Leeming Joshua P, Elkassih Omar M, Oyebode Damilola T, Woodward Joshua J, Tang Qing
Department of Biology, University of Texas at Arlington, Arlington, TX, 76019, USA.
Department of Microbiology, University of Washington, Seattle, WA, 98105, USA.
bioRxiv. 2025 May 14:2025.05.14.654034. doi: 10.1101/2025.05.14.654034.
Anti-folate antibiotics are used to treat meningitis and refractory listeriosis caused by drug-resistant (). 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 anti-folate antibiotics. In this study, we found that elevated c-di-AMP is required to inhibit TLD in . Conversely, reducing c-di-AMP levels in the Δ mutant led to increased bacterial cell death under thymidine starvation and significant reduction in intracellular growth. Furthermore, we found that Δ 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 Δ in establishing infection. Notably, decreasing c-di-AMP levels abolished the infection capacity of Δ 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 in the Δ background rescued the elevated cell death caused by c-di-AMP depletion both 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 insight into potential combined therapeutic strategies for listeriosis targeting both anti-folate and c-di-AMP metabolic pathways.
抗叶酸抗生素用于治疗由耐药（）引起的脑膜炎和难治性李斯特菌病。它们的杀菌活性归因于胸苷酸合成酶（ThyA）的失活，当胸苷耗尽时，这随后会诱导细菌细胞死亡——这一过程称为无胸腺死亡（TLD）。尽管经过了数十年的研究，但TLD的机制，尤其是在感染期间，仍不清楚。环二磷酸腺苷（c-di-AMP）是一种常见的细菌第二信使，可调节细菌应激反应，它会因抗叶酸抗生素而升高。在这项研究中，我们发现升高的c-di-AMP是抑制（）中TLD所必需的。相反，降低Δ突变体中的c-di-AMP水平会导致在胸苷饥饿下细菌细胞死亡增加，并且细胞内生长显著减少。此外，我们发现与静脉感染相比，Δ在口腔感染期间表现出更明显的生长缺陷，这是由于胆囊中胸苷可用性有限，胆囊是Δ建立感染的一个瓶颈。值得注意的是，降低c-di-AMP水平消除了Δ在两种感染模型中的感染能力。最后，我们确定当c-di-AMP浓度较低时，c-di-AMP结合蛋白PstA会导致细菌细胞死亡。在Δ背景中缺失（）可挽救由c-di-AMP耗竭在（）和小鼠感染期间引起的细胞死亡增加。我们的研究确定了一种以前未被认识的由c-di-AMP介导的TLD调节机制。这扩展了在感染背景下对TLD的基础知识，并为针对抗叶酸和c-di-AMP代谢途径的李斯特菌病潜在联合治疗策略提供了见解。
