PBP4 Mediates ?-Lactam Resistance by Altered Function.
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ABSTRACT:
SUBMITTER: Chatterjee SS
PROVIDER: S-EPMC5655059 | biostudies-literature | 2017 Nov
REPOSITORIES: biostudies-literature
Publications
PBP4 Mediates β-Lactam Resistance by Altered Function.
Chatterjee Som S SS Chen Liang L Gilbert Aubre A da Costa Thaina M TM Nair Vinod V Datta Sandip K SK Kreiswirth Barry N BN Chambers Henry F HF
Antimicrobial agents and chemotherapy 20171024 11
Penicillin binding protein 4 (PBP4) can provide high-level β-lactam resistance in <i>Staphylococcus aureus</i> A series of missense and promoter mutations associated with <i>pbp4</i> were detected in strains that displayed high-level resistance. We show here that the missense mutations facilitate the β-lactam resistance mediated by PBP4 and the promoter mutations lead to overexpression of <i>pbp4</i> Our results also suggest a cooperative interplay among PBPs for β-lactam resistance. ...[more]
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Project description: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.
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Project description:BackgroundPBP4, a low-molecular-weight PBP in Staphylococcus aureus, is not considered to be a classical mediator of β-lactam resistance. Previous studies carried out by our group with laboratory strains of S. aureus demonstrated the ability of PBP4 to produce β-lactam resistance through mutations associated with the pbp4 promoter and/or gene. Recent studies of β-lactam-resistant clinical isolates of S. aureus have reported similar mutations associated with pbp4.ObjectivesTo determine if pbp4-associated mutations reported among clinical strains of S. aureus mediate β-lactam resistance.MethodsThe pbp4 promoters and genes bearing mutations from clinical isolates were cloned into a heterologous host. Reporter, growth and Bocillin assays were performed to assess their role in β-lactam resistance. X-ray crystallography was used to obtain acyl-enzyme intermediate structures of the WT and mutant PBP4 with nafcillin and cefoxitin.ResultsOf the five strains that contained pbp4 promoter mutations, three strains exhibited enhanced expression of PBP4. The R200L mutation in pbp4 resulted in increased survival in the presence of the β-lactams nafcillin and cefoxitin. Further, introduction of either a promoter or a gene mutation into the genome of a WT host increased the ability of the strains to resist the action of β-lactams. The four high-resolution X-ray structures presented demonstrate the binding pose of the β-lactams tested and provide hints for further drug development.ConclusionsMutations associated with the pbp4 promoter and pbp4 gene altered protein activity and mediated β-lactam resistance among the clinically isolated strains that were studied.
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