Abstract Staphylococcus aureus is the most common invasive human pathogen with associated infections originating in multiple settings and patient types. Infections due to methicillin-resistant S. aureus (MRSA) increase patient morbidity and mortality in part due to limited therapeutic options and increasing antibiotic resistance to primary antibiotics. Recent evidence suggests that certain b- lactams, traditionally considered inactive against MRSA, can enhance clinical efficacy against both MRSA and MSSA infections by synergizing with daptomycin (DAP) and cationic host defense peptides (HDPs) of white cell and platelet origins. Mechanistically, this apparent “synergy” was initially attributed to enhancement of binding of these peptide antibiotics to the cell membrane targets in the presence of b-lactams. Other mechanisms appear to play a role, and we have identified that discriminative inhibition of penicillin-binding proteins (PBPs) with b-lactams results in differential daptomycin synergy. Our preliminary screening indicates that blockade of the action of PBP-1 (either specifically or promiscuously) is essential to this DAP-b-lactam synergy outcome. Given these findings, we posit that b-lactams with either selective or nonselective PBP-1 blocking activity provide multi-mechanistic and synergistic killing against S. aureus when used in combination with DAP. To test this hypothesis, we will conduct studies using three integrated Aims. In Aim 1, we identify mechanistic interactions of PBP inhibition underlying DAP/HDP synergy with b-lactams through manipulating PBP function and defining the compensatory impacts of DAP-b-lactam combinations on key cell wall and cell membrane functional metrics that traditionally link to antimicrobial potency. In Aim 2, we determine optimal b-lactam strategies in combination with DAP or HDPs against MRSA using discriminative in vitro modeling with the hollow fiber bioreactor system. Aim 3 establishes the optimal combined b-lactam-DAP treatment regimens in vivo, utilizing a prototypical endovascular MRSA infection model, experimental infective endocarditis. At the conclusion of these studies, our results will identify new mechanisms associated with DAP’s lethal pathway and provide a potentially unique option for rescuing continued use of DAP in clinical practice. Ultimately, this project may well provide clinicians with improved treatment strategies with smart targeted combinations for complex and refractory MRSA infections.