Structural and Regulatory Changes in PBP4 Trigger Decreased β-Lactam Susceptibility in <named-content content-type="genus-species">Enterococcus faecalis</named-content>

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 prosthet...

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Autores principales:	Louis B. Rice, Charlene Desbonnet, Amelia Tait-Kamradt, Monica Garcia-Solache, John Lonks, Thomas M. Moon, Éverton D. D’Andréa, Rebecca Page, Wolfgang Peti
Formato:	article
Lenguaje:	EN
Publicado:	American Society for Microbiology 2018
Materias:	antibiotic resistance Enterococcus penicillin-binding proteins Microbiology QR1-502
Acceso en línea:	https://doaj.org/article/4a29d6e56744499296c4f18b97663da3
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spelling	oai:doaj.org-article:4a29d6e56744499296c4f18b97663da32021-11-15T15:53:26ZStructural and Regulatory Changes in PBP4 Trigger Decreased β-Lactam Susceptibility in <named-content content-type="genus-species">Enterococcus faecalis</named-content>10.1128/mBio.00361-182150-7511https://doaj.org/article/4a29d6e56744499296c4f18b97663da32018-05-01T00:00:00Zhttps://journals.asm.org/doi/10.1128/mBio.00361-18https://doaj.org/toc/2150-7511ABSTRACT 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. 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 β-lactam antibiotics with low-affinity PBP4. These insights will help inform future efforts to develop therapeutics capable of inhibiting clinical enterococcal strains.Louis B. RiceCharlene DesbonnetAmelia Tait-KamradtMonica Garcia-SolacheJohn LonksThomas M. MoonÉverton D. D’AndréaRebecca PageWolfgang PetiAmerican Society for Microbiologyarticleantibiotic resistanceEnterococcuspenicillin-binding proteinsMicrobiologyQR1-502ENmBio, Vol 9, Iss 2 (2018)
institution	DOAJ
collection	DOAJ
language	EN
topic	antibiotic resistance Enterococcus penicillin-binding proteins Microbiology QR1-502
spellingShingle	antibiotic resistance Enterococcus penicillin-binding proteins Microbiology QR1-502 Louis B. Rice Charlene Desbonnet Amelia Tait-Kamradt Monica Garcia-Solache John Lonks Thomas M. Moon Éverton D. D’Andréa Rebecca Page Wolfgang Peti Structural and Regulatory Changes in PBP4 Trigger Decreased β-Lactam Susceptibility in <named-content content-type="genus-species">Enterococcus faecalis</named-content>
description	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. 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 β-lactam antibiotics with low-affinity PBP4. These insights will help inform future efforts to develop therapeutics capable of inhibiting clinical enterococcal strains.
format	article
author	Louis B. Rice Charlene Desbonnet Amelia Tait-Kamradt Monica Garcia-Solache John Lonks Thomas M. Moon Éverton D. D’Andréa Rebecca Page Wolfgang Peti
author_facet	Louis B. Rice Charlene Desbonnet Amelia Tait-Kamradt Monica Garcia-Solache John Lonks Thomas M. Moon Éverton D. D’Andréa Rebecca Page Wolfgang Peti
author_sort	Louis B. Rice
title	Structural and Regulatory Changes in PBP4 Trigger Decreased β-Lactam Susceptibility in <named-content content-type="genus-species">Enterococcus faecalis</named-content>
title_short	Structural and Regulatory Changes in PBP4 Trigger Decreased β-Lactam Susceptibility in <named-content content-type="genus-species">Enterococcus faecalis</named-content>
title_full	Structural and Regulatory Changes in PBP4 Trigger Decreased β-Lactam Susceptibility in <named-content content-type="genus-species">Enterococcus faecalis</named-content>
title_fullStr	Structural and Regulatory Changes in PBP4 Trigger Decreased β-Lactam Susceptibility in <named-content content-type="genus-species">Enterococcus faecalis</named-content>
title_full_unstemmed	Structural and Regulatory Changes in PBP4 Trigger Decreased β-Lactam Susceptibility in <named-content content-type="genus-species">Enterococcus faecalis</named-content>
title_sort	structural and regulatory changes in pbp4 trigger decreased β-lactam susceptibility in <named-content content-type="genus-species">enterococcus faecalis</named-content>
publisher	American Society for Microbiology
publishDate	2018
url	https://doaj.org/article/4a29d6e56744499296c4f18b97663da3
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Structural and Regulatory Changes in PBP4 Trigger Decreased β-Lactam Susceptibility in <named-content content-type="genus-species">Enterococcus faecalis</named-content>

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