Localization of neutralizing epitopes in the leukotoxin of Pastuerella haemolytica

Pasteurella haemolytica A1 is the organism most often isolated from calves with pneumonic pasteurellosis. The primary virulence factor of P. haemolytica is a secreted cytolytic leukotoxin. The leukotoxin (LKT) is a pore forming toxin whose action is restricted to the membranes of ruminant leukocytes. As a virulence factor LKT promotes bacterial multiplication by damaging phagocytes infiltrating the airways of infected calves. LKT-mediated phagocyte damage further enhances the inflammatory tissue damage associated with pneumonic pasteurellosis. Several functional domains of the leukotoxin have been identified and hybridomas secreting toxin-neutralizing mAbs have been isolated. This study attempted to elucidate the epitope of a potent neutralizing murine mAb, ltx-2. This mAb prevents target cell association of LKT but appears to bind a region of the toxin that has not been ascribed importance in target cell binding. The first objective of this study was to map the ltx-2 epitope. This was done by the construction of eight fusion proteins generated from the carboxyl one-third of the IktA gene. Using these constructs, the epitope recognized by ltx-2 was shown to reside between amino acid 876-939. The second objective was to evaluate each fusion protein for its ability to elicit anti-LKT antibodies as well as LKT-neutralizing antibodies. Results from these experiments showed that LKT-reactive antibodies can be stimulated by the minimal peptide used, amino acids 876-953, but toxin-neutralizing antibodies were not generated with peptides that did not include amino acids 713-939. This study also proved that LKTC modification of LKTA is not required for ltx-2 recognition nor for the ability of the protein to generate toxin neutralizing antibodies.