Cyclic di-AMP inhibits Listeria monocytogenes thymineless death during infection

Antifolate antibiotics are used to treat meningitis and refractory listeriosis caused by drug-resistant Listeria monocytogenes (Lm). Their bactericidal activity is attributed to the deactivation of thymidylate synthase (ThyA), which subsequently induces bacterial cell death when thymidine is depleted, a process known as thymineless death (TLD). Despite decades of study, the mechanisms of TLD, especially during infection, remain unclear. Cyclic di-AMP (c-di-AMP), a common bacterial second messenger that regulates bacterial stress responses, is elevated in response to antifolate antibiotics. In this study, we found that elevated c-di-AMP is required to inhibit TLD in Lm. Conversely, reducing c-di-AMP levels in the ΔthyA mutant led to increased bacterial cell death under thymidine starvation and significant reduction in intracellular growth. Furthermore, we found that ΔthyA exhibited a more pronounced growth defect during oral infection compared to intravenous infection, due to limited thymidine availability in the gallbladder, which acts as a bottleneck for ΔthyA in establishing infection. Notably, decreasing c-di-AMP levels abolished the infection capacity of ΔthyA in both infection models. Finally, we identified that the c-di-AMP-binding protein PstA contributes to bacterial cell death when c-di-AMP concentrations are low. Deletion of pstA in the ΔthyA background rescued the elevated cell death caused by c-di-AMP depletion both in vitro and during mouse infections. Our study identifies a previously unrecognized mechanism of TLD regulation mediated by c-di-AMP. This expands fundamental knowledge of TLD in the context of infection and provides insights into potential combined therapeutic strategies for listeriosis targeting both antifolate and c-di-AMP metabolic pathways.IMPORTANCEConsuming food contaminated with Listeria monocytogenes (Lm) can cause severe listeriosis, a leading foodborne illness with a 20% fatality rate. Most cases require hospitalization, and 25% of pregnancy-associated cases result in fetal or neonatal death. Antibiotics, especially β-lactams, are the main treatment, but alternatives like antifolates are used when resistance or allergies occur. Still, over 30% of patients experience treatment failure, the causes of which remain poorly understood due to limited knowledge of antibiotic action within Lm's intracellular niches and how the pathogen adapts during infection. This gap hinders the development of effective therapies. Our study bridges this gap by using a thymidine auxotroph mutant of Lm (ΔthyA) to investigate thymineless death both in vitro and in vivo. Notably, antifolate-resistant Lm strains, many of which are thymidine auxotrophs, are often found in food, posing a public health risk. Our study on how ΔthyA strains survive will provide insights into novel therapeutic targets.