Structural and Regulatory Changes in PBP4 Trigger Decreased β-Lactam Susceptibility in Enterococcus faecalis

Abstract

Enterococcus faecalis strains resistant to penicillin and ampicillin are rare and have been associated with increases in quantities of low-affinity penicillin-binding protein 4 (PBP4) or with amino acid substitutions in PBP4. We report an E. faecalis strain (LS4828) isolated from a prosthetic knee joint that was subjected to long-term exposure to aminopenicillins. Subsequent cultures yielded E. faecalis with MICs of penicillins and carbapenems higher than those for wild-type strain E. faecalis JH2-2. Sequence analysis of the pbp4 gene of LS4828 compared to that of JH2-2 revealed two point mutations with amino acid substitutions (V223I, A617T) and deletion of an adenine from the region upstream of the predicted pbp4 -35 promoter sequence (UP region). Purified PBP4 from LS4828 exhibited less affinity for Bocillin FL than did PBP4 from JH2-2, which was recapitulated by purified PBP4 containing only the A617T mutation. Differential scanning fluorimetry studies showed that the LS4828 and A617T variants are destabilized compared to wild-type PBP4. Further, reverse transcription-PCR indicated increased transcription of pbp4 in LS4828 and Western blot analysis with polyclonal PBP4 antibody revealed greater quantities of PBP4 in LS4828 than in JH2-2 lysates and membrane preparations. Placing the promoter regions from LS4828 or JH2-2 upstream of a green fluorescent protein reporter gene confirmed that the adenine deletion was associated with increased transcription. Together, these data suggest that the reduced susceptibility to β-lactam antibiotics observed in E. faecalis LS4828 results from a combination of both increased expression and remodeling of the active site, resulting in reduced affinity for penicillins and carbapenems.IMPORTANCEEnterococcus faecalis is an important cause of community-acquired and nosocomial infections and creates therapeutic dilemmas because of its frequent resistance to several classes of antibiotics. We report an E. faecalis strain with decreased ampicillin and imipenem susceptibility isolated after prolonged courses of aminopenicillin therapy for a prosthetic joint infection. Its reduced susceptibility is attributable to a combination of increased quantities of low-affinity PBP4 and an amino acid substitution in proximity to the active site that destabilizes the protein. Our findings provide a cautionary tale for clinicians who elect to "suppress" infections in prosthetic joints and offer novel insights into the interaction of β-lactam antibiotics with low-affinity PBP4. These insights will help inform future efforts to develop therapeutics capable of inhibiting clinical enterococcal strains.

Keywords: Enterococcus; antibiotic resistance; penicillin-binding proteins.

FIG 1

Comparison of PBP4 amino acid sequences of from JH2-2 and LS4828 and alignment with S. aureus PBP2a. Catalytic motifs (green) and sequence differences in PBP4 (red) are highlighted.

FIG 2

Comparative affinities of PBP4 from JH2-2 and LS4828 and variants thereof. LS4828 PBP4 and the A617T variant show reduced Bocillin FL affinities and apparent acylation rates. (A) Michaelis-Menten (K_m) curves were derived from the Bocillin FL binding of each PBP4 enzyme by using SDS-PAGE data and graphing relative fluorescence intensity versus the substrate concentration. Error bars indicate the standard deviation of the mean of two independent experiments. (B) Apparent rates of 10 µM Bocillin FL acylation over time are graphically presented for all PBP4 variants with error bars indicating the standard deviation of the mean of two independent experiments. The slopes of these progress curves were determined in GraphPad Prism version 5 and represent the rate of formation of the stable PBP-Bocillin FL (acyl) complex. The A617T and LS4828 PBP4-Bocillin FL complexes are formed at similar lower rates than those formed by JH2-2 and V223I.

FIG 3

Expression of the pbp4 gene is higher in penicillin-resistant isolate LS4828 than in the penicillin-sensitive control strain. Cultures were grown in BHI broth to mid-log phase, and cells were harvested and frozen for RNA preparation. 16S rRNA gene expression levels were used as a reference. Error bars indicate the standard deviation of the mean of three independent experiments. *, P < 0.0001.

FIG 4

Transcriptional upregulation of pbp4 in LS4828 is due to a single base deletion in the promoter region of the gene. (A) Sequence comparison of the 200-bp region between pbp4 and the gene immediately upstream of it. There is a single base change, loss of a single A in a string of seven, upstream of a putative −35 sequence. (B) Relative expression of egfp driven by the pbp4 upstream sequence is higher from the LS4828 promoter than from the JH2-2 promoter. *, P < 0.0001.

FIG 5

Quantitation of PBP4 in the lysate and membrane fractions of JH2-2 and LS4828 shows increased expression in strain LS4828. (A, top) Coomassie-stained 4 to 12% NuPAGE gel and Western blot analysis of E. faecalis samples. Lanes: 1, MagicMark XP Standards; 2, JH2-2 cell lysate; 3, LS4828 cell lysate; 4, JH2-2 membranes; 5, LS4828 membranes. (A, bottom) Western blot analysis of Coomassie-stained gel (above) with polyclonal PBP4 antibody. (B) Analysis of a Western blot assay by densitometry shows 4- and 7-fold increases in PBP4 expression from LS4828 cell lysates and membranes, respectively, and is representative of two independent experiments.

FIG 6

A617 defines the center of an extensive hydrophobic pocket that is adjacent to the PBP4 catalytic pocket. (A) Model of PBP4 based on the domain organization of PBP2a. The locations of the catalytic serine (S424; red) and the two residues mutated in PBP4 from LS4828, V223 and A617 (both cyan), are shown. (B, left) A617 (cyan, sticks) in a model of PBP4 based on the structure of S. aureus PBP2a (PDB code 1MWR; generated with FFAS), with the residues within 5 Å of A617 shown as spheres (coral). (B, right) Same as panel A, except that A617 is shown as spheres. (C, left) PBP4 with the A617T mutation, illustrating the increased space occupied by A617T in this hydrophobic pocket. (C, right) Same as panel B, except that A617T is shown as spheres.