<i>Listeria monocytogenes</i> adenosine auxotrophs are impaired for intracellular and extracellular growth but retain potent immunogenicity

<i>Listeria monocytogenes</i> is a facultative intracellular pathogen that has garnered attention as a potential cancer therapeutic due to its ability to induce robust cell-mediated immunity. To ensure safe clinical administration, deletion of certain genes, such as <i>actA</i>, has been used to attenuate <i>L. monocytogenes</i>-based vaccine strains while preserving immunogenicity. Here we explored the potential inclusion of a <i>purA</i> gene deletion to enhance the development of <i>L. monocytogenes</i>-based immunotherapy. The <i>purA</i> gene encodes adenylosuccinate synthetase, which catalyzes the conversion of inosine monophosphate to adenosine monophosphate (AMP), a critical step in the <i>de novo</i> biosynthesis of purines. Since nucleotide biosynthesis is critical for the survival and pathogenesis of many bacterial pathogens, we examined the requirements of <i>L. monocytogenes</i> AMP synthesis in tissue culture and animal infection models. The <i>purA</i> mutants were able to escape from phagosomes of bone marrow-derived macrophages but were highly defective for subsequent growth in the host cell cytosol. In contrast to wild-type bacteria, the mutants did not grow in human serum or sheep blood. In intravenously infected mice, <i>purA</i> mutants were highly attenuated, similar to <i>actA</i> mutants, but displayed distinct growth kinetics during the course of infection. Remarkably, the <i>purA</i> mutants exhibited different localization patterns across splenic immune cells and elicited a more potent CD8⁺ T-cell response compared to <i>actA</i> mutants. These results underscore the essentiality of AMP biosynthesis for <i>L. monocytogenes</i> pathogenesis and provide new avenues for developing safe <i>L. monocytogenes</i>-based vaccines and therapeutics.