<italic toggle="yes">Klebsiella pneumoniae</italic> Carbapenemase-2 (KPC-2), Substitutions at Ambler Position Asp179, and Resistance to Ceftazidime-Avibactam: Unique Antibiotic-Resistant Phenotypes Emerge from β-Lactamase Protein Engineering

ABSTRACT 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, r...

Descripción completa

Guardado en:

Detalles Bibliográficos
Autores principales:	Melissa D. Barnes, Marisa L. Winkler, Magdalena A. Taracila, Malcolm G. Page, Eric Desarbre, Barry N. Kreiswirth, Ryan K. Shields, Minh-Hong Nguyen, Cornelius Clancy, Brad Spellberg, Krisztina M. Papp-Wallace, Robert A. Bonomo
Formato:	article
Lenguaje:	EN
Publicado:	American Society for Microbiology 2017
Materias:	KPC-2 avibactam beta-lactam beta-lactamase carbapenemase ceftazidime Microbiology QR1-502
Acceso en línea:	https://doaj.org/article/062e9bcecccb41e7b5c7a796418333b1
Etiquetas:	Agregar Etiqueta Sin Etiquetas, Sea el primero en etiquetar este registro!

id	oai:doaj.org-article:062e9bcecccb41e7b5c7a796418333b1
record_format	dspace
spelling	oai:doaj.org-article:062e9bcecccb41e7b5c7a796418333b12021-11-15T15:51:51Z<italic toggle="yes">Klebsiella pneumoniae</italic> Carbapenemase-2 (KPC-2), Substitutions at Ambler Position Asp179, and Resistance to Ceftazidime-Avibactam: Unique Antibiotic-Resistant Phenotypes Emerge from β-Lactamase Protein Engineering10.1128/mBio.00528-172150-7511https://doaj.org/article/062e9bcecccb41e7b5c7a796418333b12017-11-01T00:00:00Zhttps://journals.asm.org/doi/10.1128/mBio.00528-17https://doaj.org/toc/2150-7511ABSTRACT 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. IMPORTANCE Antibiotic resistance is emerging at unprecedented rates and threatens to reach crisis levels. One key mechanism of resistance is the breakdown of β-lactam antibiotics by β-lactamase enzymes. KPC-2 is a β-lactamase that inactivates carbapenems and β-lactamase inhibitors (e.g., clavulanate) and is prevalent around the world, including in the United States. Resistance to the new antibiotic ceftazidime-avibactam, which was designed to overcome KPC resistance, had already emerged within a year. Using protein engineering, we uncovered a mechanism by which resistance to this new drug emerges, which could arm scientists with the ability to forestall such resistance to future drugs.Melissa D. BarnesMarisa L. WinklerMagdalena A. TaracilaMalcolm G. PageEric DesarbreBarry N. KreiswirthRyan K. ShieldsMinh-Hong NguyenCornelius ClancyBrad SpellbergKrisztina M. Papp-WallaceRobert A. BonomoAmerican Society for MicrobiologyarticleKPC-2avibactambeta-lactambeta-lactamasecarbapenemaseceftazidimeMicrobiologyQR1-502ENmBio, Vol 8, Iss 5 (2017)
institution	DOAJ
collection	DOAJ
language	EN
topic	KPC-2 avibactam beta-lactam beta-lactamase carbapenemase ceftazidime Microbiology QR1-502
spellingShingle	KPC-2 avibactam beta-lactam beta-lactamase carbapenemase ceftazidime Microbiology QR1-502 Melissa D. Barnes Marisa L. Winkler Magdalena A. Taracila Malcolm G. Page Eric Desarbre Barry N. Kreiswirth Ryan K. Shields Minh-Hong Nguyen Cornelius Clancy Brad Spellberg Krisztina M. Papp-Wallace Robert A. Bonomo <italic toggle="yes">Klebsiella pneumoniae</italic> Carbapenemase-2 (KPC-2), Substitutions at Ambler Position Asp179, and Resistance to Ceftazidime-Avibactam: Unique Antibiotic-Resistant Phenotypes Emerge from β-Lactamase Protein Engineering
description	ABSTRACT 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. IMPORTANCE Antibiotic resistance is emerging at unprecedented rates and threatens to reach crisis levels. One key mechanism of resistance is the breakdown of β-lactam antibiotics by β-lactamase enzymes. KPC-2 is a β-lactamase that inactivates carbapenems and β-lactamase inhibitors (e.g., clavulanate) and is prevalent around the world, including in the United States. Resistance to the new antibiotic ceftazidime-avibactam, which was designed to overcome KPC resistance, had already emerged within a year. Using protein engineering, we uncovered a mechanism by which resistance to this new drug emerges, which could arm scientists with the ability to forestall such resistance to future drugs.
format	article
