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Listeria monocytogenes is an intracellular bacterial pathogen that elicits a strong cellular immune response following infection and therefore has potential use as a vaccine vector. However, while infections byL. monocytogenes are fairly rare and can readily be controlled by a number of antibiotics, the organism can nevertheless cause meningitis and death, particularly in immunocompromised or pregnant patients. We therefore have endeavored to isolate a highly attenuated strain of this organism for use as a vaccine vector.d -Alanine is required for the synthesis of the mucopeptide component of the cell walls of virtually all bacteria and is found almost exclusively in the microbial world. We have found inL. monocytogenes two genes that control the synthesis of this compound, an alanine racemase gene (dal ) and ad -amino acid aminotransferase gene (dat ). By inactivating both genes, we produced an organism that could be grown in the laboratory when supplemented withd -alanine but was unable to grow outside the laboratory, particularly in the cytoplasm of eukaryotic host cells, the natural habitat of this organism during infection. In mice, the double-mutant strain was completely attenuated. Nevertheless, it showed the ability, particularly under conditions of transient suppression of the mutant phenotype, to induce cytotoxic T-lymphocyte responses and to generate protective immunity against lethal challenge by wild-typeL. monocytogenes equivalent to that induced by the wild-type organism.

Pathogenicity and Immunogenicity of aListeria monocytogenesStrain That Requiresd-Alanine for Growth