Molecular characterization of a carboxy-terminal eukaryotic-cell-binding domain of intimin from enteropathogenic Escherichia coli.
A eukaryotic cell-binding domain from the intimin (Int) polypeptide of enteropathogenic Escherichia coli O127 (EPEC) was investigated. Derivatives of the carboxy-terminal 280-amino-acid domains of Int (Int-EPEC280) and the Int homolog invasin (Inv) from Yersinia pseudotuberculosis (InvYP280) were fused to the E. coli maltose-binding protein (MBP), expressed, and purified. The smallest MBP-IntEPEC fusion protein that efficiently mediated binding to HEp-2 cells, monitored by using purified fusion proteins in fluorescence activated cell sorter analysis or by using fluorescent Covaspheres coated with purified fusions, contained the carboxy-terminal 150 amino acids of Int. Replacement of Cys-937 with Ser (IntEPEC280CS) destroyed the cell-binding activity of IntEPEC280. Covaspheres coated with MBP-IntEPEC280 were associated with HEp-2 cell microvilli but failed to induce actin accumulation underneath bound particles or cell spreading on coated plastic surfaces. MBP-IntEPEC280, but not MBP, MBP-IntEPEC280CS, or MBP-InvYP280, inhibited EPEC entry into HEp-2 cells.
Frankel
G
G
Department of Biochemistry, Imperial College of Science, Technology and Medicine, London, United Kingdom.
Candy
D C
DC
Fabiani
E
E
Adu-Bobie
J
J
Gil
S
S
Novakova
M
M
Phillips
A D
AD
Dougan
G
G
D018829:Adhesins, Bacterial; D001422:Bacterial Adhesion; D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry*; D001426:Bacterial Proteins / Q000737:chemistry; D001483:Base Sequence; D002352:Carrier Proteins; D002478:Cells, Cultured; D017931:DNA Primers / Q000737:chemistry; D004926:Escherichia coli / Q000737:chemistry* / Q000472:pathogenicity; D004927:Escherichia coli Infections / Q000382:microbiology; D029968:Escherichia coli Proteins; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins; D017384:Sequence Deletion; D013329:Structure-Activity Relationship
D018829:Adhesins, Bacterial; D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; D002352:Carrier Proteins; D017931:DNA Primers; D029968:Escherichia coli Proteins; D011993:Recombinant Fusion Proteins; C053476:invasin, Yersinia; C073819:eaeA protein, E coli