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The structures of penicillin-binding protein 4 (PBP4) and PBP5 from Enterococci provide structural insights into ?-lactam resistance.

ABSTRACT: The final steps of cell-wall biosynthesis in bacteria are carried out by penicillin-binding proteins (PBPs), whose transpeptidase domains form the cross-links in peptidoglycan chains that define the bacterial cell wall. These enzymes are the targets of ?-lactam antibiotics, as their inhibition reduces the structural integrity of the cell wall. Bacterial resistance to antibiotics is a rapidly growing concern; however, the structural underpinnings of PBP-derived antibiotic resistance are poorly understood. PBP4 and PBP5 are low-affinity, class B transpeptidases that confer antibiotic resistance to Enterococcus faecalis and Enterococcus faecium, respectively. Here, we report the crystal structures of PBP4 (1.8 Å) and PBP5 (2.7 Å) in their apo and acyl-enzyme complexes with the ?-lactams benzylpenicillin, imipenem, and ceftaroline. We found that, although these three ?-lactams adopt geometries similar to those observed in other class B PBP structures, there are small, but significant, differences that likely decrease antibiotic efficacy. Further, we also discovered that the N-terminal domain extensions in this class of PBPs undergo large rigid-body rotations without impacting the structure of the catalytic transpeptidase domain. Together, our findings are defining the subtle functional and structural differences in the Enterococcus PBPs that allow them to support transpeptidase activity while also conferring bacterial resistance to antibiotics that function as substrate mimics.

SUBMITTER: Moon TM

PROVIDER: S-EPMC6290140 | biostudies-literature | 2018 Nov

REPOSITORIES: biostudies-literature

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The structures of penicillin-binding protein 4 (PBP4) and PBP5 from <i>Enterococci</i> provide structural insights into β-lactam resistance.

Moon Thomas M TM D'Andréa Éverton D ÉD Lee Christopher W CW Soares Alexei A Jakoncic Jean J Desbonnet Charlene C Garcia-Solache Monica M Rice Lou B LB Page Rebecca R Peti Wolfgang W

The Journal of biological chemistry 20181024 48

The final steps of cell-wall biosynthesis in bacteria are carried out by penicillin-binding proteins (PBPs), whose transpeptidase domains form the cross-links in peptidoglycan chains that define the bacterial cell wall. These enzymes are the targets of β-lactam antibiotics, as their inhibition reduces the structural integrity of the cell wall. Bacterial resistance to antibiotics is a rapidly growing concern; however, the structural underpinnings of PBP-derived antibiotic resistance are poorly un ...[more]

PMID: 30355734

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Project description: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.

Dataset Information

The structures of penicillin-binding protein 4 (PBP4) and PBP5 from Enterococci provide structural insights into ?-lactam resistance.

Publications

The structures of penicillin-binding protein 4 (PBP4) and PBP5 from <i>Enterococci</i> provide structural insights into β-lactam resistance.

Similar Datasets

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