{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,27]],"date-time":"2026-10-27T20:58:46Z","timestamp":1793134726364,"version":"4.5.0"},"reference-count":27,"publisher":"American Society for Microbiology","issue":"2","license":[{"start":{"date-parts":[[2018,4,3]],"date-time":"2018-04-03T00:00:00Z","timestamp":1522713600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"},{"start":{"date-parts":[[2018,5,2]],"date-time":"2018-05-02T00:00:00Z","timestamp":1525219200000},"content-version":"tdm","delay-in-days":0,"URL":"https:\/\/journals.asm.org\/non-commercial-tdm-license"}],"content-domain":{"domain":["journals.asm.org"],"crossmark-restriction":true},"short-container-title":["mBio"],"published-print":{"date-parts":[[2018,5,2]]},"abstract":"<jats:title>ABSTRACT<\/jats:title>\n          <jats:p>\n            <jats:named-content content-type=\"genus-species\">Enterococcus faecalis<\/jats:named-content>\n            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\n            <jats:named-content content-type=\"genus-species\">E.\u00a0faecalis<\/jats:named-content>\n            strain (LS4828) isolated from a prosthetic knee joint that was subjected to long-term exposure to aminopenicillins. Subsequent cultures yielded\n            <jats:named-content content-type=\"genus-species\">E.\u00a0faecalis<\/jats:named-content>\n            with MICs of penicillins and carbapenems higher than those for wild-type strain\n            <jats:named-content content-type=\"genus-species\">E.\u00a0faecalis<\/jats:named-content>\n            JH2-2. Sequence analysis of the\n            <jats:italic>pbp4<\/jats:italic>\n            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\n            <jats:italic>pbp4<\/jats:italic>\n            \u221235 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\n            <jats:italic>pbp4<\/jats:italic>\n            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 \u03b2-lactam antibiotics observed in\n            <jats:named-content content-type=\"genus-species\">E.\u00a0faecalis<\/jats:named-content>\n            LS4828 results from a combination of both increased expression and remodeling of the active site, resulting in reduced affinity for penicillins and carbapenems.\n          <\/jats:p>\n          <jats:p>\n            <jats:bold>IMPORTANCE<\/jats:bold>\n            <jats:named-content content-type=\"genus-species\">Enterococcus faecalis<\/jats:named-content>\n            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\n            <jats:named-content content-type=\"genus-species\">E.\u00a0faecalis<\/jats:named-content>\n            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 \u201csuppress\u201d infections in prosthetic joints and offer novel insights into the interaction of \u03b2-lactam antibiotics with low-affinity PBP4. These insights will help inform future efforts to develop therapeutics capable of inhibiting clinical enterococcal strains.\n          <\/jats:p>","DOI":"10.1128\/mbio.00361-18","type":"journal-article","created":{"date-parts":[[2018,4,2]],"date-time":"2018-04-02T10:12:53Z","timestamp":1522663973000},"update-policy":"https:\/\/doi.org\/10.1128\/asmj-crossmark-policy-page","source":"Crossref","is-referenced-by-count":53,"title":["Structural and Regulatory Changes in PBP4 Trigger Decreased \u03b2-Lactam Susceptibility in Enterococcus faecalis"],"prefix":"10.1128","volume":"9","author":[{"given":"Louis B.","family":"Rice","sequence":"first","affiliation":[{"name":"Department of Medicine, Warren Alpert School of Medicine of Brown University, Providence, Rhode Island, USA"},{"name":"Department of Microbiology and Immunology, Warren Alpert School of Medicine of Brown University, Providence, Rhode Island, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Charlene","family":"Desbonnet","sequence":"additional","affiliation":[{"name":"Department of Medicine, Warren Alpert School of Medicine of Brown University, Providence, Rhode Island, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Amelia","family":"Tait-Kamradt","sequence":"additional","affiliation":[{"name":"Department of Medicine, Warren Alpert School of Medicine of Brown University, Providence, Rhode Island, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Monica","family":"Garcia-Solache","sequence":"additional","affiliation":[{"name":"Department of Medicine, Warren Alpert School of Medicine of Brown University, Providence, Rhode Island, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"John","family":"Lonks","sequence":"additional","affiliation":[{"name":"Department of Medicine, Warren Alpert School of Medicine of Brown University, Providence, Rhode Island, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Thomas M.","family":"Moon","sequence":"additional","affiliation":[{"name":"Department of Chemistry and Biochemistry, College of Medicine, University of Arizona, Tucson, Arizona, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"\u00c9verton D.","family":"D\u2019Andr\u00e9a","sequence":"additional","affiliation":[{"name":"Department of Chemistry and Biochemistry, College of Medicine, University of Arizona, Tucson, Arizona, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Rebecca","family":"Page","sequence":"additional","affiliation":[{"name":"Department of Chemistry and Biochemistry, College of Medicine, University of Arizona, Tucson, Arizona, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Wolfgang","family":"Peti","sequence":"additional","affiliation":[{"name":"Department of Chemistry and Biochemistry, College of Medicine, University of Arizona, 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