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

Enteropathogenic Escherichia coli (EPEC) and enterohemorrhagic E. coli (EHE C) produce the characteristic "attaching and effacing" (A/E) lesion of the brush border. Intimin, an outer membrane protein encoded by eae, is respons ible for the tight association of both pathogens with the host cell. Severa l eae have been cloned from different EPEC and EHEC strains isolated from h umans and animals. These sequences are conserved in the N-terminal region b ut highly variable in the last C-terminal 280 amino acids (aa), where the c ell binding activity is localized. Based on these considerations, we develo ped a panel of specific primers to investigate the ene heterogeneity of the variable 3' region by using PCR amplification, We then investigated the di stribution of the known intimin types in a large collection of EPEC and EHE C strains isolated from humans and different animal species. The existence of a yet-unknown family of intimin was suspected because several EHEC strai ns, isolated from human and cattle, did not react with any of the specific primer pairs, although these strains were eae positive when primers amplify ing the conserved 5' end were used. We then cloned and sequenced the ene pr esent in one of these strains (EHEC of serotype O103:H2) and subsequently d esigned a PCR primer that recognizes in a specific manner the variable 3' r egion 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 E is the largest intimin cloned to date (948 aa) a nd shares the greatest overall sequence identity with intimin beta, althoug h analysis of the last C-terminal 280 aa suggests a greater similarity with intimins alpha and gamma.