Kumar, S.; Dang, H.; Huynh, T. N.

Cyclic di-AMP (c-di-AMP) is an essential second messenger in Listeria monocytogenes, but its accumulation is detrimental as it disrupts cell wall homeostasis and attenuates virulence. The mechanisms underlying this toxicity remain poorly understood. To understand the molecular basis of this toxicity, we performed a forward genetic screen to identify suppressor mutations that restore {beta}-lactam resistance in a {Delta}pdeA {Delta}pgpH ({Delta}PDE) mutant, which accumulates high c-di-AMP and is susceptible to cell wall-targeting {beta}-lactam antibiotics. We found that the majority of suppressor mutants carried mutations in the mreB gene, which encodes the bacterial actin-like cytoskeletal protein, MreB, that directs lateral peptidoglycan synthesis during cell elongation. These mutations restored {beta}-lactam resistance and ex vivo virulence while still retaining high intracellular c-di-AMP levels. Microscopy analyses indicate that these suppressor mutations reduce MreB activity, as evidenced by cell widening, and that they phenocopy sublethal treatment with the MreB inhibitor A22. Consistently, A22 treatment also rescued {beta}-lactam sensitivity in the {Delta}PDE mutant, supporting a functional link between MreB activity and c-di-AMP toxicity. Mechanistically, c-di-AMP accumulation impaired cell division/septation and reduced peptidoglycan synthesis under cell wall stress, whereas MreB mutations restored both transglycosylation and transpeptidation activities and promoted cell division. These effects were independent of potassium homeostasis, suggesting a distinct pathway linking c-di-AMP to cell wall regulation in L. monocytogenes. Together, our findings demonstrate that dysregulated MreB activity contributes to cell wall defects at elevated c-di-AMP levels and highlight the importance of coordinating cytoskeletal dynamics with cell division to maintain cell envelope integrity.