Cell surface-localized nucleolin is a eukaryotic receptor for the adhesin intimin-gamma of enterohemorrhagic Escherichia coli O157:H7.
Intimin-gamma is an outer membrane protein of enterohemorrhagic Escherichia coli (EHEC) O157:H7 that is required for the organism to adhere tightly to HEp-2 cells and to colonize experimental animals. Another EHEC O157:H7 protein, the Transferred intimin receptor (Tir), is considered the primary receptor for intimin-gamma. Nevertheless, Tir-independent binding of intimin-gamma to HEp-2 cells has been reported. This observation suggests the existence of a eukaryotic receptor(s) for intimin-gamma. In this study, we sought to identify that receptor(s). First, we determined by equilibrium binding titration that the association of purified intimin-gamma with HEp-2 cells was specific and consistent with a single host cell receptor. Second, we isolated a protein from lysates of HEp-2 cells that bound intimin-gamma and subsequently identified this molecule as nucleolin, a protein involved in cell growth regulation that can be cell surface-expressed. Third, we established that purified intimin-gamma and nucleolin were co-localized on the surface of HEp-2 cells and that the site of EHEC O157:H7 attachment was associated with regions of nucleolin expression. Finally, we demonstrated that mouse anti-nucleolin sera significantly decreased the adherence of EHEC O157:H7 to HEp-2 cells. From this, we conclude that nucleolin is the HEp-2 cell receptor for intimin-gamma expressed by EHEC O157:H7.
Sinclair
James F
JF
Department of Microbiology and Immunology, Uniformed Services University of the Health Sciences, Bethesda, Maryland 20814-4799, USA.
O'Brien
Alison D
AD
D018829:Adhesins, Bacterial / Q000378:metabolism*; D000818:Animals; D001426:Bacterial Proteins / Q000378:metabolism; D002352:Carrier Proteins / Q000378:metabolism*; D002448:Cell Adhesion; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D004847:Epithelial Cells / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins; D019084:Fluorescent Antibody Technique, Indirect; D006367:HeLa Cells; D006801:Humans; D015151:Immunoblotting; D051379:Mice; D008856:Microscopy, Fluorescence; D008954:Models, Biological; D010750:Phosphoproteins / Q000096:biosynthesis* / Q000378:metabolism; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D016601:RNA-Binding Proteins / Q000096:biosynthesis* / Q000378:metabolism; D000097796:Nucleolin
D018829:Adhesins, Bacterial; D001426:Bacterial Proteins; D002352:Carrier Proteins; D029968:Escherichia coli Proteins; D010750:Phosphoproteins; D016601:RNA-Binding Proteins; D000097796:Nucleolin; C053476:invasin, Yersinia; C073819:eaeA protein, E coli