A Single Salt Bridge in VIM-20 Increases Protein Stability and Antibiotic Resistance under Low-Zinc Conditions

ABSTRACT To understand the evolution of Verona integron-encoded metallo-β-lactamase (VIM) genes (blaVIM) and their clinical impact, microbiological, biochemical, and structural studies were conducted. Forty-five clinically derived VIM variants engineered in a uniform background and expressed in Esch...

Descripción completa

Guardado en:

Detalles Bibliográficos
Autores principales:	Zishuo Cheng, Ben A. Shurina, Christopher R. Bethel, Pei W. Thomas, Steven H. Marshall, Caitlyn A. Thomas, Kundi Yang, Robert L. Kimble, Jonathan S. Montgomery, Matthew G. Orischak, Callie M. Miller, Jordan L. Tennenbaum, Jay C. Nix, David L. Tierney, Walter Fast, Robert A. Bonomo, Richard C. Page, Michael W. Crowder
Formato:	article
Lenguaje:	EN
Publicado:	American Society for Microbiology 2019
Materias:	VIM VIM-20 carbapenem resistance metallo-β-lactamase protein stability salt bridge Microbiology QR1-502
Acceso en línea:	https://doaj.org/article/456a2252f5b9467492680466c100f2cc
Etiquetas:	Agregar Etiqueta Sin Etiquetas, Sea el primero en etiquetar este registro!

