isknown that, depending on the specific infection site, maximalgene expression occurs at different osmolarities (32). There-fore, experiments were performed to establish the effect ofosmolarity on activation of the esp promoter. Preliminary stud-ies demonstrated (data not shown) that the maximal effect wasobserved at 1,120 mosmol/kg (430 mM NaCl). Concentrationsbelow or above this value resulted in a suboptimal level ofinduction. To avoid interference with potential osmopro-tectants present in Luria-Bertani medium, strain EDL933(pUJ3) was grown in M9-glucose medium in the presence of430 mM NaCl or an equimolar concentration of sucrose.

As shown in Fig. 6b and c, when bacteria were grown at 37and 42°C, high osmolarity resulted in an 8- to 10-fold increasedb-galactosidase activity during exponential and stationaryphases. A similar activation pattern was observed when NaClwas replaced by sucrose (data not shown). This demonstratesthat induction of the esp promoter depends on osmolarityrather than on an indirect stress effect due to elevated concen-trations of NaCl. It has been frequently observed that promot-

ers sensitive to osmolarity are also induced in the stationaryphase. However, the activation of the esp promoter was inde-pendent of bacterial entrance into the stationary phase and wastriggered immediately following inoculation in high-osmolaritymedium. Furthermore, when bacteria were grown on low-os-molarity medium (10 mM NaCl), no increment in enzymaticactivity was observed in the stationary phase (Fig. 6). Temper-ature and osmolarity are thought to play key roles in the

FIG. 4. Expression of b-galactosidase by EDL933(pUJ3) after infection ofHeLa cells. At different time intervals after infection, enzymatic activity wasdetermined in bacteria present in supernatants (■) or attached to HeLa cells (F)and compared to that produced by EDL933(pUJ3) grown in DMEM (Œ). Thebasal values of b-galactosidase obtained from EDL933 containing the promot-erless plasmid under matching conditions were at least 10-fold lower than thebasal levels of the tested clones and were subtracted from each sample. b-Ga-lactosidase activities are expressed as relative light units (rlu) per 105 bacteriaand are means of three independent experiments; standard deviations werelower than 5%. Open symbols indicate numbers of CFU at each time point.

FIG. 5. b-Galactosidase induction in response to different media and micro-nutrients. EDL933(pUJ3) was grown in DMEM (Œ) or DMEM supplementedwith 100 mM HEPES (pH 7) (}) (a) or in M9-glucose medium supplementedwith either CaCl2 (}, 0 mM; F, 0.01 mM; ■, 0.1 mM; Œ, 1 mM) (b) or MnSO4(}, 0 mM; F, 0.0033 mM; ■, 0.33 mM; Œ, 3.3 mM) (c), and b-galactosidaseactivities were determined at different time intervals. Growth rate is indicated byopen symbols (OD600). Results are expressed as relative light units (rlu) per 105

bacteria and are means of three independent experiments; standard deviationswere lower than 5%. The background values for EDL933 containing the pro-moterless plasmid under matching conditions were at least 10-fold lower than thebasal values at the tested conditions and were subtracted from each sample.



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expression of virulence genes in many enteropathogenic bac-teria (32). Since EHEC can also face any of these individualconditions outside the host, we analyzed whether at suboptimal(nonphysiologic) temperatures the promoter was activated athigh osmolarities. Despite bacteria being grown at optimal

osmolarity, the esp promoter was not induced at 25°C (Fig. 6),whereas minimal differences in activation were observed atbetween 37 and 42°C. These results suggest that the promoteris optimally activated by a combination of temperature andosmolarity.

To assess the contribution of the regions located upstreamfrom the start of transcription, the pUJ3 derivative pUJ3-285,which contains a 285-bp deletion, was generated (Fig. 3). Anincrement in the enzymatic activity of EDL933(pUJ3-285) withrespect to EDL933(pUJ3) was observed when strains weregrown at 37°C at either low or high osmolarity (Fig. 6b). Thissuggests the presence of a binding site for a negative regulatorin the deleted region. The differences were less evident at thesuboptimal temperatures of 25 and 42°C.

We then generated a hybrid plasmid (pUJ3-56) in which thefragment located downstream from the ATG start codon wasdeleted (Fig. 3). The resulting construct was transformed intoEDL933 to determine b-galactosidase activity under differentconditions. The obtained results showed 60 to 80% reductionsin enzymatic activity when bacteria were grown in high- andlow-salt medium (data not shown). This suggests that the initialpart of the espA ORF is essential for allowing optimal trans-lation efficiency, as has been previously reported for othergenes of E. coli (40).

