PATHOGENICITY AND IMMUNOGENICITY OF A LISTERIA-MONOCYTOGENES STRAIN THAT REQUIRES D-ALANINE FOR GROWTH

Listeria monocytogenes is an intracellular bacterial pathogen that eli cits a strong cellular immune response following infection and therefo re has potential use as a vaccine vector. However, while infections by L. monocytogenes are fairly rare and can readily be controlled by a n umber of antibiotics, the organism can nevertheless cause meningitis a nd death, particularly in immunocompromised or pregnant patients. We t herefore have endeavored to isolate a highly attenuated strain of this organism for use as a vaccine vector. D-Alanine is required for the s ynthesis of the mucopeptide component of the cell walls of virtually a ll bacteria and is found almost exclusively in the microbial world. We have found in L, monocytogenes two genes that control the synthesis o f this compound, an alanine racemase gene (dal) and a D-amino acid ami notransferase gene (dat). By inactivating both genes, we produced an o rganism that could be grown in the laboratory when supplemented with D -alanine but was unable to grow outside the laboratory, particularly i n the cytoplasm of eukaryotic host cells, the natural habitat of this organism during infection. In mice, the double-mutant strain was compl etely attenuated. Nevertheless, it showed the ability, particularly un der conditions of transient suppression of the mutant phenotype, to in duce cytotoxic T-lymphocyte responses and to generate protective immun ity against lethal challenge by wild-type L. monocytogenes equivalent to that induced by the wild-type organism.