MOLECULAR ANALYSIS OF THE ACTINOBACILLUS-PLEUROPNEUMONIAE RTX TOXIN-III GENE-CLUSTER

Actinobacillus pleuropneumonia strains that secrete three different ex otoxins (ApxI, ApxII, and ApxIII) have been implicated in the etiology of porcine pleuropneumonia. To understand the role of these toxins in the pathogenesis of this disease, we have previously reported the clo ning of the hemolysin gene (apxII) (Chang et al., 1989a), which encode s a 110-kD polypeptide with hemolytic and cytotoxic activity. To clone the third toxin gene (apxIII), a new genomic library using A. pleurop neumoniae serotype 2 chromosomal DNA was constructed. A series of five overlapping recombinant phage clones carrying the gene (aPXIII) for t his 120-kD antigen were identified using a DNA probe containing sequen ces from the Pasteurella haemolytica lktBD genes. Sequence analysis of a region of the cloned DNA reveals four open reading frames encoding proteins with predicted masses of 20.4, 112.5, 80.3, and 54.7 kD. Thes e genes, designated apxIIC, apxIIIA, apxIIIB, and apxIIID, respectivel y, are similar in sequence to the RTX (repeat of toxin) toxin family. The toxin produced by the cloned gene kills BL-3 cells and is not hemo lytic in vitro.