Transcription of the esp operon is dependent on the pres-ence of a functional sS factor. It has been shown that sS

controls a regulon of more than 30 genes expressed in responseto starvation or during the transition to stationary phase andinfluences the response to osmotic stress (15). Interestingly,motifs located upstream from the start of transcription of theesp promoter exhibit similarity with sS-dependent promoters(see above). Therefore, to assess whether transcription of theesp operon is dependent on the sS factor, the pUJ3 plasmidwas introduced into E. coli MC4100 and its sS-deficient deriv-ative, RH90. As shown in Fig. 7a, the expression levels of thereporter gene were dramatically reduced in the mutant strain(10-fold) under both inducing (430 mM NaCl) and noninduc-ing (10 mM NaCl) conditions. The differences were more strik-ing at high osmolarity and in the early stationary phase. Inter-estingly, when the wild-type strain MC4100 was tested, theb-galactosidase activities under both growth conditions wereapproximately two- to fourfold lower than that observed inEDL933(pUJ3), suggesting that additional factors are requiredto trigger full activation of the esp promoter in EDL933.

Transcription of the esp operon is dependent on the pres-ence of a functional H-NS protein. The global negative regu-lator H-NS is also involved in osmoregulation and can acteither indirectly, through the maintenance of low sS levels inexponentially growing (nonstressed) bacteria, or directly, in asS-independent manner (reviewed in reference 2). To analyzewhether the H-NS protein was also involved in regulation ofthe esp promoter, plasmids pUJ3 and pUJ3-285 were intro-duced into E. coli GM37 and its hns derivative, GM230. Whenthe production of b-galactosidase of strains GM37(pUJ3) andGM230(pUJ3) were compared, a 10- to 20-fold increment wasobserved in the hns mutant grown in the presence of either lowor high levels of NaCl (Fig. 7b and c). The strong increase intranscription can be explained by an overexpression of sS or anindirect H-NS-mediated effect in the plasmid copy and linkingnumbers (17).

H-NS has the strongest effect under conditions in whichexpression of the target gene is not induced by positive regu-lators, whereas under inducing conditions H-NS-mediated re-pression is almost eliminated. Therefore, by comparing theosmotic induction ratios (induction at high/low osmolarity) inthe hns1 and hns strains, a direct effect of H-NS can be dem-

FIG. 6. Activation of the esp promoter in response to changes in temperatureand osmolarity. EDL933 containing either pUJ3 (F) or its deletion derivative,pUJ3-285 (■), was grown in minimal medium (pH 7) supplemented with 10 mM(dotted lines) or 430 mM (solid lines) NaCl at 25°C (a), 37°C (b), or 42°C (c), andb-galactosidase activities were determined at different time intervals. Results areexpressed as relative light units (rlu) per 105 bacteria and are means of threeindependent experiments; standard deviations were lower than 5%. The back-ground values for EDL933 containing the promoterless plasmid were at least10-fold lower than the values at the tested conditions and were subtracted fromeach sample.



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onstrated (the greater the ratio, the more complete the repres-sion). Ratios of 2.5 and 4 were observed for GM230(pUJ3) andGM37(pUJ3) after 4 h of incubation, suggesting that the ob-served activation was directly dependent on H-NS. This hy-pothesis was further supported by the results obtained withplasmid pUJ3-285. When strains harboring this plasmid weretested, both basal and induced levels of b-galactosidase wereincreased up to 10-fold in GM37(pUJ3-285) with respect toGM37(pUJ3), whereas in the hns mutant the basal and in-duced levels of the strain harboring pUJ3-285 were onlyslightly affected in comparison to those of GM230(pUJ3) (Fig.7b and c). Abolition of H-NS-mediated repression in pUJ3-285suggests that the deleted region encompasses binding motifsfor this protein. This hypothesis is further supported by thepresence of several stretches containing the H-NS binding con-sensus sequence (59-TNTNAN-39) (38) upstream and down-stream from the EcoRV site present in the esp promoter region(Fig. 3).