author	Melissa D. Barnes Marisa L. Winkler Magdalena A. Taracila Malcolm G. Page Eric Desarbre Barry N. Kreiswirth Ryan K. Shields Minh-Hong Nguyen Cornelius Clancy Brad Spellberg Krisztina M. Papp-Wallace Robert A. Bonomo
author_facet	Melissa D. Barnes Marisa L. Winkler Magdalena A. Taracila Malcolm G. Page Eric Desarbre Barry N. Kreiswirth Ryan K. Shields Minh-Hong Nguyen Cornelius Clancy Brad Spellberg Krisztina M. Papp-Wallace Robert A. Bonomo
author_sort	Melissa D. Barnes
title	<italic toggle="yes">Klebsiella pneumoniae</italic> 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	<italic toggle="yes">Klebsiella pneumoniae</italic> 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	<italic toggle="yes">Klebsiella pneumoniae</italic> 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	<italic toggle="yes">Klebsiella pneumoniae</italic> 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	<italic toggle="yes">Klebsiella pneumoniae</italic> 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	<italic toggle="yes">klebsiella pneumoniae</italic> carbapenemase-2 (kpc-2), substitutions at ambler position asp179, and resistance to ceftazidime-avibactam: unique antibiotic-resistant phenotypes emerge from β-lactamase protein engineering
publisher	American Society for Microbiology
publishDate	2017
url	https://doaj.org/article/062e9bcecccb41e7b5c7a796418333b1
work_keys_str_mv	AT melissadbarnes italictoggleyesklebsiellapneumoniaeitaliccarbapenemase2kpc2substitutionsatamblerpositionasp179andresistancetoceftazidimeavibactamuniqueantibioticresistantphenotypesemergefromblactamaseproteinengineering AT marisalwinkler italictoggleyesklebsiellapneumoniaeitaliccarbapenemase2kpc2substitutionsatamblerpositionasp179andresistancetoceftazidimeavibactamuniqueantibioticresistantphenotypesemergefromblactamaseproteinengineering AT magdalenaataracila italictoggleyesklebsiellapneumoniaeitaliccarbapenemase2kpc2substitutionsatamblerpositionasp179andresistancetoceftazidimeavibactamuniqueantibioticresistantphenotypesemergefromblactamaseproteinengineering AT malcolmgpage italictoggleyesklebsiellapneumoniaeitaliccarbapenemase2kpc2substitutionsatamblerpositionasp179andresistancetoceftazidimeavibactamuniqueantibioticresistantphenotypesemergefromblactamaseproteinengineering AT ericdesarbre italictoggleyesklebsiellapneumoniaeitaliccarbapenemase2kpc2substitutionsatamblerpositionasp179andresistancetoceftazidimeavibactamuniqueantibioticresistantphenotypesemergefromblactamaseproteinengineering AT barrynkreiswirth italictoggleyesklebsiellapneumoniaeitaliccarbapenemase2kpc2substitutionsatamblerpositionasp179andresistancetoceftazidimeavibactamuniqueantibioticresistantphenotypesemergefromblactamaseproteinengineering AT ryankshields italictoggleyesklebsiellapneumoniaeitaliccarbapenemase2kpc2substitutionsatamblerpositionasp179andresistancetoceftazidimeavibactamuniqueantibioticresistantphenotypesemergefromblactamaseproteinengineering AT minhhongnguyen italictoggleyesklebsiellapneumoniaeitaliccarbapenemase2kpc2substitutionsatamblerpositionasp179andresistancetoceftazidimeavibactamuniqueantibioticresistantphenotypesemergefromblactamaseproteinengineering AT corneliusclancy italictoggleyesklebsiellapneumoniaeitaliccarbapenemase2kpc2substitutionsatamblerpositionasp179andresistancetoceftazidimeavibactamuniqueantibioticresistantphenotypesemergefromblactamaseproteinengineering AT bradspellberg italictoggleyesklebsiellapneumoniaeitaliccarbapenemase2kpc2substitutionsatamblerpositionasp179andresistancetoceftazidimeavibactamuniqueantibioticresistantphenotypesemergefromblactamaseproteinengineering AT krisztinampappwallace italictoggleyesklebsiellapneumoniaeitaliccarbapenemase2kpc2substitutionsatamblerpositionasp179andresistancetoceftazidimeavibactamuniqueantibioticresistantphenotypesemergefromblactamaseproteinengineering AT robertabonomo italictoggleyesklebsiellapneumoniaeitaliccarbapenemase2kpc2substitutionsatamblerpositionasp179andresistancetoceftazidimeavibactamuniqueantibioticresistantphenotypesemergefromblactamaseproteinengineering
_version_	1718427316734918656

<italic toggle="yes">Klebsiella pneumoniae</italic> Carbapenemase-2 (KPC-2), Substitutions at Ambler Position Asp179, and Resistance to Ceftazidime-Avibactam: Unique Antibiotic-Resistant Phenotypes Emerge from β-Lactamase Protein Engineering

Ejemplares similares