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 (Inv(YP280)) were fused to the E. call maltose-binding protein (MBP), expressed, and purified. The smallest MBP-Int(EPEC) 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 (Int(EPEC280CS)) destroyed the cell-binding activity of Int(EPEC280). Covaspheres coated with MBP-Int(EPEC280) were associated with HEp-2 cell microvilli but failed to induce actin accumulation underneath bound particles or cell spreading on coated plastic surfaces, MBP-Int(EPEC280), but not MBP, MBP-Int(EPEC280CS), or MBP-Inv(YP280), inhibited EPEC entry into HEp-2 cells.