Influence of DNA supercoiling in transcription of the espoperon. Osmoinduction of several promoters is determined bychanges in the degree of DNA supercoiling (32). Since EHECshould face an anaerobic environment in the intestinal niche,and anaerobicity can also affect DNA supercoiling (45), weinvestigated whether the degree of supercoiling influences ac-tivation of the esp promoter. Novobiocin was used to inhibitthe DNA gyrase, which facilitates initiation of transcription byintroducing negative supercoils. EDL933(pUJ3) was grown inthe presence of subinhibitory concentrations of novobiocin,and b-galactosidase activity was measured. The obtained re-sults showed that novobiocin reduced the levels of b-galacto-sidase in a dose-dependent manner when EDL933 was grownin the presence of 430 mM NaCl (Fig. 8), suggesting that thedegree of supercoiling is critical in regulation of the esp pro-moter.

DISCUSSION

The products encoded by LEE confer upon EHEC andEPEC their distinctive virulence property, namely, the abilityto produce A/E lesions. We studied transcriptional regulation

FIG. 7. Transcription of the esp operon is dependent on sS and H-NS. (a)Plasmid pUJ3 was transformed into E. coli MC4100 (F) and its sS-deficientderivative, RH90 (■), and b-galactosidase activities were determined underinducing (430 mM NaCl [solid lines]) and noninducing (10 mM NaCl [dottedlines]) conditions. (b and c) To investigate the role of the H-NS protein, plasmidspUJ3 (F) and pUJ3-285 (■) were transformed into E. coli GM37 (b) and its hnsderivative GM230 (c). Strains were grown in M9-glucose medium supplementedwith 10 mM (dotted lines) or 430 mM (solid lines) NaCl, and production ofb-galactosidase was determined after different time intervals. Results are ex-pressed as relative light units (rlu) per 105 bacteria and are means of threeindependent experiments; standard deviations were lower than 5%. The back-ground values for strains containing the promoterless plasmid were at least10-fold lower than the basal values at the tested conditions and were subtractedfrom each sample. No differences in growth were observed.

FIG. 8. Influence of the degree of supercoiling on transcription of the esppromoter. EDL933(pUJ3) was grown in M9-glucose medium supplemented with10 mM (open symbols) or 430 mM (solid symbols) NaCl in the presence of 0(triangles), 5 (circles), 20 (squares), or 50 (diamonds) mg of the gyrase inhibitornovobiocin per ml, and b-galactosidase activities were determined at differenttime intervals. Results are expressed as relative light units (rlu) per 105 bacteriaand are means of three independent experiments; standard deviations werelower than 5%. The background values for EDL933 containing the promoterlessplasmid were at least 10-fold lower than the basal values at the tested conditionsand were subtracted from each sample. No differences in growth were observedat the novobiocin concentrations tested (not shown).



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of the genes encoding the secreted proteins EspA, EspD, andEspB, which play a key role in A/E lesion formation. Recentresults from our group and others have demonstrated that theEspA protein from STEC and EPEC is involved in the forma-tion of filamentous surface appendages that appear duringearly infection and seem to be critical for bacterial adherence(11, 27). Although these studies strongly suggested that EspAis involved in the first steps of infection, they provided nodefinitive proof about the kinetics of appearance of EspA andthe potential induction mechanism. Northern blot and primerextension analyses showed that espA is cotranscribed with espDand espB and permitted identification of a promoter located 94bp upstream of the espA gene. A 5- to 10-fold induction of theesp promoter was observed upon bacterial attachment to HeLacells. The fact that the esp promoter was switched off laterduring infection is consistent with the lack of EspA productionby bacteria forming microcolonies (11, 27).

The esp promoter appears to be subjected to different envi-ronmental stimuli, similar to those faced by EHEC in theintestine. Previous reports showed that expression of the se-creted proteins occurred when bacteria were grown in tissueculture medium (10, 26). No induction of the esp promoter wasobserved when bacteria were grown in DMEM, whereas theaddition of 100 mM HEPES resulted in four- to fivefold-increased transcription. Therefore, the previously reported ef-fect on protein secretion seems to be due to the presence ofHEPES rather than to specific components of the tissue cul-ture medium. The presence of Ca21 also resulted in strongactivation of the esp promoter over the broad range of con-centrations tested. Therefore, calcium seems to play an impor-tant role not only in the signal transduction events leading tothe rearrangement of cytoskeletal proteins (20) but also in theearly interactions of EHEC with enterocytes via induction ofthe esp promoter. This is in agreement with the general roleplayed by Ca21 in regulation of virulence genes from severalpathogenic microorganisms (26, 32, 37). Similar activation lev-els were observed when media were supplemented with Mn21.Interestingly, Mn21 is involved in regulation of expression ofmetal transporter systems in Streptococcus spp. and Yersiniaspp. (3, 7). Although the molecular mechanism by which Mn21

exerts its effect on the esp promoter is unclear, surface proteinsare affected in EHEC, Streptococcus pneumoniae, and Yersiniapestis, suggesting common underlying processes in unrelatedpathogens. Temperature has a weak effect on induction of theesp promoter; however, increased levels of activation areachieved when it acts together with osmolarity. AlthoughEHEC can be confronted with any of these stimuli outside thehost, the combination of 37°C and high osmotic pressure rep-resents an excellent indicator that bacteria have reached theirtarget within the host intestine.

