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Characterization of the locus of enterocyte effacement (LEE) in different enteropathogenic Escherichia coli (EPEC) and Shiga-toxin producing Escherichia coli (STEC) serotypes

Vanessa Sperandio , James B Kaper , Mafalda Regina Bortolini , Bianca Cruz Neves , Rogeria Keller , Luiz R Trabulsi
DOI: http://dx.doi.org/10.1111/j.1574-6968.1998.tb13078.x 133-139 First published online: 1 July 1998

Abstract

All proteins involved in the attachment and effacement lesion produced by enteropathogenic Escherichia coli (EPEC) and Shiga-toxin producing E. coli (STEC) are encoded by the locus of enterocyte effacement (LEE). We studied the presence and insertion site of the LEE in different EPEC and STEC strains. In serotypes O119:H6/H, O55:H6, O55:H7, O142:H6, O111ac:H9/H, O111ab:H9/H LEE is inserted downstream of selC as previously described for EPEC O127:H6 and STEC O157:H7. In serotypes O111ac:H8/H and O26:H11/H the LEE is inserted in pheU as previously described for STEC O26:H. However in EPEC from serotype O111ab:H25 the LEE is not inserted in either site suggesting a third insertion site in the K12 chromosome. We also cloned fragments of 2.3 kb and 1.0 kb from the right and left hand sides of the LEE of a O111ac:H strain and identified additional insertion sequences on these LEE fragments, suggesting that the LEE may be larger and may have undergone more recombination events in these serotypes.

Key words
  • Enteropatogenic Escherichia coli
  • Pathogenicity island
  • Attachment and effacement

1 Introduction

The locus of enterocyte effacement (LEE) is a 35 kb pathogenicity island that encodes gene products required for the attaching and effacing (AE) lesions produced by EPEC on epithelial cells [1, 2]. These lesions are marked by the degenerated microvilli and ‘pedestals' of densely clustered cytoskeletal proteins, including polymerized actin, that protrude from the apical membrane and intimately cup individual bacteria [3, 4]. Attaching and effacing E. coli pathogenic for humans and animals (EPEC, Shiga-toxin producing E. coli (STEC) or enterohemorrhagic E. coli (EHEC) and rabbit diarrheagenic E. coli (REDEC)) as well as Citrobacter rodentium and Hafnia alvei all produce AE lesions and have a LEE region [1]. The LEE encodes a type III secretion system [5], an adhesin (intimin), responsible for the intimate attachment of the bacteria to the cell [6, 7], and at least three secreted proteins (EspA, EspB and EspD) involved in signal transduction [1, 810].

In EPEC E2348/69 (O127:H6) and STEC O157:H7 the LEE is inserted at 82 min in the E. coli chromosome downstream of the gene encoding the tRNA for selenocysteine (selC) [1]. In STEC from serotype O26:H the LEE is inserted at 94 min in the gene that encodes the tRNA for phenylalanine (pheU) [11].

In this study, we assayed strains from different EPEC and STEC serotypes for the presence and site of insertion of the LEE, and our results indicate that some strains have previously undescribed LEE DNA sequences and that there appears to be a third site in the E. coli chromosome where the LEE has been inserted.

2 Materials and methods

2.1 Bacterial strains

Bacterial strains from EPEC and STEC serotypes O119 (H6 and H), O111 (H2, H25, H8, H9 and H), O142:H6 and O55 (H6, H7 and H) were isolated from cases of diarrhea; genotypic and phenotypic features of these strains were previously determined [1215] and are described in Table 1. EPEC E2348/69 (O127:H6) is a prototypic strain which has been extensively characterized in volunteer studies [16, 17].

View this table:
1

Phenotypic and genotypic features of the studied E. coli strains from serogroups O111, O119, O26, O55 and O142

2.2 Hybridizations with LEE DNA probes

All strains were hybridized by a colony blot procedure [18] with LEE probe A, a 2.87 kb MluI/EcoRI fragment of pCVD453 which has the left hand side of LEE (we arbitrarily used right and left sides as described by McDaniel et al., 1995); probe B, a 2.95 kb SalI/EcoRI fragment of pCVD461 which has part of escV and escN; probe C, a 1 kb SalI/StuI fragment of pCVD443 which has part of eae; and probe D, a 2.3 kb BglII fragment of pCVD460 which has part of espA and espB[1]. All fragments were electrophoresed and excised from 0.7% agarose gels, purified with a QIAquick Gel extraction Kit (QIAGEN), labeled with α-32P-dCTP by random priming with Ready to Go DNA labelling Beads (Pharmacia Biotech) according to the manufacturer's instructions and hybridized with high stringent conditions [18].

