Alanine Uptake Is Required to Maintain Staphylococcus aureus Cell Envelope Stability Under Magnesium and Calcium Limitation

The cell envelope of Gram-positive bacteria is a primary target of host immune defenses and Antibiotics, and its stability is influenced by environmental factors, including the availability of the divalent cations Mg²⁺ and CA²⁺. Alanine also plays a critical role in cell envelope integrity, contributing to peptidoglycan cross-linking, D-alanine modification of teichoic acids, and protein synthesis. However, how these factors functionally interact to maintain envelope stability in S. aureus remains unclear. Here, we demonstrate that growth of S. aureus under Mg²⁺-limited and CA²⁺-limited conditions requires increased alanine uptake mediated by the transporter AapA. Loss of AapA results in increased Cell Lysis and impaired growth under cation-limited conditions, and removing alanine from the growth medium phenocopies these aapA mutant defects. Alanine limitation increases susceptibility to the detergent Triton X-100 and the membrane-targeting Antibiotic daptomycin, consistent with defects in envelope stability. Furthermore, aapA function contributes to Bacterial fitness in insect and murine Infection models. Together, these findings indicate that Mg²⁺, CA²⁺, and alanine play overlapping roles in stabilizing the S. aureus cell envelope, pointing to AapA as a target that may leveraged to enhance antimicrobial efficacy.