Structural and kinetic analyses of penicillin-binding protein 4 (PBP4)-mediated antibiotic resistance in .

Methicillin-resistant (MRSA) causes serious community-acquired and nosocomial infections worldwide. MRSA strains are resistant to a variety of antibiotics, including the classic penicillin and cephalosporin classes of β-lactams, making them intractable to treatment. Although β-lactam resistance in MRSA has been ascribed to the acquisition and activity of penicillin-binding protein 2a (PBP2a, encoded by ), it has recently been observed that resistance can also be mediated by penicillin-binding protein 4 (PBP4). Previously, we have shown that broad-spectrum β-lactam resistance can arise following serial passaging of a -negative COL strain of creating the CRB strain. This strain has two missense mutations in and a mutation in the promoter, both of which play an instrumental role in β-lactam resistance. To better understand PBP4's role in resistance, here we have characterized its kinetics and structure with clinically relevant β-lactam antibiotics. We present the first crystallographic PBP4 structures of apo and acyl-enzyme intermediate forms complexed with three late-generation β-lactam antibiotics: ceftobiprole, ceftaroline, and nafcillin. In parallel, we characterized the structural and kinetic effects of the PBP4 mutations present in the CRB strain. Localized within the transpeptidase active-site cleft, the two substitutions appear to have different effects depending on the drug. With ceftobiprole, the missense mutations impaired the value 150-fold, decreasing the proportion of inhibited PBP4. However, ceftaroline resistance appeared to be mediated by other factors, possibly including mutation of the promoter. Our findings provide evidence that CRB has at least two PBP4-mediated resistance mechanisms.