Translocated intimin receptors (Tir) of Shiga-toxigenic Escherichia coli isolates belonging to serogroups O26, O111, and O157 react with sera from patients with hemolytic-uremic syndrome and exhibit marked sequence heterogeneity

Abstract

The capacity to form attaching and effacing (A/E) lesions on the surfaces of enterocytes is an important virulence trait of several enteric pathogens, including enteropathogenic Escherichia coli (EPEC) and Shiga-toxigenic E. coli (STEC). Formation of such lesions depends upon an interaction between a bacterial outer membrane protein (intimin) and a bacterially encoded receptor protein (Tir) which is exported from the bacterium and translocated into the host cell membrane. Intimin, Tir, and several other proteins necessary for generation of A/E lesions are encoded on a chromosomal pathogenicity island termed the locus for enterocyte effacement (LEE). Reports of sequence heterogeneity and antigenic variation in the region of intimin believed to be responsible for receptor binding raise the possibility that the receptor itself is also heterogeneous. We have examined this by cloning and sequencing tir genes from three different STEC strains belonging to serogroups O26, O111, and O157. The deduced amino acid sequences for the Tir homologues from these strains varied markedly, exhibiting only 65.4, 80.2, and 56.7% identity, respectively, to that recently reported for EPEC Tir. STEC Tir is also highly immunogenic in humans. Western blots of E. coli DH5alpha expressing the various STEC tir genes cloned in pBluescript [but not E. coli DH5alpha(pBluescript)] reacted strongly with convalescent sera from patients with hemolytic-uremic syndrome (HUS) caused by known LEE-positive STEC. Moreover, no reaction was seen when the various clone lysates were probed with serum from a patient with HUS caused by a LEE-negative STEC or with serum from a healthy individual. Covariation of exposed epitopes on both intimin and Tir may be a means whereby STEC avoid host immune responses without compromising adhesin-receptor interaction.

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.

FIG. 2

Western immunoblot analysis of Tir-expressing clones using convalescent sera from HUS patients. Lysates of E. coli DH5α 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.

FIG. 3

Reactivity of HUS patient serum with STEC and EPEC strains and Tir-producing E. coli DH5α clones. The same serum used in Fig. 2A was used to probe Western blots of the indicated E. coli strains. Lanes: 1, E. coli DH5α(pJCP580); 2, STEC 95NR1; 3, E. coli DH5α(pJCP581); 4, STEC 95ZG1; 5, E. coli DH5α(pJCP582); 6, STEC 95SF2; 7, E. coli DH5α(pJCP583); 8, EPEC 87A. The mobility of protein mass markers is indicated to the left.