Leeming, J. P. | Search bioRxiv

bioRxiv2026

A c-di-AMP-controlled glutamine synthesis pathway promotes persistence of Staphylococcus aureus thymidine-dependent small colony variants in the lung

Children with cystic fibrosis (CF) commonly harbor Staphylococcus aureus thymidine-dependent small-colony variants (TD-SCVs), which are associated with reduced lung function and increased respiratory exacerbations. How TD-SCVs survive in the thymidine-limited CF lung is unknown. Here, we show that TD-SCVs exhibit impaired glutamine uptake and depend on c-di-AMP-regulated de novo glutamine synthesis for survival in the murine lung. We found that transcription of the glutamine synthetase gene glnA is cooperatively repressed by the transcriptional regulator GlnR, the c-di-AMP-binding protein PstA, and GlnA itself. Glutamine starvation elevates c-di-AMP levels, relieving repression by this ternary complex and promoting glutamine synthesis. Reducing c-di-AMP levels causes a profound growth defect in TD-SCVs under low-thymidine conditions, which is rescued by glnA overexpression. Moreover, pharmacological inhibition of GlnA markedly impairs TD-SCV growth in murine lung. These findings elucidate the molecular mechanism underlying S. aureus TD-SCV survival during infection and identify glutamine synthesis as a promising therapeutic target for treating infections caused by antifolate-resistant bacteria.

microbiology↗

bioRxiv2025

Cyclic di-AMP inhibits Listeria monocytogenes thymineless death during infection

Anti-folate 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 anti-folate 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 {Delta}thyA mutant led to increased bacterial cell death under thymidine starvation and significant reduction in intracellular growth. Furthermore, we found that {Delta}thyA 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 {Delta}thyA in establishing infection. Notably, decreasing c-di-AMP levels abolished the infection capacity of {Delta}thyA 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 {Delta}thyA 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 insight into potential combined therapeutic strategies for listeriosis targeting both anti-folate and c-di-AMP metabolic pathways.

microbiology↗