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Since their initial recognition over 20 years ago (20), Shiga-toxigenic strains of Escherichia coli (STEC) have emerged as an important cause of serious human gastrointestinal disease, which may result in life-threatening complications such as hemolytic-uremic syndrome (HUS) (16). Food-borne outbreaks of STEC disease appear to be increasing and, when mass-produced and mass-distributed foods are involved, can affect large numbers of people. Development of therapeutic and preventative strategies to combat STEC disease requires a thorough understanding of the mechanisms by which STEC colonize the human intestinal tract and cause local and systemic pathology. While our knowledge remains incomplete, recent studies have improved our understanding of these processes (for recent reviews, see references 24 and 28).

It has been recognized for a number of years that STEC strains causing human disease may belong to a very broad range of O serogroups (16). However, many of the STEC strains found in the gastrointestinal tracts of domestic animals (the principal source of human infections) may be of low virulence for humans. Within the human disease-associated strains, those belonging to a limited range of serogroups (notably O157, O111, and O26) are responsible for the majority of serious infections (16, 24, 28). Previous studies have compared properties of STEC from human and animal sources in order to identify the traits which distinguish human-pathogenic strains from those of lesser clinical significance. The capacity to produce attaching and effacing (A/E) lesions on intestinal mucosa, production of a plasmid-encoded enterohemolysin, and production of Shiga toxin type 2 (Stx2) rather than Shiga toxin type 1 (Stx1) have all been associated preferentially with human STEC isolates or with more-severe cases (4, 6, 19, 25, 32). Compared with STEC isolates from cases of uncomplicated diarrhea or isolates from nonhuman sources (27), STEC associated with HUS cases also have an enhanced capacity to adhere to intestinal epithelial cells in vitro.

The capacity to produce A/E lesions was initially recognized in enteropathogenic E. coli (EPEC) strains, and recent studies have elucidated the molecular events involved in their generation, as reviewed by Donnenberg et al. (9). The mechanism whereby STEC strains generate A/E lesions is less well characterized but is essentially analogous to that for EPEC (24). All of the genes necessary for generation of A/E lesions in EPEC are located on a 35.5-kb pathogenicity island termed the locus for enterocyte effacement (LEE). LEE encodes proteins with a range of functions, including a type III secretion system, various secreted effector proteins and their chaperons, and the outer membrane protein intimin, which mediates intimate attachment to the enterocyte cell surface (9). Interestingly, Kenny et al. (17) have recently reported that the receptor for intimin is also encoded by LEE. This protein was previously referred to as Hp90 but has now been renamed Tir (for translocated intimin receptor). Tir is secreted from EPEC as a 78-kDa species, and efficient delivery into the host cell is dependent upon the type III secretion system and other LEE-encoded secreted proteins. Tyrosine phosphorylation of Tir after insertion into the epithelial cell membrane increases its apparent mass to 90 kDa, but this is not essential for intimin binding, at least in vitro (17). Moreover, it does not appear to occur in STEC-infected cultures (at least those belonging to serogroup O157) (14).

Interestingly, the sequence of the C-terminal portion of intimin is known to vary markedly between EPEC and STEC and between different STEC strains (5, 15, 22, 31, 33). This region includes the putative Tir-binding domain (11), and it is possible that such heterogeneity influences the interaction between the bacterium and the enterocyte. It may also be antigenically significant, as we have recently shown that sera from several HUS patients infected with a O111:H STEC reacted with intimin from an enteropathogenic E. coli O111 strain, as well as several other eaeA-positive STEC isolates, but not with an eaeA-positive STEC belonging to serotype O157:H. The latter strain did, however, react with serum from a patient infected with both O111:H and O157:H STEC (31). Variation of exposed intimin epitopes may be a means whereby STEC avoid host immune responses, but significant changes could be potentially deleterious unless compatible variation also occurred in Tir. In the present study, we have cloned and sequenced the tir genes from STEC belonging to diverse serogroups to determine the degree of heterogeneity of this protein. We have also examined the reactivity of Tir-producing E. coli clones with convalescent sera from patients with HUS.

Bacterial strains and cloning vectors. STEC strains 95NR1 (O111:H), 95SF2 (O157:H), and 95ZG1 (O26:H) were isolated at the Women's and Children's Hospital (WCH), North Adelaide, South Australia, Australia, as previously described (26). The O111 EPEC strain 87A was also isolated at WCH and has been described previously (27). E. coli K-12 strain DH5 was obtained from Gibco-BRL, Gaithersburg, Md., and the phagemid pBluescript SK was obtained from Stratagene, La Jolla, Calif. All E. coli strains were routinely grown in Luria-Bertani (LB) medium (23) with or without 1.5% Bacto Agar (Difco Laboratories, Detroit, Mich.). Where appropriate, ampicillin was added to growth medium at a concentration of 50 µg/ml.

