INTRODUCTION

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Many pathogenic Escherichia coli share a conserved region inserted into the chromosome known as the locus of enterocyte effacement (LEE). This pathogenicity island encodes bacterial products which are required for the production of the attaching and effacing (A/E) lesions (24). Recently, Perna et al. published the DNA sequence of the LEE of enterohemorrhagic E. coli (EHEC) EDL933, which comprises 41 non-prophage 933L open reading frames (ORFs) (30). The comparison between the LEE of the enteropathogenic E. coli (EPEC) strain E2348/69 (10) and that of the EHEC strain EDL933 shows that they are highly conserved, containing ORFs with identities ranging from 100% (escS and escF) to 66.48% (tir and espE). While all the identified components of the type III secretion apparatus encoded within the LEE, except the sepZ gene, reveal fairly high homologies (98 to 100%), the secreted proteins EspA, EspB, EspD, and EspE are more diverse (84.63, 74.01, 80.36, and 66.48% homology, respectively).

Type III secretion systems are widespread in a variety of pathogenic bacteria and encoded by at least 20 genes (for reviews, see references 4 and 12). The disruption of any of these genes results in the abolishment of signal transduction events that are essential for bacterial interactions with eukaryotic cells during the infection process (11, 22, 32). However, differences have been observed between EPEC and Shiga toxin-producing E. coli (STEC) when the involvement of secreted proteins in the infection of eukaryotic cells was assessed. While the attachment of strains of EPEC and rabbit EPEC (REPEC) with mutations in espA to the host cells was only slightly affected, if at all (1, 19), the adhesion of strains of STEC with mutations in espA was strongly reduced (3, 9), suggesting that EspA plays a key role in the pathogenicity of STEC. The secreted protein EspA is a major component of the recently described surface appendages which are required for localized bacterial adherence, the formation of microcolonies, and the induction of A/E lesions (9, 21). In addition, EspA is necessary for the translocation of the potential effector protein EspB, which in turn is found in the cytosol and the cytoplasmic membranes of infected cells (38).

The esp operon encodes EspA, EspB, and a third protein, EspD (3). Although the ability to accumulate actin underneath bacteria of an EPEC espD mutant was abolished, the ability to adhere to eukaryotic cells was maintained (23). In order to assess the role played by EspD in the pathogenesis of EHEC, a nonpolar espD deletion derivative of the prototype EHEC strain EDL933 was generated. The characterization of this clone demonstrated that, as has been demonstrated for EspA (3, 9), EspD plays a more significant role in the pathogenic process of EHEC than in that of EPEC. The obtained results suggest that EspD is essential for the formation of surface appendages, is integrated in the cytoplasmic membranes of target cells, and might participate in the translocation of effector molecules.

	MATERIALS AND METHODS

Top Abstract Introduction Materials and Methods Results and Discussion References

Bacterial strains, plasmids, and media. The strains and plasmids used in this study are described in Table 1. Bacteria were grown in Luria-Bertani (LB) broth (33) or LB agar and in serum-free Dulbecco's modified Eagle's medium (DMEM; GIBCO, Karlruhe, Germany) supplemented with 100 mM HEPES (pH 7.4). Plasmids were maintained in E. coli DH5, and the INVF' strain was used as a recipient for cloning fragments amplified by PCR into the pCR2.1 vector. Media were supplemented with chloramphenicol (50 µg ml¹), ampicillin (200 µg ml¹), or nalidixic acid (50 µg ml¹) when required.

DNA manipulations. Plasmid DNA isolation, restriction endonuclease digestion, ligation, transformation, agarose gel electrophoresis, and other standard DNA techniques were carried out as described by Sambrook et al. (33). Oligonucleotides (Table 2) were synthesized by GIBCO. Colony PCR, extraction of PCR products, and cloning experiments were performed according to standard protocols (33). DNA sequencing was performed with a Taq dyedeoxy terminator cycle sequencing kit and an automatic DNA sequencer (model 373A; Applied Biosystems) according to the manufacturer's instructions. Restriction and modification enzymes were purchased from New England Biolabs (Schwalbach, Germany). Electroporation was carried out with a gene pulser (Bio-Rad Laboratories) as described by O'Callaghan and Charbit (28). Searches in databases for nucleotide and amino acid sequence homologies were performed with the BLASTP (2), BLASTP plus BEAUTY (2, 39), NNPP (31), and PSORT (26) algorithms.

