The mechanosensitive channel MscS links lipoteichoic acid synthesis alterations to beta-lactam sensitivity in Staphylococcus aureus

Lipoteichoic acid is an essential cell envelope polymer in Staphylococcus aureus and contributes to beta-lactam resistance in methicillin-resistant S. aureus. Mutations that alter lipoteichoic acid synthesis sensitize MRSA to beta-lactam antibiotics, but the mechanism connecting envelope polymer defects to antibiotic susceptibility has remained unclear. Here, we used suppressor genetics, genome sequencing, transcriptomics, inducible gene expression, antibiotic susceptibility assays, and measurements of intracellular potassium, cyclic di-AMP, and penicillin-binding protein production to define this connection. We found that lipoteichoic acid synthesis defects increased expression of a ParB-like protein and the mechanosensitive channel MscS. Repression of this locus in RNA polymerase suppressor mutants restored beta-lactam resistance, whereas induced expression of both genes reduced resistance. Increased ParBL-MscS expression was associated with decreased intracellular potassium and reduced cyclic di-AMP, linking altered membrane-envelope physiology to second messenger signaling. Lipoteichoic acid synthesis mutants also showed reduced production of penicillin-binding protein 4, an important determinant of beta-lactam resistance. Modest restoration of penicillin-binding protein 4 improved resistance in the mutant with reduced lipoteichoic acid abundance, whereas the mutant producing elongated lipoteichoic acid showed a distinct response, indicating that different lipoteichoic acid defects impose different envelope stresses. Together, these findings identify a potassium-dependent pathway connecting lipoteichoic acid synthesis, mechanosensitive channel activity, cyclic di-AMP signaling, penicillin-binding protein 4 production, and beta-lactam resistance. This work reveals how cell envelope perturbations are converted into cytoplasmic regulatory responses that control antibiotic susceptibility and suggests that ion homeostasis and cyclic di-AMP signaling may be exploitable pathways for restoring beta-lactam efficacy against MRSA. Author SummaryAntibiotic-resistant Staphylococcus aureus, including methicillin-resistant S. aureus, is difficult to treat because it can withstand many beta-lactam antibiotics, a widely used class of drugs. Previous work showed that changes in lipoteichoic acid, an important molecule in the bacterial cell envelope, make these bacteria more sensitive to beta-lactams. However, it was not clear how a change at the cell surface affects antibiotic resistance inside the cell. In this study, we found that defects in lipoteichoic acid production activate a pathway involving a predicted membrane channel. This pathway changes the level of potassium inside the bacterial cell and affects a small signaling molecule that helps coordinate cell wall maintenance. These changes also reduce the amount of an enzyme involved in building the cell wall, making the bacteria more vulnerable to beta-lactam antibiotics. Our findings suggest that antibiotic resistance depends not only on the direct targets of antibiotics, but also on how bacteria maintain the balance between their cell envelope, ion levels, and internal signaling. Understanding this connection may help identify new ways to make resistant bacteria more sensitive to existing antibiotics.