EXPRESSION AND CHARACTERIZATION OF THE EAEA GENE-PRODUCT OF ESCHERICHIA-COLI SEROTYPE O157-H7

Citation
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
Citations number
36
Categorie Soggetti
Immunology,"Infectious Diseases
Journal title
ISSN journal
00199567
Volume
61
Issue
10
Year of publication
1993
Pages
4085 - 4092
Database
ISI
SICI code
0019-9567(1993)61:10<4085:EACOTE>2.0.ZU;2-5
Abstract
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.