id	oai:doaj.org-article:456a2252f5b9467492680466c100f2cc
record_format	dspace
spelling	oai:doaj.org-article:456a2252f5b9467492680466c100f2cc2021-11-15T15:54:47ZA Single Salt Bridge in VIM-20 Increases Protein Stability and Antibiotic Resistance under Low-Zinc Conditions10.1128/mBio.02412-192150-7511https://doaj.org/article/456a2252f5b9467492680466c100f2cc2019-12-01T00:00:00Zhttps://journals.asm.org/doi/10.1128/mBio.02412-19https://doaj.org/toc/2150-7511ABSTRACT To understand the evolution of Verona integron-encoded metallo-β-lactamase (VIM) genes (blaVIM) and their clinical impact, microbiological, biochemical, and structural studies were conducted. Forty-five clinically derived VIM variants engineered in a uniform background and expressed in Escherichia coli afforded increased resistance toward all tested antibiotics; the variants belonging to the VIM-1-like and VIM-4-like families exhibited higher MICs toward five out of six antibiotics than did variants belonging to the widely distributed and clinically important VIM-2-like family. Generally, maximal MIC increases were observed when cephalothin and imipenem were tested. Additionally, MIC determinations under conditions with low zinc availability suggested that some VIM variants are also evolving to overcome zinc deprivation. The most profound increase in resistance was observed in VIM-2-like variants (e.g., VIM-20 H229R) at low zinc availability. Biochemical analyses reveal that VIM-2 and VIM-20 exhibited similar metal binding properties and steady-state kinetic parameters under the conditions tested. Crystal structures of VIM-20 in the reduced and oxidized forms at 1.25 Å and 1.37 Å resolution, respectively, show that Arg229 forms an additional salt bridge with Glu171. Differential scanning fluorimetry of purified proteins and immunoblots of periplasmic extracts revealed that this difference increases thermostability and resistance to proteolytic degradation when zinc availability is low. Therefore, zinc scarcity appears to be a selective pressure driving the evolution of multiple metallo-β-lactamase families, although compensating mutations use different mechanisms to enhance resistance. IMPORTANCE Antibiotic resistance is a growing clinical threat. One of the most serious areas of concern is the ability of some bacteria to degrade carbapenems, drugs that are often reserved as last-resort antibiotics. Resistance to carbapenems can be conferred by a large group of related enzymes called metallo-β-lactamases that rely on zinc ions for function and for overall stability. Here, we studied an extensive panel of 45 different metallo-β-lactamases from a subfamily called VIM to discover what changes are emerging as resistance evolves in clinical settings. Enhanced resistance to some antibiotics was observed. We also found that at least one VIM variant developed a new ability to remain more stable under conditions where zinc availability is limited, and we determined the origin of this stability in atomic detail. These results suggest that zinc scarcity helps drive the evolution of this resistance determinant.Zishuo ChengBen A. ShurinaChristopher R. BethelPei W. ThomasSteven H. MarshallCaitlyn A. ThomasKundi YangRobert L. KimbleJonathan S. MontgomeryMatthew G. OrischakCallie M. MillerJordan L. TennenbaumJay C. NixDavid L. TierneyWalter FastRobert A. BonomoRichard C. PageMichael W. CrowderAmerican Society for MicrobiologyarticleVIMVIM-20carbapenem resistancemetallo-β-lactamaseprotein stabilitysalt bridgeMicrobiologyQR1-502ENmBio, Vol 10, Iss 6 (2019)
institution	DOAJ
collection	DOAJ
language	EN
topic	VIM VIM-20 carbapenem resistance metallo-β-lactamase protein stability salt bridge Microbiology QR1-502
spellingShingle	VIM VIM-20 carbapenem resistance metallo-β-lactamase protein stability salt bridge Microbiology QR1-502 Zishuo Cheng Ben A. Shurina Christopher R. Bethel Pei W. Thomas Steven H. Marshall Caitlyn A. Thomas Kundi Yang Robert L. Kimble Jonathan S. Montgomery Matthew G. Orischak Callie M. Miller Jordan L. Tennenbaum Jay C. Nix David L. Tierney Walter Fast Robert A. Bonomo Richard C. Page Michael W. Crowder A Single Salt Bridge in VIM-20 Increases Protein Stability and Antibiotic Resistance under Low-Zinc Conditions
description	ABSTRACT To understand the evolution of Verona integron-encoded metallo-β-lactamase (VIM) genes (blaVIM) and their clinical impact, microbiological, biochemical, and structural studies were conducted. Forty-five clinically derived VIM variants engineered in a uniform background and expressed in Escherichia coli afforded increased resistance toward all tested antibiotics; the variants belonging to the VIM-1-like and VIM-4-like families exhibited higher MICs toward five out of six antibiotics than did variants belonging to the widely distributed and clinically important VIM-2-like family. Generally, maximal MIC increases were observed when cephalothin and imipenem were tested. Additionally, MIC determinations under conditions with low zinc availability suggested that some VIM variants are also evolving to overcome zinc deprivation. The most profound increase in resistance was observed in VIM-2-like variants (e.g., VIM-20 H229R) at low zinc availability. Biochemical analyses reveal that VIM-2 and VIM-20 exhibited similar metal binding properties and steady-state kinetic parameters under the conditions tested. Crystal structures of VIM-20 in the reduced and oxidized forms at 1.25 Å and 1.37 Å resolution, respectively, show that Arg229 forms an additional salt bridge with Glu171. Differential scanning fluorimetry of purified proteins and immunoblots of periplasmic extracts revealed that this difference increases thermostability and resistance to proteolytic degradation when zinc availability is low. Therefore, zinc scarcity appears to be a selective pressure driving the evolution of multiple metallo-β-lactamase families, although compensating mutations use different mechanisms to enhance resistance. IMPORTANCE Antibiotic resistance is a growing clinical threat. One of the most serious areas of concern is the ability of some bacteria to degrade carbapenems, drugs that are often reserved as last-resort antibiotics. Resistance to carbapenems can be conferred by a large group of related enzymes called metallo-β-lactamases that rely on zinc ions for function and for overall stability. Here, we studied an extensive panel of 45 different metallo-β-lactamases from a subfamily called VIM to discover what changes are emerging as resistance evolves in clinical settings. Enhanced resistance to some antibiotics was observed. We also found that at least one VIM variant developed a new ability to remain more stable under conditions where zinc availability is limited, and we determined the origin of this stability in atomic detail. These results suggest that zinc scarcity helps drive the evolution of this resistance determinant.