Construction of a nonpolar mutation. By overlap extension PCR (16) an in-frame deletion in the espD gene from the EHEC strain EDL933 was generated (Fig. 1). Two PCR fragments were obtained by colony PCR with an Expand High Fidelity kit (Boehringer, Mannheim, Germany) with the primer pairs ANKA288-ANKA289 and ANKA290-ANK7191, followed by mixing of the PCR products and a second PCR with the primer pair ANKA288-ANK7191. The resulting 875-bp fragment contained the first 218 bp and the last 52 bp of the espD ORF and coded for a polypeptide (99 amino acids [aa]) in which 275 aa of the wild-type EspD protein (374 aa) are deleted, herein called the espD gene. After being cloned into the vector pCR2.1 and after the DNA sequence was checked, the espD fragment was subcloned into KpnI- and XbaI-digested pANK1 (a pMAK700oriT derivative [35]), thereby generating pANK155. This plasmid was transformed into the S17-1 (pir) strain and then transferred by conjugation (15) into the recipient EHEC strain E32511/0 Nal^r. Plasmid pANK155 was recovered from E32511/0 and subsequently electroporated into the EHEC strain EDL933. The cointegration and excision of the suicide vector were performed as previously described (22). The in-frame deletion contained in the EDL933espD mutant resulting from the allelic exchange was confirmed by PCR analysis with the primer pair ANKA288-ANK7191 or ANK14-ANK16, which are homologous to adjacent external sequences (data not shown).

View larger version (24K): [in this window] [in a new window]   FIG. 1.   Construction of an in-frame deletion mutant of the espD gene in EHEC. (A) The ORF of espD in the wild-type EHEC strain EDL933 (arrow a) and the recombinant ORF of the espD mutant EDL933espD (broken arrow b) are schematically shown. The corresponding positions (according to the published sequence [EMBL accession no. Y13068]) are also shown. (B) Plasmids used for the complementation studies performed with the EHEC strain EDL933espD. Plasmid pANK84 contains an insert from the 3' end of eaeA (dotted line) to the 5' end of espB, and plasmid pAKSK78 contains a sequence from the 3' end of eaeA (dotted line) to the 5' end of espD.

Tissue culture methods and analysis by immunofluorescence microscopy. HeLa cells (ATCC CCL2) were maintained in DMEM supplemented with 25 mM HEPES, 10% (vol/vol) fetal calf serum (FCS), and glutamine (GIBCO) in an atmosphere containing 5% CO₂ at 37°C. To study the reorganization of cellular actin underneath bacteria upon EHEC infection, cells were seeded at a concentration of approximately 5 × 10⁴ per well onto 12-mm-diameter glass coverslips (InterMed Nunc, Roskilde, Denmark) in 24-well Nunclon Delta tissue culture plates (InterMed Nunc). Cell monolayers were infected with bacteria grown overnight and resuspended in DMEM-HEPES at a cell/bacterium ratio of 1:100. After 4 h of incubation, monolayers were washed to remove unattached bacteria, fixed with 3.7% (vol/vol) p-formaldehyde in phosphate-buffered saline (PBS), and permeabilized with 0.2% Triton X-100 in PBS and bacteria were stained with a rabbit polyclonal antiserum against O157 K (Behring, Marburg, Germany). After being washed, the primary antibody was labeled with fluorescein isothiocyanate (FITC)-conjugated goat anti-rabbit antibodies (Dianova, Hamburg, Germany), and F-actin was stained (20) with tetramethyl rhodamine isothiocyanate (TRITC)-labeled phalloidin (Sigma, Deisenhofen, Germany). Then coverslips were washed and mounted and cells were examined by epifluorescence with a Zeiss axiophot microscope (Carl Zeiss, Jena, Germany).

