title: Analysis of toxinogenic functions associated with the RTX repeat region and monoclonal antibody D12 epitope of Escherichia coli hemolysin.
abstract: Amino acids (aa) 550 through 850 of the Escherichia coli hemolysin (HlyA) contain sequences important for several steps in cytolysis. These include the Ca(2+)-binding glycine-rich tandem repeats recognized by the monoclonal antibody A10, the putative HlyC-dependent acylation site that corresponds to the monoclonal antibody D12 epitope, and the erythrocyte specificity domain which confers erythrolytic activity to the Pasteurella haemolytica leukotoxin. To further investigate the toxinogenic functions associated with this region of HlyA, we constructed mutants in the hlyA sequences coding for the repeat region and the D12 epitope. Mutants were analyzed for anti-HlyA antibody reactivity, cytolytic activities, target cell binding, Ca2+ requirements, and virulence. The D12 epitope was mapped to aa 673 through 726, with portions of the epitope both amino terminal and carboxy terminal to aa 700. This region was necessary, but not sufficient, for toxin binding to erythrocytes. A substitution at aa 684 resulted in loss of the D12 epitope, while cytolytic activity was retained. The nature of the D12 epitope and its associated functions are discussed. The A10 epitope mapped to residues 745 through 829, corresponding to repeats 4 through 11. Insertions within the glycine-rich repeats resulted in mutant forms of HlyA which retained A10 reactivity but required increased Ca2+ for lytic activity. These in vitro effects on cytolysis corresponded to a significant decrease in HlyA-mediated virulence in mice. HlyA from one insertion mutant was able to associate with leukocyte membranes under conditions that were Ca2+ deficient for cytolysis. The role of the glycine-rich repeats and Ca2+ in HlyA activity are discussed.
authors.affiliation: Department of Medical Microbiology and Immunology, University of Wisconsin Medical School, Madison 53706.
authors.forename: G E
authors.initials: GE
authors.lastname: Rowe
authors.forename: S
authors.initials: S
authors.lastname: Pellett
authors.forename: R A
authors.initials: RA
authors.lastname: Welch
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal; D000942:Antigens, Bacterial / Q000235:genetics; D001426:Bacterial Proteins / Q000235:genetics* / Q000276:immunology* / Q000502:physiology; D001483:Base Sequence; D002118:Calcium / Q000378:metabolism; D002417:Cattle; D003602:Cytotoxicity, Immunologic; D004269:DNA, Bacterial / Q000235:genetics; D000939:Epitopes / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics* / Q000276:immunology* / Q000502:physiology; D029968:Escherichia coli Proteins; D005260:Female; D005798:Genes, Bacterial; D006460:Hemolysin Proteins / Q000235:genetics* / Q000276:immunology* / Q000502:physiology; D006461:Hemolysis; D066298:In Vitro Techniques; D007962:Leukocytes / Q000276:immunology; D051379:Mice; D008969:Molecular Sequence Data; D009154:Mutation; D012091:Repetitive Sequences, Nucleic Acid; D014774:Virulence / Q000235:genetics
chemical_list: D000911:Antibodies, Monoclonal; D000942:Antigens, Bacterial; D001426:Bacterial Proteins; D004269:DNA, Bacterial; D000939:Epitopes; D029968:Escherichia coli Proteins; D006460:Hemolysin Proteins; C036507:Hlya protein, E coli; D002118:Calcium