Cloning and expression of the Pasteurella multocida toxin gene, toxA, in Escherichia coli

Abstract

A chromosomal DNA library of a toxigenic type D strain of Pasteurella multocida subsp. multocida was established in Escherichia coli. From this library two clones, SPE308 and SPE312, were identified by using a monoclonal antibody against the osteoclast-stimulating P. multocida toxin (PMT). Extracts of these clones showed cytopathic activity identical to that of extracts of toxigenic P. multocida. The recombinant plasmids, pSPE308 and pSPE312, directed the synthesis of a protein with an apparent molecular weight of 143,000 which could be specifically detected by anti-PMT antibody. The recombinant toxin, which was located in the cytoplasm of E. coli, was purified by affinity chromatography with immobilized monoclonal antibody and was shown to react in a manner identical to that of PMT in a quantitative structural test using a series of monoclonal antibodies as well as in all quantitative functional tests used, i.e., tests for dermonecrotic activity and mouse lethality and the embryonic bovine lung cell test for cytopathic activity. The gene encoding this toxic activity was named toxA and was found to be present in the chromosome of toxigenic strains only of P. multocida. A probe spanning the toxA gene therefore has potential in the diagnosis and surveillance of progressive atrophic rhinitis in pigs.