Detection of secreted and cellular proteins in and cellular fractioning of infected HeLa cells. To enhance expression and secretion of Esp proteins, bacteria were grown in DMEM-HEPES until they reached an absorbance at 600 nm of 0.6 (18). Then the proteins present in the supernatant fluids were precipitated by the addition of 10% (vol/vol) trichloroacetic acid, overnight incubation at 4°C, and subsequent centrifugation at 4,000 × g for 30 min. The dry pellet was resuspended in 1.5 M Tris (pH 8.8). To obtain whole-cell extracts, bacteria were pelleted, and after resuspension in electrophoresis sample buffer (33), they were boiled at 100°C for 10 min. Bacteria were fractionated to obtain cytoplasmic and periplasmic and outer- and inner-membrane extracts according to standard protocols (34). Proteins (30 µg/lane) were fractionated by discontinuous sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with a 12.5% separating gel (33). Then they were transferred onto a positively charged Biodyne B nylon membrane (Pall, Dreieich, Germany) with a semidry device (Bio-Rad Laboratories). Nonspecific binding sites were saturated with 5% (vol/vol) low-fat milk (1.5%) in PBS-Tween 20 (0.1%, vol/vol). The EspA, EspB, EspD, and EspE proteins were detected with mouse monoclonal antibodies (MAbs) specific for EspA (MAb B71), EspB (MAb A289), EspD (MAb anti-EspD), and EspE (MAb B51) (6, 8, 9) as first antibodies and with horseradish peroxidase-conjugated rabbit anti-mouse immunoglobulin G and immunoglobulin M as second antibodies (Bio-Rad Laboratories). Antigen-antibody complexes were visualized by chemiluminescence with the ECL system (Amersham Life Science, Braunschweig, Germany). Cellular fractioning of infected HeLa cells was carried out according to the methods described by Wolff et al. (38).

SEM. For scanning electron microscopy (SEM), infected cells grown on round 12-mm-diameter Thermanox glass coverslips were fixed in cacodylate buffer (0.1 M cacodylate, 0.01 M MgCl₂, 0.01 M CaCl₂ [pH 6.9]) containing 3% (vol/vol) glutaraldehyde and 5% (vol/vol) p-formaldehyde for 45 min on ice, washed with PBS, dehydrated in a graded series of acetone, and subjected to critical-point drying with CO₂. Samples were then sputtered with a 10-nm-thick gold film and examined with a Zeiss DSM 982 Gemini field emission SEM.

Quantitative determination of bacterial attachment and invasion. HeLa cells were seeded into 24-well plates (5 × 10⁴ cells/well) and grown overnight in DMEM-HEPES with 10% FCS. Prior to infection, each well was washed and the medium was replaced by DMEM-HEPES supplemented or not supplemented with FCS. Monolayers were infected at a bacterium/cell ratio of 100:1 for 3.5 h. Supernatant fluids were subsequently discarded, the wells were washed with PBS to remove nonadherent bacteria, DMEM supplemented with gentamicin (100 µg ml¹) was added, and cells were further incubated for 2.5 h. Then the wells were washed with PBS, HeLa cells were lysed by adding 500 µl of 0.25% (vol/vol) Triton X-100, and the number of CFU recovered from each well was determined by plating appropriate dilutions on LB agar plates with a spiral plater (Autoplate model 3000; Spiral Biotech, Inc., Bethesda, Md.). For the quantification of attached bacteria, cells were infected for 6 h with antibiotic-free DMEM-HEPES (during this period monolayers were washed several times to remove nonadherent bacteria). Then cells were washed and lysed and the total number of bacteria recovered per well was determined. Values were corrected by subtracting the number of viable intracellular bacteria, as determined from matching controls pretreated with gentamicin. Reported results are mean values from three independent experiments. The statistical significance of the obtained results has been evaluated by the Student t test, and differences were considered significant at a P of 0.05.

