title: Intimin types alpha, beta, and gamma bind to nucleolin with equivalent affinity but lower avidity than to the translocated intimin receptor.
abstract: The outer membrane adhesins of enteropathogenic Escherichia coli, Citrobacter rodentium, and enterohemorrhagic E. coli (EHEC) O157:H7 that mediate attach and efface intestinal lesions are classified as intimin alpha, beta, and gamma, respectively. Each of these intimin types binds to its cognate, bacterially encoded receptor (called Tir for translocated intimin receptor) to promote tight adherence of the organism to the host-cell plasma membrane. We previously reported that gamma intimin of EHEC O157:H7 also bound to a eucaryotic receptor that we determined was nucleolin. The objective of this study was to investigate in vitro and in vivo the interactions of intimins alpha, beta, and gamma with nucleolin in the presence of Tir from EHEC O157:H7. Protein binding experiments demonstrated that intimin of types alpha, beta, and gamma bound nucleolin with similar affinity. Moreover, all three intimin types co-localized with regions of nucleolin expressed on the surface of HEp-2 cells. When intimin alpha, beta, or gamma bound to Tir in vitro, the intimin interaction with nucleolin was blocked. Both Tir and nucleolin accumulated beneath intimin-presenting bacteria that had attached to the surface of HEp-2 cells. Taken together, these findings suggest that nucleolin is involved in bacterial adherence promoted by all intimin types and that Tir and nucleolin compete for intimin during adherence.
authors.affiliation: Department of Microbiology and Immunology, Uniformed Services University of the Health Sciences, Bethesda, Maryland 20814, USA.
authors.forename: James F
authors.initials: JF
authors.lastname: Sinclair
authors.forename: Alison D
authors.initials: AD
authors.lastname: O'Brien
mesh_terms: D000199:Actins / Q000032:analysis / Q000378:metabolism; D018829:Adhesins, Bacterial / Q000235:genetics / Q000378:metabolism*; D001422:Bacterial Adhesion / Q000502:physiology; D001667:Binding, Competitive; D019921:Biotinylation; D002460:Cell Line; D004847:Epithelial Cells; D019453:Escherichia coli O157 / Q000737:chemistry*; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D005455:Fluorescent Antibody Technique; D017353:Gene Deletion; D006801:Humans; D007830:Larynx; D009154:Mutation; D010750:Phosphoproteins / Q000032:analysis / Q000378:metabolism*; D011108:Polymers / Q000378:metabolism; D011485:Protein Binding; D016601:RNA-Binding Proteins / Q000032:analysis / Q000378:metabolism*; D011956:Receptors, Cell Surface / Q000378:metabolism*; D000097796:Nucleolin
chemical_list: D000199:Actins; D018829:Adhesins, Bacterial; D029968:Escherichia coli Proteins; D010750:Phosphoproteins; D011108:Polymers; D016601:RNA-Binding Proteins; D011956:Receptors, Cell Surface; D000097796:Nucleolin; C109287:Tir protein, E coli; C073819:eaeA protein, E coli