title: Expression and characterization of the eaeA gene product of Escherichia coli serotype O157:H7.
abstract: In enteropathogenic Escherichia coli, the eaeA gene produces a 94-kDa outer membrane protein called intimin which has been shown to be necessary but not sufficient to produce the attaching-and-effacing lesion. The purpose of this study was to characterize the intimin specified by the eaeA allele of the enterohemorrhagic E. coli (EHEC) serotype O157:H7 strain CL8 and to determine its role in adherence. The carboxyl-terminal 266 amino acids of the CL8 intimin were expressed as a protein fusion with glutathione S-transferase, which was used to raise antiserum in rabbits. The antiserum reacted in Western immunoblots with a 97-kDa outer membrane protein of EHEC strains of serogroups O5, O26, O111, and O157 and enteropathogenic E. coli strains of serogroups O55 and O127. Surface labelling of CL8 with 125I showed that intimin was surface exposed. An eaeA insertional inactivation mutant of CL8 was produced and was designated CL8-KO1. Total adherence of CL8-KO1 to HEp-2 cells was not significantly different from that of CL8, but CL8-KO1 gave a negative result in the fluorescent actin staining test. The eaeA gene expressed alone in E. coli HB101 also gave a negative fluorescent actin staining test result. The eaeA gene of CL8 was able to complement the eaeA deletion mutation in CVD206. We conclude that the product of the EHEC eaeA gene is a 97-kDa surface-exposed protein and propose that it be designated intiminO157. Sherman et al. described a 94-kDa outer membrane protein which played an important role in adherence of E. coli O157:H7 (Infect. Immun. 59:890-899, 1991). Western immunoblotting and indirect fluorescent antibody studies showed that the protein described by Sherman et al. is not intimin.
authors.affiliation: Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, Ontario, Canada.
authors.forename: M
authors.initials: M
authors.lastname: Louie
authors.forename: J C
authors.initials: JC
authors.lastname: de Azavedo
authors.forename: M Y
authors.initials: MY
authors.lastname: Handelsman
authors.forename: C G
authors.initials: CG
authors.lastname: Clark
authors.forename: B
authors.initials: B
authors.lastname: Ally
authors.forename: M
authors.initials: M
authors.lastname: Dytoc
authors.forename: P
authors.initials: P
authors.lastname: Sherman
authors.forename: J
authors.initials: J
authors.lastname: Brunton
mesh_terms: D008841:Actin Cytoskeleton / Q000648:ultrastructure; D000199:Actins / Q000378:metabolism; D018829:Adhesins, Bacterial; D000942:Antigens, Bacterial / Q000235:genetics; D000954:Antigens, Surface / Q000235:genetics; D001422:Bacterial Adhesion; D001425:Bacterial Outer Membrane Proteins / Q000235:genetics* / Q000276:immunology / Q000378:metabolism; D001483:Base Sequence; D002352:Carrier Proteins; D004926:Escherichia coli / Q000235:genetics / Q000472:pathogenicity*; D029968:Escherichia coli Proteins; D005798:Genes, Bacterial; D006367:HeLa Cells; D006801:Humans; D066298:In Vitro Techniques; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D011993:Recombinant Fusion Proteins
chemical_list: D000199:Actins; D018829:Adhesins, Bacterial; D000942:Antigens, Bacterial; D000954:Antigens, Surface; D001425:Bacterial Outer Membrane Proteins; D002352:Carrier Proteins; D029968:Escherichia coli Proteins; D009838:Oligodeoxyribonucleotides; D011993:Recombinant Fusion Proteins; C073819:eaeA protein, E coli