Nucleotide sequence accession number. The nucleotide sequence of espD (22) is available in the EMBL database under the accession no. Y13068. This sequence is identical to the one deposited and published by Perna et al. (30) (EMBL accession no. AF071034), with the exception of cytosin residues at positions 751 and 967 of the espD gene.

	RESULTS AND DISCUSSION

Top Abstract Introduction Materials and Methods Results and Discussion References

Sequence analysis of espD from EHEC EDL933. The gene encoding the secreted protein EspD from the EHEC strain EDL933 is localized 13 bp downstream of the stop codon of espA and 21 bp upstream of espB, exposing a potential Shine-Dalgarno sequence 5 bp upstream of espD (GGAGA). The 1,124 bp of the espD gene encodes a protein product of 374 aa with an isoelectric point of 5.23, and no signal peptide cleavage site was detected. The analysis using the algorithm of the topology prediction program TMPred (17) resulted in the identification of three certain transmembrane (TM) stretches (I₁₇₁ to N₁₉₁, L₁₉₄ to M₂₁₄, and V₂₂₈ to S₂₄₈) and three putative TM stretches (D₇ to S₂₇, T₄₈ to A₆₈, and P₁₀₈ to S₁₂₈), whereas the PSORT algorithm (26) detected two certain TM regions (V₁₈₁ to M₂₀₅ and V₂₂₈ to G₂₄₄). Based on the predicted topology, the EspD protein is localized to either the bacterial inner membrane (P = 0.215) or the eukaryotic plasma membrane (P = 0.440). Using the BLASTP plus BEAUTY algorithm (2, 39) we found homologies to the EspD protein in EPEC and diffusely adhering E. coli strains (between 73 and 85% identity and 82 and 90% similarity; EMBL accession no. Y09228, Y13859, Y17875, and Y17874), the translocator protein YopB from Yersinia pestis and Yersinia enterolitica (25 and 24% identity and 42 and 40% similarity, respectively; EMBL accession no. Q06114 and P37131), and flagellin from Pseudomonas putida (EMBL accession no. L15366). With the FASTA algorithm (29), homologies were also detected for IpaB from Shigella flexneri and Shigella dysenteriae (22% identity and 39% similarity, respectively; EMBL accession no. P18011 and Q03945). The degree of homology between EspD and the YopB protein, as well as their conserved structural features (36), suggests that EspD might be involved in the formation of a translocation apparatus.

Generation and characterization of an espD deletion mutant. In former studies, we have given proof that EHEC EDL933 secretes EspD via the type III secretion system, because a mutant which was defective in an essential component of the type III secretion apparatus (pas) no longer secreted EspD (22). To characterize the role played by EspD in the pathogenicity of EHEC, a mutant with an in-frame deletion in espD (EDL933espD) was generated as described in Materials and Methods (Fig. 1). The deletion of the espD gene in EDL933espD was expected to result in the loss of production and secretion of EspD but not that of the proteins EspA and EspB, which are encoded by ORFs located upstream and downstream of espD, respectively. Therefore, EDL933 and its espD derivative were grown in DMEM-HEPES; bacterial cultures were fractionated into supernatant, cytoplasmic and periplasmic, and outer- and inner-membrane fractions; and the resulting extracts were separated by SDS-PAGE and analyzed by immunoblotting with MAb anti-EspD to determine the expression and secretion of EspD. EDL933 produced and secreted EspD as expected (only secretion shown in Fig. 2A), whereas no signals were obtained in any of the fractions for EDL933espD, showing that the mutant was deficient in the production of EspD (Fig. 2B to E).

