Typing of intimin genes in human and animal enterohemorrhagic and enteropathogenic Escherichia coli: characterization of a new intimin variant

Abstract

Enteropathogenic Escherichia coli (EPEC) and enterohemorrhagic E. coli (EHEC) produce the characteristic "attaching and effacing" (A/E) lesion of the brush border. Intimin, an outer membrane protein encoded by eae, is responsible for the tight association of both pathogens with the host cell. Several eae have been cloned from different EPEC and EHEC strains isolated from humans and animals. These sequences are conserved in the N-terminal region but highly variable in the last C-terminal 280 amino acids (aa), where the cell binding activity is localized. Based on these considerations, we developed a panel of specific primers to investigate the eae heterogeneity of the variable 3' region by using PCR amplification. We then investigated the distribution of the known intimin types in a large collection of EPEC and EHEC strains isolated from humans and different animal species. The existence of a yet-unknown family of intimin was suspected because several EHEC strains, isolated from human and cattle, did not react with any of the specific primer pairs, although these strains were eae positive when primers amplifying the conserved 5' end were used. We then cloned and sequenced the eae present in one of these strains (EHEC of serotype O103:H2) and subsequently designed a PCR primer that recognizes in a specific manner the variable 3' region of this new intimin type. This intimin, referred to as "epsilon," was present in human and bovine EHEC strains of serogroups O8, O11, O45, O103, O121, and O165. Intimin epsilon is the largest intimin cloned to date (948 aa) and shares the greatest overall sequence identity with intimin beta, although analysis of the last C-terminal 280 aa suggests a greater similarity with intimins alpha and gamma.

FIG. 1

CLUSTAL W alignment of intimin ɛ sequence (AF116899) from the EHEC O103:H2 strain PMK5 with α, β, γ and δ intimins cloned from different pathogenic E. coli (EPEC or EHEC), an intimin homologue from H. alvei, and an intimin homologue from C. rodentium. The accession numbers in GenBank for these sequences and the names of the bacterial strains are given in Materials and Methods. Of note, a recent study (35) suggests that the H. alvei strains containing the intimin gene are, in fact, unusual biotypes of E. coli or represent a new species in the genus Escherichia. The multiple alignment was based on the last C-terminal amino acids of the different intimin (starting with alanine 658). The two cysteine residues necessary for the formation of a disulfide bond and the binding activity are indicated by asterisks.

FIG. 2

Phylogenetic tree based on the last C-terminal amino acids of the different intimin (starting with alanine 658) of different α, β, γ, δ, and ɛ intimins of pathogenic E. coli, an intimin homologue from H. alvei, and an intimin homologue from C. rodentium. The accession numbers in GenBank for these sequences and the names of the bacterial strains are given in Materials and Methods. Of note, a recent study (35) suggests that the H. alvei strains containing the intimin gene are, in fact, unusual biotypes of E. coli or represent a new species in the genus Escherichia.

FIG. 3

PstI RFLP analysis of the eae PCR products obtained with the different type-specific primers from E. coli strains representative of the different intimin types. M, molecular size marker; lane 1, type α1 (EPEC O127:H6); lane 2, type α2 (EPEC O125:H6); lane 3, type β1 (EHEC O26:H11); lane 4, type β2 (EPEC O86:H34); lane 5, type γ1 (EHEC O157:H7); lane 6, type γ2 (EHEC O111:H−); lane 7, type ɛ (EHEC O103:H2).