Listeria monocytogenes adenosine auxotrophs are impaired for intracellular and extracellular growth but retain potent immunogenicity.

Listeria monocytogenes 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 actA, has been used to attenuate L. monocytogenes-based vaccine strains while preserving immunogenicity. Here we explored the potential inclusion of a purA gene deletion to enhance the development of L. monocytogenes-based immunotherapy. The purA gene encodes adenylosuccinate synthetase, which catalyzes the conversion of inosine monophosphate to adenosine monophosphate (AMP), a critical step in the de novo biosynthesis of purines. Since nucleotide biosynthesis is critical for the survival and pathogenesis of many bacterial pathogens, we examined the requirements of L. monocytogenes AMP synthesis in tissue culture and animal infection models. The purA 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, purA mutants were highly attenuated, similar to actA mutants, but displayed distinct growth kinetics during the course of infection. Remarkably, the purA mutants exhibited different localization patterns across splenic immune cells and elicited a more potent CD8⁺ T-cell response compared to actA mutants. These results underscore the essentiality of AMP biosynthesis for L. monocytogenes pathogenesis and provide new avenues for developing safe L. monocytogenes-based vaccines and therapeutics.