Pathogenicity and Immunogenicity of a Listeria monocytogenes Strain That Requires d-Alanine for Growth

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 by L. 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 in L. monocytogenes two genes that control the synthesis of this compound, an alanine racemase gene (dal) and a d-amino acid aminotransferase gene (dat). By inactivating both genes, we produced an organism that could be grown in the laboratory when supplemented with d-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-type L. monocytogenes equivalent to that induced by the wild-type organism.Listeria monocytogenes is a gram-positive facultative intracellular microorganism which has been used for decades as a model pathogen for the study of cell-mediated immunity (24). Immunization of mice with a sublethal L. monocytogenes infection results in the generation of immunity which is largely major histocompatibility complex (MHC) class I mediated. Such infections generate CD8⁺ T cells, which can adoptively transfer immunity and specifically recognize and kill Listeria-infected target cells (3, 6, 17, 19).Recently, the cell biology of L. monocytogenes intracellular growth has been defined (47). Subsequent to internalization, the bacteria escape from a phagocytic vacuole and replicate in the host cell cytosol. Hence, secreted proteins of L. monocytogenes are delivered directly into the cytosol and into the MHC class I pathway of antigen processing and presentation (1, 5). Mutants of L. monocytogenes which are unable to enter the cytosol are absolutely avirulent and fail to immunize mice, and cells infected by such mutants are not recognized by L. monocytogenes-immune CD8⁺ T cells (6, 28).The natural properties of L. monocytogenes make it particularly attractive as a potential live vaccine vector for the induction of cell-mediated immunity to foreign antigens. Indeed, recombinant L. monocytogenes expressing such antigens successfully has been used to protect mice against lymphocytic choriomeningitis virus (15, 40) and influenza virus (22) infections and against lethal tumor cell challenge (32, 33). We have suggested the use of L. monocytogenes for the induction of cytolytic T cells directed against human immunodeficiency virus (HIV) antigens and have shown that strong cell-mediated immune responses against HIV-1 Gag protein can be induced in mice infected with recombinant L. monocytogenes carrying a chromosomal copy of the HIV-1 gag gene (13).Because of the potential broad use of this organism as a vaccine vector in infectious disease and cancer, the safety of L. monocytogenes becomes an important issue. While infections by L. 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. An ideal vaccine strain of L. monocytogenes would be absolutely avirulent but fully immunogenic. We therefore sought to isolate a mutant which could enter the cytosol but have limited growth potential both in vivo and in the environment.d-Alanine is required for the synthesis of the mucopeptide component of the cell walls of virtually all bacteria, including L. monocytogenes (21, 23, 43), and is also found in the lipoteichoic acids of this and some other gram-positive organisms (11, 37). However, it is present in only trace quantities and fails to accumulate in vertebrates; the likely origin of these trace quantities is the breakdown products of intestinal and food bacteria (16, 20, 25, 29). We hypothesized that a strain of L. monocytogenes that is unable to synthesize this compound could be grown in the laboratory when supplemented with d-alanine but should be unable to grow outside the laboratory, particularly in the cytoplasm of eukaryotic host cells, the natural habitat of this organism during infection. The isolation of such a mutant of L. monocytogenes required the identification and inactivation of two genes, dal and dat. dal encodes alanine racemase, which catalyzes the reaction: l-alanine↔d-alanine. dat encodes d-amino acid aminotransferase, which catalyzes the reaction d-glutamic acid + pyruvate↔α-ketoglutaric acid + d-alanine. The dal dat double-mutant strain had the anticipated phenotype and in addition showed the ability to induce cytotoxic T-lymphocyte (CTL) responses and to generate protective immunity against lethal challenge by wild-type L. monocytogenes in infected mice under restricted conditions.