Characterization of epitopes involved in the neutralization of Pasteurella haemolytica serotype A1 leukotoxin.
Defined segments of the leukotoxin A gene (lktA) from an A1 serotype of Pasteurella haemolytica were cloned into a plasmid vector and expressed as LacZ alpha fusion proteins. These fusion proteins were electrophoresed in SDS-PAGE gels and their immunoblotting reactivities with several monoclonal antibodies characterized. The epitope recognized by a strongly neutralizing monoclonal antibody was localized to a 32 amino acid region near the C terminus of the leukotoxin A (LktA) molecule. The epitope recognized by a non-neutralizing antibody was localized to a 33 amino acid region immediately adjacent. Smaller recombinant peptides containing these epitopes were not antigenic, but a polypeptide encompassing 229 amino acids at the C terminus evoked neutralizing antibodies when used to immunize specific-pathogen-free lambs. The distributions of linear epitopes recognized by this antiserum and by antisera raised to full-length recombinant LktA and to native LktA produced by P. haemolytica serotype A1 were determined by their reactivities with a set of overlapping 10 amino acid synthetic peptides. This revealed a complex distribution of linear epitopes at the C-terminal end of LktA. Toxin-neutralizing antibodies in convalescent sheep serum were shown to be directed against conformational epitopes by selective absorption of antibodies directed against linear epitopes.
Lainson
F A
FA
Moredun Research Institute, Edinburgh, UK.
Murray
J
J
Davies
R C
RC
Donachie
W
W
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000276:immunology; D001426:Bacterial Proteins; D003001:Cloning, Molecular; D003602:Cytotoxicity, Immunologic; D018604:Epitope Mapping; D000939:Epitopes / Q000235:genetics* / Q000276:immunology*; D005098:Exotoxins / Q000235:genetics* / Q000276:immunology*; D015964:Gene Expression Regulation, Bacterial; D006460:Hemolysin Proteins / Q000235:genetics* / Q000276:immunology*; D015151:Immunoblotting; D016978:Mannheimia haemolytica / Q000235:genetics* / Q000276:immunology*; D008969:Molecular Sequence Data; D009500:Neutralization Tests; D010455:Peptides / Q000138:chemical synthesis / Q000276:immunology; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000276:immunology
D000911:Antibodies, Monoclonal; D001426:Bacterial Proteins; D000939:Epitopes; D005098:Exotoxins; D006460:Hemolysin Proteins; D010455:Peptides; D011994:Recombinant Proteins; C102677:lktA protein, bacteria