Invasive infections due to the “superbug” methicillin-resistant Staphylococcus aureus (MRSA) have poor outcomes that are worsened by reduced susceptibility to first-line agents, vancomycin (glycopeptide; GP), and daptomycin (lipopeptide; LP). The long-acting lipoglycopeptide (LGP), dalbavancin, is an alternative that can be given weekly or as a single dose, which can facilitate discharge and reduce costs. However, we have shown that its long half-life may increase its resistance selection potential and can select for cross-resistance to vancomycin and daptomycin. Thus, there is a critical need to understand the mechanism(s) of cross-resistance among cell envelope-targeting drugs in MRSA and to investigate strategies to mitigate or overcome such resistance. Our work from the previous grant periods found that 75% of GP/LP/LGP non-susceptible isolates from in vitro PK/PD models simulating dalbavancin exposures acquired mutations related to the essential two-component regulatory system walKR. Furthermore, we recently published a case in which dalbavancin treatment selected for GP/LP/LGP-resistant MRSA in a patient with endocarditis, via a walK mutation. These recent findings led to the goal for this renewal: to elucidate the multiple mechanisms through which walKR mutations lead to GP/LP/LGP cross-resistance and reveal how beta-lactams and other metabolic modulators interact with WalKR-regulated metabolic networks to synergize with GP/LP/LGP and prevent resistance. We hypothesize that walKR mutations underlie GP/LP/LGP cross-resistance phenotypes through modulation of both cell envelope and nucleotide metabolism, and metabolic modulators can re-sensitize these strains to GP/LP/LGP or prevent resistance by further altering cell envelope or nucleotide metabolism. In AIM 1, we will measure the contribution of reduced WalKR function to cross-resistance phenotypes in MRSA using genetic, lipidomic, metabolomic, transcriptomic, and proteomic approaches in combination with susceptibility testing and quantitative biophysical assessment of the cell envelope properties. In AIM 2, we will test the hypothesis that beta-lactams and other metabolic modulators can re-sensitize walK-knockdown strains to GP/LP/LGP. We will examine the synergistic effects of cell wall inhibitors beta-lactams and fosfomycin, lipid synthesis inhibitors, and anti-folate drugs, trimethoprim/sulfamethoxazole, which will inform metabolic pathways that are important for walKR mutation- caused resistance. In AIM 3, we will evaluate the potential of beta-lactams and other metabolic modulators to prevent the selection of GP/LP/LGP resistance by dalbavancin in vitro using serial passage and PK/PD models. This work is significant because dalbavancin exposures readily select for vancomycin and daptomycin-resistant S. aureus and a strategy to prevent resistance and/or re-sensitize MRSA to GP/LG/LGP is critical to preserve these drugs, especially in the current context of increasing dalbavancin use. ...