Genetic and molecular basis for the specificity of the RTX toxins from Pasteurella haemolytica and Actinobacillus pleuropneumoniae

Abstract

The leukotoxin (LktA) from Pasteurella haemolytica and the hemolysin (AppA) from Actinobacillus pleuropneumoniae are members of a highly conserved family of cytolytic proteins produced by gram negative bacteria. Their toxins are characterized by the presence of gly/asp rich nonapeptide repeats in the carboxyl-terminal third of the toxin proteins. Despite their extensive homology, these two toxins exhibit striking different target cell specificities. The leukotoxin is specific for ruminant leukocytes while the hemolysin lyses erythrocytes and a variety of nucleated cells including ruminant leukocytes. Both proteins require activation (acylation) facilitated by the product of an accessory RTX C gene for optimal biological activity. I have constructed several hybrid genes encoding hybrid toxins by recombining domains of IktA and appA and have examined the target cell specificities of the resulting hybrid proteins. Activation of the parental toxins by cognate and noncognate C proteins revealed that the C proteins are not functionally equivalent. My results also indicate that the leukotoxic potential of AppA, like that of LktA, maps to the central region (300 residues) of the toxin, proximal to and may include the repeat region of the protein. The leukotoxic potential of AppA was found to be physically separable from the region specifying erythrocyte lysis. The ability to lyse erythrocytes map to the N-terminal half of the toxin protein. The specificity of one hybrid was found to be dependent upon the RTX C protein used for activation. With AppC acylation, this hybrid lysed both erythrocytes and leukocytes while LktC acylation produced a toxin which could only attack leukocytes. This is the first demonstration that the specificity of an RTX toxin can be determined by the process of C-mediated acylation. The location of the porcine leukocyte specificity determinant of AppA was determined by generating leukotoxin/hemolysin hybrids by forced recombination between the central regions of the cloned toxin genes. The biological activity of these hybrids indicates that the ability of the hemolysin to lyse porcine leukocytes maps to a small, 50 codon region of the hemolysin gene.