Thymidine starvation promotes c-di-AMP-dependent inflammation during pathogenic bacterial infection

Antimicrobials can impact bacterial physiology and host immunity with negative treatment outcomes. Extensive exposure to antifolate antibiotics promotes thymidine-dependent Staphylococcus aureus small colony variants (TD-SCVs), commonly associated with worse clinical outcomes. We show that antibiotic-mediated disruption of thymidine synthesis promotes elevated levels of the bacterial second messenger cyclic di-AMP (c-di-AMP), consequently inducing host STING activation and inflammation. An initial antibiotic screen in Firmicutes revealed that c-di-AMP production was largely driven by antifolate antibiotics targeting dihydrofolate reductase (DHFR), which promotes folate regeneration required for thymidine biosynthesis. Additionally, TD-SCVs exhibited excessive c-di-AMP production and STING activation in a thymidine-dependent manner. Murine lung infection with TD-SCVs revealed STING-dependent elevation of proinflammatory cytokines, causing higher airway neutrophil infiltration and activation compared with normal-colony S. aureus and hemin-dependent SCVs. Collectively, our results suggest that thymidine metabolism disruption in Firmicutes leads to elevated c-di-AMP-mediated STING-dependent inflammation, with potential impacts on antibiotic usage and infection outcomes.

Automated summary

Antimicrobials can have negative effects on bacterial physiology and host immunity. Antifolate antibiotics can lead to the formation of thymidine-dependent Staphylococcus aureus small colony variants (TD-SCVs), resulting in worse clinical outcomes. Disruption of thymidine synthesis by antibiotics can increase the production of the bacterial second messenger cyclic di-AMP (c-di-AMP), leading to host STING activation and inflammation.