View larger version (12K): [in this window] [in a new window]   FIG. 2.   Expression and secretion of EspD. The supernatant fluids of the EHEC strain EDL933 (lane A) and its espD derivative EDL933espD (lane B) were concentrated with trichloroacetic acid, and cultures from EDL933espD were fractionated into the cytoplasmic and periplasmic (lane C), inner-membrane (lane D), and outer-membrane (lane E) fractions. Samples (30 µg) of proteins were separated by SDS-PAGE and analyzed by immunoblotting with MAb anti-EspD. The expected size of the EspD protein (40 kDa) is indicated.

The EDL933espD mutant exhibits an impaired attachment to HeLa cells. Knutton et al. found that an espA mutant of the EPEC strain E2348/69 exhibited a significant reduction in its adherence to epithelial cells (21). However, the deletion of espA had a less dramatic effect on the attachment of EPEC or REPEC (1, 19) than on that of STEC (3, 9). It seems that in EPEC and REPEC the presence of another adhesin(s) compensates for the lack of EspA-containing filaments, resulting in conserved attachment. To assess the role played by EspD in the adherence of STEC strains to epithelial cells, the attachment of the EDL933espD mutant to HeLa cells was analyzed. The adhesion of EDL933espD was reduced by 88% when compared to that of the parental strain (Fig. 3). Interestingly, the invasiveness of the espD derivative was not affected (Fig. 3). This suggests that bacterial invasion is EspD independent.

View larger version (33K): [in this window] [in a new window]   FIG. 3.   Adherence and invasiveness of the EHEC strain EDL933espD. The ability of EHEC strains EDL933 and EDL933espD to attach to and invade HeLa cells was determined as described in Materials and Methods. As a control, the attachment and invasion of E. coli K-12 were also analyzed (0.1 and 0%, respectively, of the values for EDL933 [not shown]). Differences between EDL933 and EDL933espD were statistically significant at a P of 0.05 (*).

EspD is required for actin accumulation underneath adherent bacteria. The infection of eukaryotic cells with the EHEC strain EDL933 does not result in the tyrosine phosphorylation of the translocated receptor for intimin EspE (6). Therefore, to investigate whether the signal transduction pathway was impaired in the mutant strain, actin accumulation underneath adherent bacteria was studied by immunofluorescence microscopy. Staining of F-actin with TRITC-labeled phalloidin revealed that in contrast to the parental strain EDL933, the mutant strain EDL933espD was unable to trigger actin accumulation (Fig. 4). Therefore, the involvement of the EspD protein from EHEC in the generation of A/E lesions appears to be similar to that of the EspD protein from EPEC (23). The capacity to accumulate the actin of the EDL933espD mutant was restored when this strain was transformed with a plasmid containing the espD gene under the control of its natural promoter (pANK84) (Fig. 4C1 to C3). The fact that the wild-type phenotype was not restored by providing in trans a pANK84 derivative (pAKSK78) in which the espD gene was deleted (not shown) ruled out any contribution of the upstream sequences in the observed complementation.

View larger version (80K): [in this window] [in a new window]   FIG. 4.   Immunofluorescence microscopy of HeLa cells infected with EHEC. HeLa cells were infected for 4 h with EDL933espD (A1 to A3), EDL933 (B1 to B3), or EDL933espD[pANK84] (C1 to C3). Bacteria were labeled with O157-specific antiserum and TRITC-conjugated secondary antibodies (column 1), and actin was labeled with FITC-conjugated phalloidin (column 3). An overlay of series 1 and 3 is shown (column 2). Arrows indicate adherent EDL933espD bacteria, which do not accumulate actin.

