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

Leeming, J. P.; Kang, S.; Thakkar, P. P.; Gorige, P.; Patel, D. R.; Pan, Y.; Villasana Espinosa, O. E.; Wolter, D. J.; Boutte, C. C.; Tang, Q.

doi:10.64898/2026.07.30.741254

bioRxiv · 10.64898/2026.07.30.741254

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

Leeming, J. P.·Kang, S.·Thakkar, P. P.·Gorige, P.·Patel, D. R.·Pan, Y.·Villasana Espinosa, O. E.·Wolter, D. J.·Boutte, C. C.·Tang, Q.

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Abstract

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.

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Leeming, J. P., Kang, S., Thakkar, P. P., Gorige, P., Patel, D. R., Pan, Y., Villasana Espinosa, O. E., Wolter, D. J., Boutte, C. C., Tang, Q.. 2026-07-31. A c-di-AMP-controlled glutamine synthesis pathway promotes persistence of Staphylococcus aureus thymidine-dependent small colony variants in the lung. https://doi.org/10.64898/2026.07.30.741254

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