2.3 PCR reactions for LEE insertion downstream of selC

Insertion of the LEE downstream of selC was assayed using primers K255 and K260 for the right junction; K296 and K295 for the left junction; and K261 and K260 for the intact selC gene (Table 2). Primers K255 and K296 are inside the LEE and primers K260, K261 and K295 are in K12 chromosomal sequences. When LEE is inserted downstream of selC, primers K255 and K260 as well as K295 and K296 amplify fragments of 418 bp each, and primers K260 and K261 do not amplify any detectable fragment, as determined for the LEE insertion in EPEC strain E2348/69 (Accession numbers AF031371 and AF031372). In the absence of LEE, primers K260 and K261 amplify a 402 bp fragment since selC is intact (Accession number AE000443) [1]. All three reactions were performed with 200 ng of purified genomic DNA template, 100 ng of each primer (K255 and K260 for the right junction; K296 and K295 for the left junction; and K260 and K261 for the intact selC gene), 200 μM deoxynucleoside triphosphates (dNTPs), 1 U Taq DNA polymerase, and 1.5 mM MgCl2 in Taq polymerase buffer (Life Technologies, Gaithersburg, MD). After denaturing the template at 94°C for 10 min, a total of 30 cycles were performed at 94°C for 1 min, 52°C for 1 min, and 72°C for 3 min.

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2

Primers used in PCR reactions

2.4 PCR reaction to amplify the pheU gene

Primers K913 and K914 (Table 2) were used to amplify the pheU gene. Both primers were used in a PCR reaction to amplify a 300 bp fragment, corresponding to the pheU gene in E. coli (Accession number S67565), in the same conditions described above with 30 cycles of 94°C for 1 min, 52°C for 1 min and 72°C for 2 min.

2.5 Determination of PCR fragment size

Agarose gel electrophoresis was performed to determined the size of the PCR fragments amplified using different primers. We electrophoresed 10 μl of 50 μl PCRs in 1% agarose gels in Tris-Acetate/EDTA electrophoresis buffer. The sizes of the fragments were determined by electrophoretic mobility compared to the 1 kb DNA ladder from Gibco BRL.

2.6 DNA probes for the right and left hand sides of LEE

Based on the DNA sequence of the LEE a set of four primers were designed to amplify the right and the left hand sides of LEE to be used as DNA probes. The primers used to amplify the right hand side of LEE were K723 and K724 (Table 2). The primers used to amplify the left hand side of LEE were K721 and K722 (Table 2). Both reactions were performed with 200 ng of purified genomic DNA of EPEC E2348/69, 100 ng of each primer, 200 μM deoxynucleoside triphosphates (dNTPs), 1 U Taq DNA polymerase, and 1.5 mM MgCl2 in Taq polymerase buffer (Life Technologies, Gaithersburg, MD). After an initial denaturation of the template at 94°C for 10 min, a total of 30 cycles were performed at 94°C for 1 min, 50°C for 1 min and 72°C for 2 min. Fragments of 800 bp and 1000 bp were amplified for the right and left side of LEE respectively (data not shown). Both fragments were electrophoresed and excised from 1% agarose gels, purified with a QIAquick Gel extraction Kit (QIAGEN), labeled with α-32P-dCTP by random priming with Ready to Go DNA labelling Beads (Pharmacia Biotech) and used as probes to detect LEE's right and left hand sides [18].

2.7 Cloning and sequencing of the right and left sides of the LEE from strain 172

Southern blots of genomic DNA of strain 172 (O111ac:H) digested with different endonucleases were performed. A BglII-PstI DNA fragment of 2.3 kb hybridized with the right hand side LEE probe and a 1.0 kb EcoRV fragment with the left hand side probe. Both fragments were cloned into pBluescript SKII (Stratagene) digested with BamHI-PstI and EcoRV, respectively, generating plasmids pVS15 (right) and pVS16 (left) [18]. The nucleotide sequences of pVS15 and pVS16 were determined using Ready Reaction Dye Dideoxy Terminator Cycle Sequencing Kit (Applied Biosystems) and an Applied Biosystems model 373A automated sequencer.

3 Results and discussion

A total of 80 EPEC and STEC strains were studied and all of them hybridized with LEE probes A, B, C and D (Table 3). This result was expected since all the strains were positive for the fluorescein-actin staining test (FAS) [19] (Table 1), which is diagnostic of the AE lesion.

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3

Presence of the LEE in EPEC and EHEC strains and PCR analysis for the LEE insertion site

