Construction and characterisation of aromatic amino acid dependent mutants of Listeria monocytogenes.

The severe forms of listeriosis, have a very high mortality rate. In farm animals, especially sheep, these losses can be of considerable economic importance. The increase in cases of listeriosis in both man and animals over the last decade has stimulated research to develop an effective vaccine to protect against Listeria monocytogenes. However, attempts at protection using killed or chemically attenuated live vaccines have been disappointing. An alternative to these procedures is the development of strains with a defined mechanism of attenuation. Attempts were made to construct aromatic amino acid dependent mutant strains of L. monocytogenes and to investigate their efficacy as a vaccine. Two strategies were used for the transposon mutagenesis of L. monocytogenes. Suicide vectors carrying transposon Tn917 and unable to replicate in Listeria were constructed. To facilitate the transformation of these vectors into Listeria species an efficient electrotransformation system was developed. However, this procedure was unsuccessful in generating Tn917 insertion mutants. Insertional mutagenesis of L. monocytogenes EGD with Tn917 was achieved using a temperature sensitive plasmid. An aromatic amino acid requiring mutant deficient in chorismate mutase activity was isolated. The multiplication of this mutant was found to be unimpaired in both mouse tissues and cultured bone marrow derived macrophages. Organisms isolated from infected tissues were found to be prototrophic while still harbouring a Tn917 insertion. It was concluded that the original mutant carried a point mutation in the gene encoding chorismate mutase and that this had reverted on passage through the mouse. A transposon induced aromatic amino acid dependent mutant of L. monocytogenes found to be deficient in prephenate dehydratase activity was obtained for investigation. The virulence and multiplication of this mutant were reduced in the mouse. Vaccination with this mutant was found to stimulate a protective immune response in mice. The results indicate that aromatic amino acid dependent mutants of L monocytogenes protect against listeric infection and offer a new approach to the development of anti-listerial vaccines.