Mutational analysis of the Actinobacillus pleuropneumoniae hemolysin gene

Actinobacillus pleuropneumoniae is one of a number of gram-negative bacteria that secrete toxins of the RTX (repeat toxin) family. The ApxIIA protein secreted from this organism is both hemolytic and cytotoxic. However, in this study, I have found that when the cloned apxII operon is expressed in Escherichia coli, two forms of the ApxIIA protein can be recovered. Toxin which remains intracellular has hemolytic and cytotoxic activities, while toxin that is secreted is cytotoxic with little or no hemolytic activity. Furthermore, by mutational analysis of the cloned toxin gene, I have successfully found non-hemolytic mutants that are leukotoxic. This is the first time that point mutants in the hemolysin gene could be found that abolished the activity against erythrocytes without disrupting activity against leukocytes. Together, these results indicate that the cytotoxicity of ApxIIA is independent of its hemolytic activity.