Cloning and characterization of the Actinobacillus pleuropneumoniae-RTX-toxin III (ApxIII) gene.

To study the role of Actinobacillus pleuropneumoniae-RTX-toxin III (ApxIII) in the pathogenesis of porcine pleuropneumonia, we cloned and characterized the gene encoding this toxin. For that purpose, we screened an expression library of genomic DNA of serotype 8 with an ApxIII-specific monoclonal antibody and isolated a 425-bp fragment of an immunoreactive clone. Using this fragment as a probe, we identified and cloned an overlapping chromosomal NsiI restriction fragment of 5.0 kbp. Escherichia coli cells that contained this fragment produced a protein similar to ApxIII. Like ApxIII, the protein had a molecular mass of approximately 120 kDa, was recognized by an ApxIII-specific antibody, killed porcine lung macrophages, and was not lytic for sheep erythrocytes. We concluded from these data that the 5.0-kbp NsiI fragment contained the ApxIII-coding gene. Nucleotide sequence analysis of the 5.0-kbp NsiI fragment revealed the presence of two genes, apxIIIC and apxIIIA. These genes coded for proteins ApxIIIC and ApxIIIA, respectively, which were 53 and 50% identical to the prototypic RTX proteins HlyC and HlyA of E. coli. We assumed that the apxIIIA gene coded for the structural RTX toxin and that the apxIIIC gene coded for its activator. In addition, we found that ApxIII could be secreted from E. coli by the heterologous RTX transporter proteins HlyB and HlyD. The deduced amino acid sequence of ApxIIIA was 50% identical to that of ApxIA and 41% identical to that of ApxIIA. We concluded that, beside ApxI and ApxII, ApxIII is the third RTX toxin produced by A. pleuropneumoniae.