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PBP4 activity and its overexpression are necessary for PBP4-mediated high-level ?-lactam resistance.

ABSTRACT: Background:PBP4 is typically considered unimportant for conferring high-level ?-lactam resistance in Staphylococcus aureus. Mutations in PBP4 have been associated with ?-lactam non-susceptibility among natural strains of S. aureus. We have previously shown that PBP4 can mediate high-level ?-lactam resistance in laboratory-generated strains passaged in ?-lactam antibiotics. Mutations in the pbp4 promoter that up-regulate its expression and missense mutations that surround PBP4's active site were detected in high frequencies among passaged strains, suggesting PBP4 plays a key role in resistance. How these mutations participate in PBP4's ability to provide high-level ?-lactam resistance is unknown. Objectives:To determine whether enzymatic activity of PBP4 is required for high-level ?-lactam resistance and to investigate how the pbp4-associated mutations provide ?-lactam resistance. Methods:The catalytic activity of PBP4 was disabled through introduction of a serine to alanine point mutation in its active site (Ser-75?Ala) in a representative and well-studied passaged strain, CRB. pbp4 promoter and missense mutations detected in CRB were reconstituted in a WT strain individually and in combination. ?-Lactam resistance of the resultant strains was evaluated by population analysis. Bacterial peptidoglycan composition of the pbp4 mutants was evaluated with and without antibiotic treatment using LC. Results:PBP4 inactivation imparted complete ?-lactam susceptibility of CRB. Reconstitution of PBP4 missense mutations alone did not impart ?-lactam resistance, but did so in synergism with pbp4 promoter mutation. A similar synergistic interaction of pbp4 mutations was observed in enhanced peptidoglycan cross-linking upon antibiotic treatment. Conclusions:PBP4's activity and overexpression both contribute to high-level ?-lactam resistance.

SUBMITTER: Basuino L

PROVIDER: S-EPMC5909641 | biostudies-literature | 2018 May

REPOSITORIES: biostudies-literature

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PBP4 activity and its overexpression are necessary for PBP4-mediated high-level β-lactam resistance.

Basuino Li L Jousselin Ambre A Alexander J Andrew N JAN Strynadka Natalie C J NCJ Pinho Mariana G MG Chambers Henry F HF Chatterjee Som S SS

The Journal of antimicrobial chemotherapy 20180501 5

<h4>Background</h4>PBP4 is typically considered unimportant for conferring high-level β-lactam resistance in Staphylococcus aureus. Mutations in PBP4 have been associated with β-lactam non-susceptibility among natural strains of S. aureus. We have previously shown that PBP4 can mediate high-level β-lactam resistance in laboratory-generated strains passaged in β-lactam antibiotics. Mutations in the pbp4 promoter that up-regulate its expression and missense mutations that surround PBP4's active si ...[more]

PMID: 29360990

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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

PBP4 activity and its overexpression are necessary for PBP4-mediated high-level ?-lactam resistance.

Publications

PBP4 activity and its overexpression are necessary for PBP4-mediated high-level β-lactam resistance.

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