Concerning the insertion site of the LEE as determined by our PCR analysis, within a specific serotype, all strains gave the same result. In strains from O119 (H6 and H), O111ac:H9, O111ab (H9 and H), O142:H6, and O55 (H6, H7 and H) serotypes, LEE is inserted downstream of selC. However, in serotypes O111ac (H8 and H), O111ab (H2 and H25) and O26 (H11 and H) LEE is inserted in a different site on the chromosome, since in these strains the selC gene is intact, as observed by the PCR reactions (Table 3). Primers K255 and K296 are inside the LEE while primers K260, K261 and K295 are in the E. coli K12 chromosome region. The PCR reactions with primers K255 and K260 amplify the right LEE junction, and K295 and K296 the left LEE junction with selC generating fragments of approximately 418 bp in both reactions. Primer K261 can only anneal in the absence of the LEE insertion, and generate a PCR fragment of 402 bp together with K260. These results are consistent with those recently reported by Wieler et al. [20], showing that the LEE insertion site varies according to the evolutionary lineage. Wieler et al. [20] showed that in the cluster of strains called EPEC1 and EHEC1, LEE is inserted downstream of selC while in EPEC2 and EHEC2 this locus is inserted elsewhere at an unknown location. Recently, Benkel et al. [11] described that in a O26:H strain the LEE was inserted at 94 min in the E. coli chromosome in the pheU gene. To determine whether the LEE insertion was in pheU in the serotypes where it is not inserted downstream of selC, we designed primers to amplify pheU and performed a PCR with these primers for all these strains. In serotypes O111ac:H, O111ac:H8, O26:H11 and O26:H (EHEC2) the pheU locus was interrupted suggesting that this would be the insertion site of LEE. However in serotype O111ab:H25 this locus was not interrupted, meaning that LEE is inserted at another site somewhere else in the chromosome of these bacteria and suggesting a third insertion site of LEE in the E. coli chromosome. A third insertion site is consistent with the fact that serotype O111ab:H25 does not belong to any of the EPEC/EHEC clusters [12, 21], and probably is from a different evolutionary lineage.

Since there is a considerable amount of variation of the LEE insertion sites based on evolutionary lineage, we decided to further study the LEE region from a O111ac:H strain (172) which had been isolated in 1977 from a diarrheal case in the United States, and in which the LEE insertion seems to interrupt the pheU gene. This serotype is evolutionarily divergent from serotype O127:H6, where the LEE was first described. For this purpose we amplified fragments very close to both ends of the LEE region from EPEC strain E2348/69 (O127:H6) to be used as probes to clone both right and left LEE sides from strain 172 (O111ac:H). We then digested strain 172 genomic DNA with different endonucleases and hybridized with LEE probes ‘right’ and ‘left’ in a Southern blot. We then cloned a 2.3 kb BglII-PstI fragment that hybridized with the ‘right’ LEE probe and a 1.0 kb EcoRV fragment that hybridized with LEE ‘left’ probe in pBluescript SKII to generate plasmids pVS15 (right) and pVS16 (left), respectively.

The right hand side of the LEE from strain 172 (O111ac:H), as determined by sequencing plasmid pVS15 (Accession number AF041809), contained the two last open reading frames of E2348/69 LEE [22]: ORFD3 which shared 94.82% identity and ORFD4 which shared 70% identity between strains E2348/69 (Accession number L76581) and 172. It also contained an IS600 homologue, YIS1-SHISO, which encodes a predicted 11 kDa protein that is also present in E2348/69 LEE, and an additional IS3 homologue element (Accession number AE000160) (Fig. 1 A and B), which is not present in E2348/69 LEE. In the recently published E. coli genome of K12 strain MG1655 sequence [23] there are 5 IS3 homologues in the chromosome and all of them contain the IS600 homologue upstream of them. Since there is an IS600 sequence on the right hand of the LEE DNA sequence of strain E2348/96 [22], this IS600 could potentially allow recombination of the LEE with the YIS1-SHISO homologue upstream of IS3.

1

A: Schematic organization of the right hand side of the LEE from strain 172 (O111ac:H) (Accession number AF041809). B: Schematic organization of the left hand side of the LEE from strain 172 (O111ac:H) (Accession number AF041810).

The DNA sequence of the left hand side of the LEE from strain 172 (O111ac:H), as determined by sequencing plasmid pVS16 (Accession number AF041810), was very similar to that of E2348/69 sharing 98% identity over 159 bp with E2348/69 LEE's 562–719 bp region [24]. It also contained two additional insertion sequence homologues: a Shigella sonnei IS630 (iso-IS2) homologue (86% identity) (Accession number X05955) and an E. coli IS2 homologue (97% identity) (Accession number AE000253) (Fig. 1 A and C) which are not present in the E2348/69 LEE.

These results show that the left hand side of the LEE seems to be more conserved than the right hand side and also suggest that in this O111ac:H strain LEE may be larger than the LEE in E2348/69 and may have undergone more recombination events, based on the amount of insertion sequences found in this region. Our results and those of Wieler et al. [20] and Benkel et al. [11], indicate considerable heterogeneity in this pathogenicity island. Further characterization of this divergence should yield additional insights into the evolution of the attaching and effacing family of pathogens.

Acknowledgements

We thank Dr. Tania Tardelli Gomes from Escola Paulista de Medicina, São Paulo, Brazil for the gift of some radioactive isotopes used in this study and Dr. Timothy McDaniel, Stanford University for the LEE probes. This work was supported by grants FAPESP 96/4148–5, FINEP/MCT/PRONEX 41.96.0881.00 and National Institutes of Health AI 21657.

References

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