Structural and Regulatory Changes in PBP4 Trigger Decreased beta-Lactam Susceptibility in Enterococcus faecalis
AuthorRice, Louis B.
Moon, Thomas M.
D’Andréa, Éverton D.
AffiliationUniv Arizona, Coll Med, Dept Chem & Biochem
MetadataShow full item record
PublisherAMER SOC MICROBIOLOGY
CitationRice LB, Desbonnet C, Tait-Kamradt A, Garcia-Solache M, Lonks J, Moon TM, D’Andréa ÉD, Page R, Peti W. 2018. Structural and regulatory changes in PBP4 trigger decreased β-lactam susceptibility in Enterococcus faecalis. mBio 9:e00361-18. https://doi.org/10.1128/mBio.00361-18.
RightsCopyright © 2018 Rice et al. This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license.
Collection InformationThis item from the UA Faculty Publications collection is made available by the University of Arizona with support from the University of Arizona Libraries. If you have questions, please contact us at firstname.lastname@example.org.
AbstractEnterococcus 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 beta-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. IMPORTANCE Enterococcus 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 beta-lactam antibiotics with low-affinity PBP4. These insights will help inform future efforts to develop therapeutics capable of inhibiting clinical enterococcal strains.
VersionFinal published version
SponsorsNational Institute of Allergy and Infectious Diseases [R56AI045626]
- Evaluation of polymorphisms in pbp4 gene and genetic diversity in penicillin-resistant, ampicillin-susceptible Enterococcus faecalis from hospitals in different states in Brazil.
- Authors: Infante VH, Conceição N, de Oliveira AG, Darini AL
- Issue date: 2016 Apr
- Mechanisms of resistance to imipenem and ampicillin in Enterococcus faecalis.
- Authors: Ono S, Muratani T, Matsumoto T
- Issue date: 2005 Jul
- Penicillin-resistant, ampicillin-susceptible Enterococcus faecalis of hospital origin: pbp4 gene polymorphism and genetic diversity.
- Authors: Conceição N, da Silva LE, Darini AL, Pitondo-Silva A, de Oliveira AG
- Issue date: 2014 Dec
- Genetic analysis of faropenem-resistant Enterococcus faecalis in urinary isolates.
- Authors: Hiraga N, Muratani T, Naito S, Matsumoto T
- Issue date: 2008 Apr
- The penicillin resistance of Enterococcus faecalis JH2-2r results from an overproduction of the low-affinity penicillin-binding protein PBP4 and does not involve a psr-like gene.
- Authors: Duez C, Zorzi W, Sapunaric F, Amoroso A, Thamm I, Coyette J
- Issue date: 2001 Sep