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

Abstract

Methicillin-resistant Staphylococcus aureus (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 mecA), 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 mecA-negative COL strain of S. aureus, creating the CRB strain. This strain has two missense mutations in pbp4 and a mutation in the pbp4 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 K_m value 150-fold, decreasing the proportion of inhibited PBP4. However, ceftaroline resistance appeared to be mediated by other factors, possibly including mutation of the pbp4 promoter. Our findings provide evidence that S. aureus CRB has at least two PBP4-mediated resistance mechanisms.

Keywords: Gram-positive bacteria; Methicillin-resistant Staphylococcus aureus (MRSA); X-ray crystallography; antibiotic resistance; enzyme kinetics; microbiology; multidrug resistance; penicillin-binding protein 4 (PBP4); peptidoglycan; resistance gene; β-lactam antibiotics.

Figure 1.

PBP4 structure and protein sequence alignment of PBP4 and PBP4^CRB from S. aureus. In each panel, the three conserved motifs in PBPs, SXXK, SXN, and KTG, are shown in red, orange, and purple, respectively. A shows PBP4 in cartoon representation. B depicts the PBP4 active site in cartoon representation with selected residues shown as sticks. A sequence alignment with PBP4 and PBP4^CRB sequences is shown in C. Areas with visable electron density are shown in color (the N-terminal transpeptidase domain sequence is shown on a blue background, whereas the C-terminal domain sequence is shown on an olive background), whereas areas with no density or not present in the construct crystalized are shown in gray. Areas of matching sequence are shown with white letters, whereas mismatches are shown with red letters.

Figure 2.

S. aureus peptidoglycan monomer structure. The PG glycan chain is characterized by repeating units of GlcNAc and N-acetyl-muramic acid. The stem peptide allows the glycan strands to be cross-linked, creating an essential protective mesh around the bacterium. There is some variation in the S. aureus stem peptide between strains with the d-iso-Gln carboxyl group being converted to an amide group in some strains.

Figure 3.

Separate structural alignments of (A) PBP4 structures and (B) PBP4^CRB structures. Apo-PBP4 and PBP4 acyl-enzyme intermediate structures in complex with ceftobiprole, ceftaroline, and nafcillin are shown in light brown, blue, green, and teal, respectively. Apo-PBP4^CRB and PBP4^CRB acyl-enzyme intermediate structures in complex with ceftobiprole, ceftaroline, and nafcillin are shown in red, dark blue, purple, and light blue, respectively. The structures are depicted as cartoons and the catalytic serine (Ser-75), Ser-139 of the SXN motif, and mutated residue in PBP4^CRB (F241R) are displayed as sticks in the bottom panel. Ligands are not shown for greater clarity.

Figure 4.

PBP4 and PBP4^CRB active-site residues in complex with ceftobiprole, ceftaroline, and nafcillin. Alignments of PBP4 and PBP4^CRB are shown in a covalent complex with (A) ceftobiprole, (B) ceftaroline, and (C) nafcillin along with 2D representations of the chemical structures of the β-lactam antibiotics used. PBP4 is shown as light gray lines and its covalently linked ligands are shown in teal, whereas PBP4^CRB is shown in dark gray and its covalently linked ligands are shown in orange. Selected residues involved in coordinating the ligands are displayed as lines with coloration according to atom type and water molecules are shown as cyan spheres. Ligands are displayed as thin stick and ball structures, whereas hydrogen bonding and electrostatic interactions are represented as black dashes.

Figure 5.

Cα alignment of PBP4 from S. aureus, PBP3 from S. pneumoniae (PDB 1XP4), and PBP5 from E. coli (PDB 3MZE) shown in light brown, blue, and green, respectively.