Cloning and sequence analysis of tir genes. We have previously described the sequence of a portion of the LEE locus from STEC strain 95NR1, which contained orfU and eaeA (31). Comparison of this sequence with that of EPEC tir (17) indicated that it also included the 3' portion of the STEC tir homologue. In order to isolate the remainder of the tir gene, we performed inverse PCR amplification on 95NR1 DNA which had been digested with BglII, recircularized, and ligated. The primers were 5'-CGTTAAGAATTCAGAGAACAACGTTGCAGC-3' and 5'-CTGGGAATTCCCCATTAACCTTCCGGTAAC-3', and amplification was performed with the Expand High Fidelity PCR System (Boehringer GmbH, Mannheim, Germany). This generated a 3.0-kb fragment which overlapped our previous sequence and yielded an additional 1,645 bp of 5'-flanking DNA. The purified PCR product was digested with EcoRI (the PCR primers contained EcoRI sites), cloned into pBluescript SK, and transformed into E. coli DH5. The sequences of both strands of the STEC DNA insert, as well as nested deletions thereof (constructed by the method of Henikoff [12]), were then determined by using dye terminator chemistry on an Applied Biosystems model 373A automated DNA sequencer. The sequence was analyzed using DNASIS and PROSIS version 7.0 software (Hitachi Software Engineering, San Bruno, Calif.). Comparison of this sequence with sequence databases with the program BLASTX (2) confirmed that the inverse PCR product contained a region of STEC 95NR1 LEE encoding a homologue of an EPEC LEE open reading frame (orf19) with as yet unknown function (10) and the first 503 amino acids of the Tir homologue.

View larger version (124K): [in this window] [in a new window]   FIG. 1.   Alignment of the deduced amino acid sequences of Tir from STEC strains 95NR1 (O111:H), 95SF2 (O157:H), and 95ZG1 (O26) and from EPEC 87A (O111) with two published sequences for Tir from EPEC strain E2348/69 (GenBank accession nos. AF013122 and AF022236, respectively [10, 17]). Residues at a given position which are identical to that for 95NR1 Tir are boxed, while dashes indicate the absence of an amino acid. Conserved tyrosine residues in the C-terminal half of Tir are shaded, and the location of the Tyr residue predicted by the PROSITE algorithm (3) to be the site for tyrosine kinase phosphorylation is indicated with an asterisk.

Western blot analysis using HUS patient sera. To examine the expression of cloned tir, lysates of E. coli DH5 carrying pBluescript, pJCP580, pJCP581, pJCP582, or pJCP583, or E. coli 95NR1 were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (21), and antigens were electrophoretically transferred to nitrocellulose filters (30). Filters were then probed with convalescent sera from various HUS patients (kindly provided by K. F. Jureidini, Renal Unit, WCH) or from a healthy donor (all sera were used at a dilution of 1:3,000), followed by goat anti-human immunoglobulin G conjugated to alkaline phosphatase. Immunoreactive bands were visualized using chromogenic substrate (4-nitroblue tetrazolium and 5-bromo-4-chloro-3-indolylphosphate). The serum used in Fig. 2A was from a patient with HUS associated with an outbreak caused by dry-fermented sausage contaminated with a range of STEC including 95NR1 (26); this patient had an O111:H STEC similar to 95NR1 isolated from feces. This serum labelled a number of protein species in the lysate of E. coli DH5(pJCP580) (Fig. 2A, lane 2), the largest and one of the most prominent having an apparent mass of 64 kDa, which is slightly higher than that predicted for 95NR1 Tir by sequence analysis (57 kDa). It also appeared to comigrate with a weakly immunoreactive species in the lysate of the wild-type STEC 95NR1 (lane 6). Other prominently labelled protein species in the E. coli DH5(pJCP580) lysate were in the range 38 to 60 kDa and presumably reflect degradation products of Tir, as the only other open reading frame in the portion of 95NR1 LEE cloned in pJCP580 encoded Orf19, which has a predicted mass of 22.5 kDa (a weak immunoreactive band is seen at this apparent mass). The HUS patient serum reacted with a very similar pattern of protein species in the lysate of E. coli DH5(pJCP582) (lane 4) which produces Tir from the O157:H STEC, in spite of the fact that there is only 59% amino acid identity between the two proteins. However, the labelling pattern in lysates of E. coli DH5(pJCP581) and DH5(pJCP583) (lanes 3 and 5), which produce Tir from the O26 STEC 95ZG1 and the O111 EPEC strain 87A, respectively, was much weaker; the principal immunoreactive species had apparent masses of 72, 64, and 60 kDa. The Tir from these two strains exhibit approximately 64% amino acid identity with 95NR1 Tir. Significantly, there were no detectable immunoreactive species in the lysate of E. coli DH5(pBluescript) (lane 1). Two other convalescent sera from patients with HUS associated with the same outbreak were also tested. The serum used in Fig. 2C was from a patient who was also infected with an O111:H STEC closely related to 95NR1, while that used in Fig. 2B yielded both O111:H and O157:H STEC isolates from feces. Both these sera exhibited labelling patterns with the various E. coli lysates similar to that seen in Fig. 2A. No immunoreactive species were detected in the various clone lysates when healthy donor serum was used (result not shown). Furthermore, when convalescent serum from a HUS patient who was infected with a LEE-negative STEC strain belonging to serotype OR:H9 was used to probe filters, there were also no immunoreactive species detected (Fig. 2D). When this serum was used to probe a lysate from the causative OR:H9 strain, very few protein bands were labelled (result not shown), but this was not unexpected, as we have recently demonstrated that the bulk of antibody responses of HUS patients with LEE-positive STEC disease appear to be directed at either lipopolysaccharide or LEE-encoded proteins (31). None of these antigens are present in the OR:H9 strain. There was also no evidence that the OR:H9-infected HUS patient had defective immune responses.