It is known that the presence of the 60-MDa plasmidpMAR2 is required in EPEC to achieve full virulence; plas-midless bacteria exhibit a reduced ability to infect HeLa cells(14). Although a 90-kb plasmid (pO157) has been identified inEHEC, its role in the infection process is still unclear (24).When the STEC strain 413.89-1 (44) and its plasmidless deriv-ative (413.89-1/6) were transformed with pUJ3, a significantimpairment (sixfold) in production of b-galactosidase was ob-served under inducing conditions when the megaplasmid wasabsent (not shown). This suggests that activation of the esppromoter is also fine tuned by a product(s) encoded by themegaplasmid.

Results obtained with an rpoS mutant and the homologybetween putative consensus sequences and the promoter ofosmE (5) suggested that transcription from the esp promoter issS dependent. Interestingly, activation of the esp promoter

preferentially occurs during the exponential phase of growth,whereas during the stationary phase it slightly decreases. How-ever, the role of sS is more complex than that of other alter-native s factors, as it plays a role under various conditions ofslow growth, such as those observed during the stationaryphase and under osmotic shock (15, 16). Although basal ex-pression of the reporter was strongly reduced in the rpoS mu-tant, osmoinduction was preserved (Fig. 7). Tanaka et al. (41)showed that several promoters can be recognized by either theEs70 or EsS RNA polymerase holoenzymes. Therefore, sS-independent transcription of the esp promoter may be directlydependent on Es70. The esp promoter also exhibits homologywith the bfpA promoter (36). Although it has been suggested,without experimental evidence, that this promoter is s70 de-pendent, it is intriguing that promoters driving expression ofproteins involved in the synthesis of surface appendages re-quired for initial attachment have common motifs. The appar-ent decreased activity of the esp promoter in the late stationaryphase might reflect a mechanism evolved by EHEC to avoidthe extra energetic cost required to synthesize products whichare required only in the initial phases of infection.

The H-NS protein is involved in regulation of many genesactivated by environmental signals (2). We have demonstratedthat the levels of transcription of the esp promoter are signif-icantly increased in an hns mutant. The presence of this regu-lator usually results in 2- to 20-fold repression, which is stron-ger when H-NS both acts at the promoter level and affects theexpression of positive regulators (2). Therefore, the observedinfluence of H-NS in activation of the esp promoter can beexplained by (i) hyperexpression of the sS factor which isrepressed by H-NS (2) and (ii) a direct effect on the promoteritself, since putative H-NS-binding regions have been identi-fied.

No vaccines able to prevent infections caused by EHEC arepresently commercially available, and antibiotics are not usefulfor therapy since they can worsen symptoms by enhancing therelease of bacterial toxins. Study of the interactions betweenbacteria and host cells may permit identification of novel mo-lecular targets for therapeutic interventions. It might be pos-sible to modulate the expression of virulence factors to makebacteria more susceptible to chemotherapeutics or host clear-ance mechanisms. Thus, an understanding of fine regulatorymechanisms may be the first step towards development of newtools to fight EHEC infections.

The data emerging from this work show that overall regu-lation of the esp promoter is an extremely complex process.During their transit across different niches, EHEC organismsmust integrate different signals to optimize and fine tune theexpression of virulence factors. The activation process is in partmodulated by factors which are also needed for regulation ofhousekeeping genes from nonpathogenic E. coli. This has beendemonstrated as well for other pathogens (12) and suggeststhat virulence genes, which were inherited later during bacte-rial evolution, exploit previously established regulatory net-works.

ACKNOWLEDGMENTS

We are grateful to F. Sasse for insight into the performance offluorometry experiments, F. Ebel for providing antibodies and strain413.89-1/6, and K. N. Timmis for generous support and encourage-ment.

Part of this work was supported by a grant from the Lower Saxony-Israel Cooperation Programme, founded by the Volkswagen Founda-tion (21.45-75/2).



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