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Klebsiella pneumoniae Carbapenemase-2 (KPC-2), Substitutions at Ambler Position Asp179, and Resistance to Ceftazidime-Avibactam: Unique Antibiotic-Resistant Phenotypes Emerge from β-Lactamase Protein Engineering

The emergence of Klebsiella pneumoniae carbapenemases (KPCs), β-lactamases that inactivate “last-line” antibiotics such as imipenem, represents a major challenge to contemporary antibiotic therapies. The combination of ceftazidime (CAZ) and avibactam (AVI), a potent β-lactamase inhibitor, represents...

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Autores principales: Barnes, Melissa D., Winkler, Marisa L., Taracila, Magdalena A., Page, Malcolm G., Desarbre, Eric, Kreiswirth, Barry N., Shields, Ryan K., Nguyen, Minh-Hong, Clancy, Cornelius, Spellberg, Brad, Papp-Wallace, Krisztina M., Bonomo, Robert A.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: American Society for Microbiology 2017
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5666153/
https://www.ncbi.nlm.nih.gov/pubmed/29089425
http://dx.doi.org/10.1128/mBio.00528-17
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author Barnes, Melissa D.
Winkler, Marisa L.
Taracila, Magdalena A.
Page, Malcolm G.
Desarbre, Eric
Kreiswirth, Barry N.
Shields, Ryan K.
Nguyen, Minh-Hong
Clancy, Cornelius
Spellberg, Brad
Papp-Wallace, Krisztina M.
Bonomo, Robert A.
author_facet Barnes, Melissa D.
Winkler, Marisa L.
Taracila, Magdalena A.
Page, Malcolm G.
Desarbre, Eric
Kreiswirth, Barry N.
Shields, Ryan K.
Nguyen, Minh-Hong
Clancy, Cornelius
Spellberg, Brad
Papp-Wallace, Krisztina M.
Bonomo, Robert A.
author_sort Barnes, Melissa D.
collection PubMed
description The emergence of Klebsiella pneumoniae carbapenemases (KPCs), β-lactamases that inactivate “last-line” antibiotics such as imipenem, represents a major challenge to contemporary antibiotic therapies. The combination of ceftazidime (CAZ) and avibactam (AVI), a potent β-lactamase inhibitor, represents an attempt to overcome this formidable threat and to restore the efficacy of the antibiotic against Gram-negative bacteria bearing KPCs. CAZ-AVI-resistant clinical strains expressing KPC variants with substitutions in the Ω-loop are emerging. We engineered 19 KPC-2 variants bearing targeted mutations at amino acid residue Ambler position 179 in Escherichia coli and identified a unique antibiotic resistance phenotype. We focus particularly on the CAZ-AVI resistance of the clinically relevant Asp179Asn variant. Although this variant demonstrated less hydrolytic activity, we demonstrated that there was a prolonged period during which an acyl-enzyme intermediate was present. Using mass spectrometry and transient kinetic analysis, we demonstrated that Asp179Asn “traps” β-lactams, preferentially binding β-lactams longer than AVI owing to a decreased rate of deacylation. Molecular dynamics simulations predict that (i) the Asp179Asn variant confers more flexibility to the Ω-loop and expands the active site significantly; (ii) the catalytic nucleophile, S70, is shifted more than 1.5 Å and rotated more than 90°, altering the hydrogen bond networks; and (iii) E166 is displaced by 2 Å when complexed with ceftazidime. These analyses explain the increased hydrolytic profile of KPC-2 and suggest that the Asp179Asn substitution results in an alternative complex mechanism leading to CAZ-AVI resistance. The future design of novel β-lactams and β-lactamase inhibitors must consider the mechanistic basis of resistance of this and other threatening carbapenemases.