View larger version (67K): [in this window] [in a new window]   FIG. 2.   Western immunoblot analysis of Tir-expressing clones using convalescent sera from HUS patients. Lysates of E. coli DH5 carrying pBluescript (lane 1), pJCP580 (lane 2), pJCP581 (lane 3), pJCP582 (lane 4), or pJCP583 (lane 5) or STEC 95NR1 (lane 6) were separated by SDS-PAGE, electroblotted, and probed with convalescent sera from HUS patients infected with O111:H STEC (A and C), both O111:H and O157:H STEC (B), or a LEE-negative OR:H9 STEC (D). The mobility of protein mass markers is indicated to the left.

View larger version (102K): [in this window] [in a new window]   FIG. 3.   Reactivity of HUS patient serum with STEC and EPEC strains and Tir-producing E. coli DH5 clones. The same serum used in Fig. 2A was used to probe Western blots of the indicated E. coli strains. Lanes: 1, E. coli DH5(pJCP580); 2, STEC 95NR1; 3, E. coli DH5(pJCP581); 4, STEC 95ZG1; 5, E. coli DH5(pJCP582); 6, STEC 95SF2; 7, E. coli DH5(pJCP583); 8, EPEC 87A. The mobility of protein mass markers is indicated to the left.

Conclusions. The capacity to produce A/E lesions on enterocytes, mediated by the outer membrane protein intimin, is clearly a significant virulence trait of both EPEC and STEC (24). However, intimin is highly immunogenic and is exposed on the bacterial cell surface, and systemic or local immune responses to this protein may be capable of blocking the interaction between the bacterium and the intestinal epithelium. Antigenic variation, particularly in the exposed intimin domains, may therefore confer a significant selective advantage upon the bacterium, assuming such variation does not compromise receptor interactions. Amino acid sequence heterogeneity, particularly in the C-terminal portion of intimin which is responsible for receptor binding (11), is now well documented (5, 15, 22, 31, 33). Furthermore, Adu-Bobie et al. (1) have recently demonstrated the existence of at least five distinct classes of intimin in EPEC and STEC strains on the basis of reactivity with polyclonal antisera raised against the C-terminal portions of intimin from various strains. We have also provided direct evidence for antigenic diversity of intimin in STEC strains, as judged by reactivity of convalescent sera from HUS patients with various STEC strains (31). The fact that the intimin receptor Tir is encoded by the bacterium rather than the host (17) provides a mechanism whereby substantial intimin sequence (and hence antigenic) variation can be tolerated without compromising ligand-receptor interactions, assuming that compensatory changes also occur in the amino acid sequence of Tir.

Nucleotide sequence accession number. The nucleotide sequences described in this study have been deposited with GenBank under accession numbers AF025311, AF070067, AF070068, and AF070069.