M. Louie et al., EXPRESSION AND CHARACTERIZATION OF THE EAEA GENE-PRODUCT OF ESCHERICHIA-COLI SEROTYPE O157-H7, Infection and immunity, 61(10), 1993, pp. 4085-4092
In enteropathogenic Escherichia coli, the eaeA gene produces a 94-kDa
outer membrane protein called intimin which has been shown to be neces
sary but not sufficient to produce the attaching-and-effacing lesion.
The purpose of this study was to characterize the intimin specified by
the eae-4 allele of the enterohemorrhagic E. coli (EHEC) serotype O15
7:H7 strain CL8 and to determine its role in adherence. The carboxyl-t
erminal 266 amino acids of the CL8 intimin were expressed as a protein
fusion with glutathione S-transferase, which was used to raise antise
rum in rabbits. The antiserum reacted in Western immunoblots with a 97
-kDa outer membrane protein of EHEC strains of serogroups O5, O26, O11
1, and O157 and enteropathogenic E. coli strains of serogroups O55 and
O127. Surface labelling of CL8 with I-125 showed that intimin was sur
face exposed. An eaeA insertional inactivation mutant of CL8 was produ
ced and was designated CL8-KO1. Total adherence of CL8-KO1 to HEp-2 ce
lls was not significantly different from that of CL8, but CL8-KO1 gave
a negative result in the fluorescent actin staining test. The eaeA ge
ne expressed alone in E. coli HB101 also gave a negative fluorescent a
ctin staining test result. The eae-4 gene of CL8 was able to complemen
t the eaeA deletion mutation in CVD206. We conclude that the product o
f the EHEC eaeA gene is a 97-kDa surface-exposed protein and propose t
hat it be designated intimin(O157). Sherman et al. described a 94-kDa
outer membrane protein which played an important role in adherence of
E. coli 0157:H7 (Infect. Immun. 59:890-899, 1991). Western immunoblott
ing and indirect fluorescent antibody studies showed that the protein
described by Sherman et al. is not intimin.