Multiple Low-Reactivity Class B Penicillin-Binding Proteins Are Required for Cephalosporin Resistance in Enterococci

Abstract

Enterococcus faecalis and Enterococcus faecium are commensals of the gastrointestinal tract of most terrestrial organisms, including humans, and are major causes of health care-associated infections. Such infections are difficult or impossible to treat, as the enterococcal strains responsible are often resistant to multiple antibiotics. One intrinsic resistance trait that is conserved among E. faecalis and E. faecium is cephalosporin resistance, and prior exposure to cephalosporins is one of the most well-known risk factors for acquisition of an enterococcal infection. Cephalosporins inhibit peptidoglycan biosynthesis by acylating the active-site serine of penicillin-binding proteins (PBPs) to prevent the PBPs from catalyzing cross-linking during peptidoglycan synthesis. For decades, a specific PBP (known as Pbp4 or Pbp5) that exhibits low reactivity toward cephalosporins has been thought to be the primary PBP required for cephalosporin resistance. We analyzed other PBPs and report that in both E. faecalis and E. faecium, a second PBP, PbpA(2b), is also required for resistance; notably, the cephalosporin ceftriaxone exhibits a lethal effect on the ΔpbpA mutant. Strikingly, PbpA(2b) exhibits low intrinsic reactivity with cephalosporins in vivo and in vitro Unlike the Δpbp5 mutant, the ΔpbpA mutant exhibits a variety of phenotypic defects in growth kinetics, cell wall integrity, and cellular morphology, indicating that PbpA(2b) and Pbp5(4) are not functionally redundant and that PbpA(2b) plays a more central role in peptidoglycan synthesis. Collectively, our results shift the current understanding of enterococcal cephalosporin resistance and suggest a model in which PbpA(2b) and Pbp5(4) cooperate to coordinately mediate peptidoglycan cross-linking in the presence of cephalosporins.

Keywords: beta-lactam resistance; cephalosporins; enterococci; penicillin-binding proteins; peptidoglycan synthesis.

FIG 1

The ΔpbpA mutant is defective at peptidoglycan synthesis. Exponentially growing E. faecalis cells were pulse-labeled with [¹⁴C] GlcNAc to monitor incorporation into SDS-insoluble peptidoglycan. For wild-type cells, an aliquot was also treated with fosfomycin to inhibit peptidoglycan synthesis. At intervals, aliquots were treated with SDS, and insoluble peptidoglycan was subjected to scintillation counting. Incorporated radioactivity (CPM) was normalized to total biomass (OD₆₀₀) and expressed as CPM/(OD₆₀₀ × 10⁴). Data represent the mean ± standard error from two independent experiments. Strains were wild-type, OG1; ΔpbpA, JL632; and Δpbp5, JL339.

FIG 2

The ΔpbpA mutant exhibits aberrant cellular morphology. E. faecalis strains growing exponentially in MHB were examined using differential interference contrast microscopy (top panels, scale bars = 1 μm) and by transmission electron microscopy (bottom panels, scale bars = 0.5 μm). Strains were wild-type, OG1; ΔpbpA, JL632; Δpbp5, JL339; and ΔpbpA_comp, DDJ241.

FIG 3

The ΔpbpA mutant is susceptible to killing by ceftriaxone. E. faecalis cells growing exponentially in MHB were treated with ceftriaxone. Samples were removed and enumerated for viability (A) and the OD₆₀₀ determined (B) at intervals. Data represent the mean ± standard error from two independent experiments. Strains were wild-type, OG1; ΔpbpA, JL632; Δpbp5, JL339; and ΔpbpA_comp, DDJ241.

FIG 4

Ceftriaxone acylates only a subset of E. faecalis PBPs on growing cells. Two evolutionarily diverse E. faecalis strains (OG1 and an erythromycin-sensitive derivative of the clinical isolate V583) were cultured in MHB to the exponential phase and treated with 2 mg/ml ceftriaxone (or not) as indicated. Cells were collected after 20 min and treated with Bocillin FL to acylate any PBPs that had not been previously acylated by ceftriaxone. Total cell lysates were subjected to SDS-PAGE and scanned to visualize fluorescently labeled PBPs. Data are representative of more than 3 independent experiments.

FIG 5

PbpA(2b) exhibits intrinsically low reactivity for ceftriaxone in vitro. Purified, recombinant E. faecalis His₆-PbpA(2b) ΔTM (3 μM) was incubated with the indicated concentrations of ceftriaxone (A) or ampicillin (B) for 20 min and then treated with excess Bocillin FL. The reactions were subjected to SDS-PAGE and scanned to visualize fluorescently labeled PbpA(2b), followed by Coomassie staining for total protein. Data are representative of 3 independent experiments.