β-Lactams are an important class of antibiotics that target penicillin-binding proteins (PBPs) essential to bacterial cell wall synthesis. They are used to treat serious bacterial infections, including those caused by the versatile pathogen Staphylococcus aureus. Some strains carry the resistance gene, mecA, encoding a β-lactam-insensitive PBP2A that allows for peptidoglycan synthesis in the presence of β-lactams such as methicillin, limiting treatment options. We previously identified BAY 11-7082 as having antibacterial activity against methicillin-resistant S. aureus (MRSA) and showed that it re-sensitizes MRSA to β-lactams such as penicillin G, making BAY 11-7082 and its analogs promising candidates for the development of an antibiotic adjuvant. Although the direct antibacterial mechanism of BAY 11-7082 remains undefined, here we aimed to better understand how it potentiates β-lactam activity. We tested BAY 11-7082 in combination with a variety of β-lactams and identified a subset that were synergistic in MRSA but not methicillin-susceptible S. aureus, suggesting that they may directly or indirectly impact the function of PBP2A. Electron and fluorescence microscopy studies revealed that unlike other β-lactam adjuvants, such as those that target the biosynthesis of wall teichoic acids (WTAs), BAY 11-7082 failed to impact cell division, disrupt PBP2 localization, or activate a sensitive reporter of cell wall damage. However, the production of WTA was necessary for BAY 11-7082-β-lactam synergy. Together, these data suggest its mechanism of β-lactam potentiation is distinct from known compounds, making BAY 11-7082 a useful tool to better understand the complexity of resistance in MRSA.
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