The importance of the carboxyl portion of the Apx toxins of actinobacillus pleuropneumoniae in eliciting toxin-neutralizing antibodies

Actinobacillus pleuropneumoniae is the organism responsible for porcine pleuropneumonia. The primary virulence factors of A. pleuropneumoniae are three secreted Apx toxins. Apx I and II are cytolytic and hemolytic, while Apx III is only cytolytic. The Apx toxins are pore-forming toxins that promote bacterial multiplication by harming phagocytes infiltrating the respiratory tract of infected swine. Apx-mediated phagocyte damage further intensifies the inflammatory tissue damage associated with porcine pleuropneumonia. The Apx toxins are members of a group of toxins known as RTX toxins. Another RTX toxin previously worked with in this laboratory is the leukotoxin of Pasteurella haemoltyica, which is known to be a primary virulence factor involved in pneumonic pasteurellosis. Several functional domains of the Apx toxins and other RTX toxins have been identified. The carboxyl region containing the repeat domain was of particular interest. This study attempted to answer some preliminary questions about the Apx toxins in relation to work previously done with P. haemolytica leukotoxin in this laboratory. This information will allow future work with the Apx toxins in this laboratory to be more focused on particular aspects of the proteins. The first objective of this study was to examine cross-recognition between the Apx toxins and leukotoxin. This was done by using murine mAbs; ltx-2, ltx-4, and ltx-35, known to neutralize leukotoxin, as well as different samples of rabbit antisera generated against GST-fused peptides of the carboxyl one-third of the IktA, Apx lA, and Apx IIA genes, respectively. Monoclonal antibodies did not cross-react with the Apx toxins. However, rabbit antisera samples did cross-react between the Apx toxins and leukotoxin. The second objective was to evaluate the ability of fusion proteins, containing peptides of the carboxyl portion of Apx I and Apx II, respectively, to elicit anti-Apx toxin antibodies as well as Apx toxin-neutralizing antibodies. Results from these experiments showed that Apx toxin-reactive antibodies can be stimulated by the peptides used, as well as toxin-neutralizing antibodies. The results support earlier reports that the hemolytic and cytotoxic activities of the Apx toxins are independent of each other, as well. This study also confirms previous work from this laboratory showing the importance of the carboxyl portion of the RTX toxins in eliciting toxin-neutralizing antibodies, as well as the finding that post-translational modification of RTX toxin proteins is not required for recognition nor for the capability of the proteins to generate toxin-neutralizing antibodies.