format	article
author	Zishuo Cheng Ben A. Shurina Christopher R. Bethel Pei W. Thomas Steven H. Marshall Caitlyn A. Thomas Kundi Yang Robert L. Kimble Jonathan S. Montgomery Matthew G. Orischak Callie M. Miller Jordan L. Tennenbaum Jay C. Nix David L. Tierney Walter Fast Robert A. Bonomo Richard C. Page Michael W. Crowder
author_facet	Zishuo Cheng Ben A. Shurina Christopher R. Bethel Pei W. Thomas Steven H. Marshall Caitlyn A. Thomas Kundi Yang Robert L. Kimble Jonathan S. Montgomery Matthew G. Orischak Callie M. Miller Jordan L. Tennenbaum Jay C. Nix David L. Tierney Walter Fast Robert A. Bonomo Richard C. Page Michael W. Crowder
author_sort	Zishuo Cheng
title	A Single Salt Bridge in VIM-20 Increases Protein Stability and Antibiotic Resistance under Low-Zinc Conditions
title_short	A Single Salt Bridge in VIM-20 Increases Protein Stability and Antibiotic Resistance under Low-Zinc Conditions
title_full	A Single Salt Bridge in VIM-20 Increases Protein Stability and Antibiotic Resistance under Low-Zinc Conditions
title_fullStr	A Single Salt Bridge in VIM-20 Increases Protein Stability and Antibiotic Resistance under Low-Zinc Conditions
title_full_unstemmed	A Single Salt Bridge in VIM-20 Increases Protein Stability and Antibiotic Resistance under Low-Zinc Conditions
title_sort	single salt bridge in vim-20 increases protein stability and antibiotic resistance under low-zinc conditions
publisher	American Society for Microbiology
publishDate	2019
url	https://doaj.org/article/456a2252f5b9467492680466c100f2cc
work_keys_str_mv	AT zishuocheng asinglesaltbridgeinvim20increasesproteinstabilityandantibioticresistanceunderlowzincconditions AT benashurina asinglesaltbridgeinvim20increasesproteinstabilityandantibioticresistanceunderlowzincconditions AT christopherrbethel asinglesaltbridgeinvim20increasesproteinstabilityandantibioticresistanceunderlowzincconditions AT peiwthomas asinglesaltbridgeinvim20increasesproteinstabilityandantibioticresistanceunderlowzincconditions AT stevenhmarshall asinglesaltbridgeinvim20increasesproteinstabilityandantibioticresistanceunderlowzincconditions AT caitlynathomas asinglesaltbridgeinvim20increasesproteinstabilityandantibioticresistanceunderlowzincconditions AT kundiyang asinglesaltbridgeinvim20increasesproteinstabilityandantibioticresistanceunderlowzincconditions AT robertlkimble asinglesaltbridgeinvim20increasesproteinstabilityandantibioticresistanceunderlowzincconditions AT jonathansmontgomery asinglesaltbridgeinvim20increasesproteinstabilityandantibioticresistanceunderlowzincconditions AT matthewgorischak asinglesaltbridgeinvim20increasesproteinstabilityandantibioticresistanceunderlowzincconditions AT calliemmiller asinglesaltbridgeinvim20increasesproteinstabilityandantibioticresistanceunderlowzincconditions AT jordanltennenbaum asinglesaltbridgeinvim20increasesproteinstabilityandantibioticresistanceunderlowzincconditions AT jaycnix asinglesaltbridgeinvim20increasesproteinstabilityandantibioticresistanceunderlowzincconditions AT davidltierney asinglesaltbridgeinvim20increasesproteinstabilityandantibioticresistanceunderlowzincconditions AT walterfast asinglesaltbridgeinvim20increasesproteinstabilityandantibioticresistanceunderlowzincconditions AT robertabonomo asinglesaltbridgeinvim20increasesproteinstabilityandantibioticresistanceunderlowzincconditions AT richardcpage asinglesaltbridgeinvim20increasesproteinstabilityandantibioticresistanceunderlowzincconditions AT michaelwcrowder asinglesaltbridgeinvim20increasesproteinstabilityandantibioticresistanceunderlowzincconditions AT zishuocheng singlesaltbridgeinvim20increasesproteinstabilityandantibioticresistanceunderlowzincconditions AT benashurina singlesaltbridgeinvim20increasesproteinstabilityandantibioticresistanceunderlowzincconditions AT christopherrbethel singlesaltbridgeinvim20increasesproteinstabilityandantibioticresistanceunderlowzincconditions AT peiwthomas singlesaltbridgeinvim20increasesproteinstabilityandantibioticresistanceunderlowzincconditions AT stevenhmarshall singlesaltbridgeinvim20increasesproteinstabilityandantibioticresistanceunderlowzincconditions AT caitlynathomas singlesaltbridgeinvim20increasesproteinstabilityandantibioticresistanceunderlowzincconditions AT kundiyang singlesaltbridgeinvim20increasesproteinstabilityandantibioticresistanceunderlowzincconditions AT robertlkimble singlesaltbridgeinvim20increasesproteinstabilityandantibioticresistanceunderlowzincconditions AT jonathansmontgomery singlesaltbridgeinvim20increasesproteinstabilityandantibioticresistanceunderlowzincconditions AT matthewgorischak singlesaltbridgeinvim20increasesproteinstabilityandantibioticresistanceunderlowzincconditions AT calliemmiller singlesaltbridgeinvim20increasesproteinstabilityandantibioticresistanceunderlowzincconditions AT jordanltennenbaum singlesaltbridgeinvim20increasesproteinstabilityandantibioticresistanceunderlowzincconditions AT jaycnix singlesaltbridgeinvim20increasesproteinstabilityandantibioticresistanceunderlowzincconditions AT davidltierney singlesaltbridgeinvim20increasesproteinstabilityandantibioticresistanceunderlowzincconditions AT walterfast singlesaltbridgeinvim20increasesproteinstabilityandantibioticresistanceunderlowzincconditions AT robertabonomo singlesaltbridgeinvim20increasesproteinstabilityandantibioticresistanceunderlowzincconditions AT richardcpage singlesaltbridgeinvim20increasesproteinstabilityandantibioticresistanceunderlowzincconditions AT michaelwcrowder singlesaltbridgeinvim20increasesproteinstabilityandantibioticresistanceunderlowzincconditions
_version_	1718427210595958784

A Single Salt Bridge in VIM-20 Increases Protein Stability and Antibiotic Resistance under Low-Zinc Conditions

Ejemplares similares