High-Level Resistance of Staphylococcus aureus to β-Lactam Antibiotics Mediated by Penicillin-Binding Protein 4 (PBP4)

Abstract

Penicillin-binding protein 4 (PBP4), a nonessential, low-molecular-weight penicillin-binding protein of Staphylococcus aureus, has been implicated in low-level resistance to β-lactam antibiotics, although the mechanism is unknown. Mutations in PBP4 and its promoter were identified in a laboratory-generated mutant strain, CRB, which expresses high-level resistance to β-lactams, including resistance to the new-generation cephalosporins active against methicillin-resistant strains of S. aureus These mutations did not appreciably alter the β-lactam antibiotic binding affinity of purified recombinant mutant PBP4 compared to that of wild-type PBP4. Compared to the susceptible parent strain, COLnex, the CRB strain produces a highly cross-linked cell wall peptidoglycan, indicative of increased transpeptidase activity. The pbp4 promoter mutation of CRB was associated with greatly increased amounts of PBP4 in membranes compared to those in the COLnex parent. Replacement of the native promoter of COLnex with the mutant promoter of CRB resulted in increased amounts of PBP4 in membranes and a highly cross-linked cell wall. PBP4 can be repurposed to provide essential transpeptidase activity in vivo and confer high-level resistance to β-lactam antibiotics, such as ceftobiprole and ceftaroline.

Keywords: PBP4; Staphylococcus aureus; ceftaroline; ceftobiprole; drug resistance mechanisms; mechanisms of resistance; penicillin-binding protein 4; penicillin-binding proteins; β-lactam antibiotics; β-lactams.

FIG 1

PBP4 amounts and pbp4 transcription in the CRB and COLnex P_CRB pbp4 strains. (A) PBPs in Bocillin-FL-labeled whole-cell lysates of parental strain COLnex, mutant CRB, and the CRB Δpbp4 strain. PBPs 1 to 4 are indicated in the left margin. (B) PBPs in Bocillin-FL-labeled whole-cell lysates of COLnex and the COLnex P_CRB pbp4 strain with the CRB promoter mutation. (C) S. aureus cells were collected at an OD₆₀₀ of 0.1 (∼10⁸ CFU/ml) and an OD₆₀₀ of 1.0 (∼10⁹ CFU/ml). Shown is the fold increase in the level of pbp4 expression relative to that in the COLnex parental strain. Data are from four independent experiments with CRB and two independent experiments with COLnex P_CRB pbp4. Each sample was run in duplicate.

FIG 2

Antibiotic binding to wild-type and mutant recombinant PBP4. Recombinant PBP4 samples were incubated with 0 to 100 μg/ml (fluorescent gels on the left) and 0 to 2,000 μg/ml (percent densities in the graphs on the right) of ceftobiprole (BPR) (A), ceftaroline (CPT) (B), or nafcillin (NAF) (C) for 15 min at 37°C and then labeled with Bocillin-FL. Band density was measured with a Typhoon Trio variable-mode imager and analyzed with ImageQuant TL software. Percent density is relative to that for the no-antibiotic control (0 μg/ml). Data are representative of those from one of two independent experiments.

FIG 3

Changes in the peptidoglycan compositions of S. aureus CRB mutant strains. (A to D) Peptidoglycans were prepared, and muropeptide patterns were analyzed by HPLC for the COLnex parental strain (A), mutant CRB (B), CRB in which pbp4 was deleted (CRB Δpbp4) (C), and the COLnex P_CRB pbp4 strain with the CRB promoter mutation (D). Peaks 5, 11, 15, 16, and 17 represent the mono-, di-, tri-, tetra-, and pentameric muropeptide derivatives, respectively. Peaks 18+ (the hump) contain highly cross-linked oligomeric components. (E) Quantitative differences in the peptidoglycan compositions of COLnex, CRB, and their derivatives. The bars show the relative amounts of muropeptides calculated from the UV absorbance of the peaks in relation to the total amount of muropeptides. The relative amounts of muropeptides for peaks 5 to 17 and 18+ (the hump) are depicted on the left and right axes, respectively.

FIG 4

Effect of antibiotics on peptidoglycan composition. Peptidoglycans were prepared, and muropeptide patterns were analyzed by HPLC for the COLnex parental strain grown with nafcillin (NAF) at 0.5 μg/ml or ceftaroline (CPT) at 0.25 μg/ml) (A) and mutant CRB grown with nafcillin at 128 μg/ml or ceftaroline at 64 μg/ml (B). Peaks 5, 11, 15, 16, and 17 represent the mono-, di-, tri-, tetra-, and pentameric muropeptide derivatives, respectively. Peaks 18+ (the hump) contain highly cross-linked oligomeric components. The bars show the relative amounts of muropeptides calculated from the UV absorbance of the peaks. The relative amounts of muropeptides for peaks 5 to 17 and 18+ (the hump) are depicted on the left and right axes, respectively.

FIG 5

PBP4 protein amounts in strains grown in the presence of antibiotic. (A and B) COLnex (A) and CRB (B) were grown to an OD₆₀₀ of 0.4 (arrow), and then no drug, nafcillin (NAF) at 0.5 μg/ml (COLnex) or 128 μg/ml (CRB), or ceftaroline (CPT) at 0.25 μg/ml (COLnex) or 64 μg/ml (CRB) was added. (C) PBPs in Bocillin-FL-labeled whole-cell lysates were prepared from cells, all of which were collected at the same time, at an OD₆₀₀ of 1.0 for the COLnex strain or the CRB strain. PBPs 1 to 4 are indicated in the left margin. Data are representative of those from one of two independent experiments.