Influence of EspD in the production of EspA filaments. Recently, our group and others (9, 21) have described the production of surface appendages by STEC and EPEC. These filaments contain EspA as a major component and seem to be translocation channels to deliver effector molecules into the cytosol of host cells. Knutton et al. reported that an espD mutant of EPEC produces shorter variants of these filaments and hypothesized that EspD might be a constituent of EspA surface appendages (21). However, experimental data supporting this speculation were not provided and whether the reported effect was due to the direct involvement of EspD in the formation of the filaments or to its indirect involvement in the synthesis, export, or assemblage of structural components of the appendages was not analyzed. Thus, we carried out initial immunofluorescence studies with MAb B71 to detect EspA filaments during EHEC infection of HeLa cells. The wild-type strain EDL933 produced EspA-containing surface appendages which established a link between bacteria and host cells, whereas the espD derivative lacked the filamentous structures and exhibited poor staining with anti-EspA antibodies (Fig. 5). However, the appendages produced by EHEC were less numerous and thinner than those produced by EPEC (30 versus 50 nm in thickness [not shown]). Interestingly, some members of the EDL933espD population showed focal accumulation of EspA on the bacterial surface, suggesting the presence of aggregates of structural proteins following an aborted formation of surface appendages (Fig. 5A to C).

View larger version (26K): [in this window] [in a new window]   FIG. 5.   Immunofluorescence microscopy of EspA filaments from EDL933. After 4 h of infection of HeLa cells with EDL933espD (A to C) or EDL933 (D), bacteria were labeled with O157-specific antiserum and FITC-conjugated secondary antibodies (A and D) and EspA was labeled with MAb B71 and TRITC-conjugated secondary antibodies (C and D). An overlay of panels A and C (B) is shown. The arrows indicate the secreted EspA protein aggregated and localized between EDL933espD bacteria (A to C) or EspA filaments of EDL933 (D).

View larger version (135K): [in this window] [in a new window]   FIG. 6.   SEM analysis of EDL933espD-infected HeLa cells. Aggregates of bacteria (A to C), which contained amorphous material (indicated by arrows) between individual bacterial cells (B and C), could be visualized. The surface appendages observed in EDL933-infected cells after 4 h (E) were not produced by EDL933espD (D). The wild-type phenotype was restored in the complemented strain EDL933espD[pANK84] (F), which also formed microcolonies (G; arrows) covered by microvilli (H) on the surfaces of HeLa cells. Arrows in panels E and F indicate EspA filaments.

Localization of the product encoded by the espD gene during EHEC infection of HeLa cells. To investigate the potential involvement of EspD in the synthesis of the surface appendages, immunofluorescence studies were performed. In contrast to what was expected, EspD was not found as a second component of the EspA filaments but rather was found to form patches underneath attached bacteria (Fig. 7A and B). However, this finding seems to be in agreement with the fact that EspD exhibits homology with the secreted protein YopB of Yersinia spp., which is delivered into the membranes of target cells to function as a translocator for effector proteins (5), and with the predicted topology of EspD in the cytoplasmic membranes of eukaryotic cells (see above).

View larger version (43K): [in this window] [in a new window]   FIG. 7.   Detection of EspD by immunofluorescence microscopy. After 4 h of infection of HeLa cells, bacteria were labeled with O157-specific antiserum and FITC-conjugated secondary antibodies (A1 to A12), actin was labeled with FITC-conjugated phalloidin (A1 to A12), and EspD was labeled with MAb anti-EspD and TRITC-conjugated secondary antibodies (B1 to B12). The analytical sectioning by confocal laser microscopy was performed from the top to the bottom of the microcolony (panels 1 to 12) in 0.02-µm steps. (C) The x,z sectioning with the same labeling shows EspD underneath attached bacteria. The bar in panel A1 indicates 5 µm, and the arrow in panel C indicates EspD within the eukaryotic cells.

View larger version (24K): [in this window] [in a new window]   FIG. 8.   Localization of the EspD protein within HeLa cells after infection with EDL933. Cells were infected with the EHEC strain EDL933 for 6 h. Eukaryotic cells were fractionated as described in Materials and Methods, and EspD was detected by Western blotting. (A) Secreted proteins of bacteria grown in DMEM-HEPES; (B) secreted protein of bacteria during infection of HeLa cells; (C) cytosolic fraction; (D) cytoplasmic membrane fraction; (E) fraction containing nuclei, cytoskeletal proteins, and proteins of adherent bacteria. The molecular mass of the main EspD protein is indicated by an arrow.