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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\n                      <jats:italic toggle=\"yes\">Lm<\/jats:italic>\n                      . Conversely, reducing c-di-AMP levels in the \u0394\n                      <jats:italic toggle=\"yes\">thyA<\/jats:italic>\n                      mutant led to increased bacterial cell death under thymidine starvation and significant reduction in intracellular growth. Furthermore, we found that \u0394\n                      <jats:italic toggle=\"yes\">thyA<\/jats:italic>\n                      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 \u0394\n                      <jats:italic toggle=\"yes\">thyA<\/jats:italic>\n                      in establishing infection. Notably, decreasing c-di-AMP levels abolished the infection capacity of \u0394\n                      <jats:italic toggle=\"yes\">thyA<\/jats:italic>\n                      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\n                      <jats:italic toggle=\"yes\">pstA<\/jats:italic>\n                      in the \u0394\n                      <jats:italic toggle=\"yes\">thyA<\/jats:italic>\n                      background rescued the elevated cell death caused by c-di-AMP depletion both\n                      <jats:italic toggle=\"yes\">in vitro<\/jats:italic>\n                      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.\n                    <\/jats:p>\n                    <jats:sec>\n                      <jats:title>IMPORTANCE<\/jats:title>\n                      <jats:p>\n                        Consuming food contaminated with\n                        <jats:italic toggle=\"yes\">Listeria monocytogenes<\/jats:italic>\n                        (\n                        <jats:italic toggle=\"yes\">Lm<\/jats:italic>\n                        ) 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 \u03b2-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\n                        <jats:italic toggle=\"yes\">Lm<\/jats:italic>\n                        \u2019s 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\n                        <jats:italic toggle=\"yes\">Lm<\/jats:italic>\n                        (\u0394\n                        <jats:italic toggle=\"yes\">thyA<\/jats:italic>\n                        ) to investigate thymineless death both\n                        <jats:italic toggle=\"yes\">in vitro<\/jats:italic>\n                        and\n                        <jats:italic toggle=\"yes\">in vivo<\/jats:italic>\n                        . Notably, antifolate-resistant\n                        <jats:italic toggle=\"yes\">Lm<\/jats:italic>\n                        strains, many of which are thymidine auxotrophs, are often found in food, posing a public health risk. Our study on how \u0394\n                        <jats:italic toggle=\"yes\">thyA<\/jats:italic>\n                        strains survive will provide insights into novel therapeutic targets.\n                      <\/jats:p>\n                    <\/jats:sec>\n                  <\/jats:sec>","DOI":"10.1128\/mbio.03351-25","type":"journal-article","created":{"date-parts":[[2025,12,11]],"date-time":"2025-12-11T14:00:09Z","timestamp":1765461609000},"update-policy":"https:\/\/doi.org\/10.1128\/asmj-crossmark-policy-page","source":"Crossref","is-referenced-by-count":2,"title":["Cyclic di-AMP inhibits\n                    <i>Listeria monocytogenes<\/i>\n                    thymineless death during infection"],"prefix":"10.1128","volume":"17","author":[{"ORCID":"https:\/\/orcid.org\/0009-0007-6085-4672","authenticated-orcid":false,"given":"Joshua P.","family":"Leeming","sequence":"first","affiliation":[{"id":[{"id":"https:\/\/ror.org\/019kgqr73","id-type":"ROR","asserted-by":"publisher"}],"name":"Department of Biology, University of Texas at Arlington","place":["Arlington, USA"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Omar M.","family":"Elkassih","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/019kgqr73","id-type":"ROR","asserted-by":"publisher"}],"name":"Department of Biology, University of Texas at Arlington","place":["Arlington, USA"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5770-6467","authenticated-orcid":false,"given":"Damilola T.","family":"Oyebode","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/019kgqr73","id-type":"ROR","asserted-by":"publisher"}],"name":"Department of Biology, University of Texas at Arlington","place":["Arlington, USA"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4630-403X","authenticated-orcid":true,"given":"Joshua J.","family":"Woodward","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/00cvxb145","id-type":"ROR","asserted-by":"publisher"}],"name":"Department of Microbiology, University of Washington","place":["Seattle, USA"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-3732-6675","authenticated-orcid":true,"given":"Qing","family":"Tang","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/019kgqr73","id-type":"ROR","asserted-by":"publisher"}],"name":"Department of Biology, University of Texas at Arlington","place":["Arlington, USA"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"235","reference":[{"key":"e_1_3_5_2_2","doi-asserted-by":"publisher","DOI":"10.3390\/pathogens9010005"},{"key":"e_1_3_5_3_2","doi-asserted-by":"publisher","DOI":"10.1038\/s41598-021-88514-y"},{"key":"e_1_3_5_4_2","doi-asserted-by":"publisher","DOI":"10.1016\/S1473-3099(14)70870-9"},{"key":"e_1_3_5_5_2","doi-asserted-by":"publisher","DOI":"10.1128\/cmr.00060-19"},{"key":"e_1_3_5_6_2","unstructured":"CDC. 2004. 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