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spelling pubmed-56661532017-11-03 Klebsiella pneumoniae Carbapenemase-2 (KPC-2), Substitutions at Ambler Position Asp179, and Resistance to Ceftazidime-Avibactam: Unique Antibiotic-Resistant Phenotypes Emerge from β-Lactamase Protein Engineering Barnes, Melissa D. Winkler, Marisa L. Taracila, Magdalena A. Page, Malcolm G. Desarbre, Eric Kreiswirth, Barry N. Shields, Ryan K. Nguyen, Minh-Hong Clancy, Cornelius Spellberg, Brad Papp-Wallace, Krisztina M. Bonomo, Robert A. mBio Research Article The emergence of Klebsiella pneumoniae carbapenemases (KPCs), β-lactamases that inactivate “last-line” antibiotics such as imipenem, represents a major challenge to contemporary antibiotic therapies. The combination of ceftazidime (CAZ) and avibactam (AVI), a potent β-lactamase inhibitor, represents an attempt to overcome this formidable threat and to restore the efficacy of the antibiotic against Gram-negative bacteria bearing KPCs. CAZ-AVI-resistant clinical strains expressing KPC variants with substitutions in the Ω-loop are emerging. We engineered 19 KPC-2 variants bearing targeted mutations at amino acid residue Ambler position 179 in Escherichia coli and identified a unique antibiotic resistance phenotype. We focus particularly on the CAZ-AVI resistance of the clinically relevant Asp179Asn variant. Although this variant demonstrated less hydrolytic activity, we demonstrated that there was a prolonged period during which an acyl-enzyme intermediate was present. Using mass spectrometry and transient kinetic analysis, we demonstrated that Asp179Asn “traps” β-lactams, preferentially binding β-lactams longer than AVI owing to a decreased rate of deacylation. Molecular dynamics simulations predict that (i) the Asp179Asn variant confers more flexibility to the Ω-loop and expands the active site significantly; (ii) the catalytic nucleophile, S70, is shifted more than 1.5 Å and rotated more than 90°, altering the hydrogen bond networks; and (iii) E166 is displaced by 2 Å when complexed with ceftazidime. These analyses explain the increased hydrolytic profile of KPC-2 and suggest that the Asp179Asn substitution results in an alternative complex mechanism leading to CAZ-AVI resistance. The future design of novel β-lactams and β-lactamase inhibitors must consider the mechanistic basis of resistance of this and other threatening carbapenemases. American Society for Microbiology 2017-10-31 /pmc/articles/PMC5666153/ /pubmed/29089425 http://dx.doi.org/10.1128/mBio.00528-17 Text en Copyright © 2017 Barnes et al. https://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license (https://creativecommons.org/licenses/by/4.0/) .
spellingShingle Research Article
Barnes, Melissa D.
Winkler, Marisa L.
Taracila, Magdalena A.
Page, Malcolm G.
Desarbre, Eric
Kreiswirth, Barry N.
Shields, Ryan K.
Nguyen, Minh-Hong
Clancy, Cornelius
Spellberg, Brad
Papp-Wallace, Krisztina M.
Bonomo, Robert A.
Klebsiella pneumoniae Carbapenemase-2 (KPC-2), Substitutions at Ambler Position Asp179, and Resistance to Ceftazidime-Avibactam: Unique Antibiotic-Resistant Phenotypes Emerge from β-Lactamase Protein Engineering
title Klebsiella pneumoniae Carbapenemase-2 (KPC-2), Substitutions at Ambler Position Asp179, and Resistance to Ceftazidime-Avibactam: Unique Antibiotic-Resistant Phenotypes Emerge from β-Lactamase Protein Engineering
title_full Klebsiella pneumoniae Carbapenemase-2 (KPC-2), Substitutions at Ambler Position Asp179, and Resistance to Ceftazidime-Avibactam: Unique Antibiotic-Resistant Phenotypes Emerge from β-Lactamase Protein Engineering
title_fullStr Klebsiella pneumoniae Carbapenemase-2 (KPC-2), Substitutions at Ambler Position Asp179, and Resistance to Ceftazidime-Avibactam: Unique Antibiotic-Resistant Phenotypes Emerge from β-Lactamase Protein Engineering
title_full_unstemmed Klebsiella pneumoniae Carbapenemase-2 (KPC-2), Substitutions at Ambler Position Asp179, and Resistance to Ceftazidime-Avibactam: Unique Antibiotic-Resistant Phenotypes Emerge from β-Lactamase Protein Engineering
title_short Klebsiella pneumoniae Carbapenemase-2 (KPC-2), Substitutions at Ambler Position Asp179, and Resistance to Ceftazidime-Avibactam: Unique Antibiotic-Resistant Phenotypes Emerge from β-Lactamase Protein Engineering
title_sort klebsiella pneumoniae carbapenemase-2 (kpc-2), substitutions at ambler position asp179, and resistance to ceftazidime-avibactam: unique antibiotic-resistant phenotypes emerge from β-lactamase protein engineering
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5666153/
https://www.ncbi.nlm.nih.gov/pubmed/29089425
http://dx.doi.org/10.1128/mBio.00528-17
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