title: Prelytic and lytic conformations of erythrocyte-associated Escherichia coli hemolysin.
abstract: Flow cytometry was developed as a method to assess the conformation of erythrocyte-bound Escherichia coli hemolysin polypeptide (HlyA). Topology of membrane-associated hemolysin (HlyA(E)) was investigated by testing surface accessibility of HlyA regions in lytic and nonlytic bound states, using a panel of 12 anti-HlyA monoclonal antibodies (MAbs). Hemolysin associates nonlytically with erythrocytes at 0 to 2 degrees C. To test the hypothesis that the nonlytic HlyA(E) conformation at 0 to 2 degrees C differs from the lytic conformation at 23 degrees C, MAb epitope reactivity profiles at the two temperatures were compared by flow cytometry. Four MAbs have distinctly increased reactivity at 0 to 2 degrees C compared to 23 degrees C. HlyA requires HlyC-dependent acylation at lysine residues 563 and 689 for lytic function. Toxin with cysteine substitution mutations at each lysine (HlyA(K563C) and HlyA(K689C)) as well as the nonacylated form of hemolysin made in a HlyC-deficient strain were examined by flow cytometry at 0 to 2 and 23 degrees C. The three mutants bind erythrocytes at wild-type toxin levels, but there are conformational changes reflected by altered MAb epitope accessibility for six of the MAbs. To test further the surface accessibility of regions in the vicinity of MAb-reactive epitopes, HlyA(E) was proteolytically treated prior to testing for MAb reactivity. Differences in protease susceptibility at 0 to 2 degrees and 23 degrees C for the reactivities of three of the MAbs further support the model of two distinct conformations of cell-associated toxin.
authors.affiliation: Department of Medical Microbiology and Immunology, University of Wisconsin-Madison, 53706, USA.
authors.forename: M
authors.initials: M
authors.lastname: Moayeri
authors.forename: R A
authors.initials: RA
authors.lastname: Welch
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism; D000939:Epitopes; D004912:Erythrocytes / Q000378:metabolism*; D004926:Escherichia coli / Q000472:pathogenicity*; D029968:Escherichia coli Proteins; D005434:Flow Cytometry; D006460:Hemolysin Proteins / Q000737:chemistry* / Q000378:metabolism; D051379:Mice; D011487:Protein Conformation; D011817:Rabbits; D013329:Structure-Activity Relationship
chemical_list: D000911:Antibodies, Monoclonal; D001426:Bacterial Proteins; D000939:Epitopes; D029968:Escherichia coli Proteins; D006460:Hemolysin Proteins; C036507:Hlya protein, E coli