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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.
MBio 2017; 8(5)MBIO

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.

Authors+Show Affiliations

Research Service, Louis Stokes Cleveland Department of Veterans Affairs, Cleveland, Ohio, USA. Department of Medicine, Case Western Reserve University, Cleveland, Ohio, USA.Research Service, Louis Stokes Cleveland Department of Veterans Affairs, Cleveland, Ohio, USA. Department of Molecular Biology and Microbiology, Case Western Reserve University, Cleveland, Ohio, USA.Research Service, Louis Stokes Cleveland Department of Veterans Affairs, Cleveland, Ohio, USA. Department of Medicine, Case Western Reserve University, Cleveland, Ohio, USA.Jacobs University, Bremen, Germany.Basilea Pharmaceutica International Ltd., Basel, Switzerland.Public Health Research Institute Center, New Jersey Medical School, Rutgers University, Newark, New Jersey, USA.Department of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania, USA.Department of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania, USA.Department of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania, USA.Los Angeles County + University California Medical Center, Los Angeles, California, USA. Division of Infectious Diseases, Keck School of Medicine at USC, Los Angeles, California, USA.Research Service, Louis Stokes Cleveland Department of Veterans Affairs, Cleveland, Ohio, USA. Department of Medicine, Case Western Reserve University, Cleveland, Ohio, USA. Department of Biochemistry, Case Western Reserve University, Cleveland, Ohio, USA. Department of Proteomics, Case Western Reserve University, Cleveland, Ohio, USA.Research Service, Louis Stokes Cleveland Department of Veterans Affairs, Cleveland, Ohio, USA robert.bonomo@va.gov. Department of Medicine, Case Western Reserve University, Cleveland, Ohio, USA. Department of Molecular Biology and Microbiology, Case Western Reserve University, Cleveland, Ohio, USA. Department of Pharmacology, Case Western Reserve University, Cleveland, Ohio, USA. Department of Biochemistry, Case Western Reserve University, Cleveland, Ohio, USA. Department of Proteomics, Case Western Reserve University, Cleveland, Ohio, USA. Department of Bioinformatics, Case Western Reserve University, Cleveland, Ohio, USA.

Pub Type(s)

Journal Article

Language

eng

PubMed ID

29089425

Citation

Barnes, Melissa D., et al. "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." MBio, vol. 8, no. 5, 2017.
Barnes MD, Winkler ML, Taracila MA, et al. 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. MBio. 2017;8(5).
Barnes, M. D., Winkler, M. L., Taracila, M. A., Page, M. G., Desarbre, E., Kreiswirth, B. N., ... Bonomo, R. A. (2017). 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. MBio, 8(5), doi:10.1128/mBio.00528-17.
Barnes MD, et al. 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. MBio. 2017 10 31;8(5) PubMed PMID: 29089425.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - 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. AU - Barnes,Melissa D, AU - Winkler,Marisa L, AU - Taracila,Magdalena A, AU - Page,Malcolm G, AU - Desarbre,Eric, AU - Kreiswirth,Barry N, AU - Shields,Ryan K, AU - Nguyen,Minh-Hong, AU - Clancy,Cornelius, AU - Spellberg,Brad, AU - Papp-Wallace,Krisztina M, AU - Bonomo,Robert A, Y1 - 2017/10/31/ PY - 2017/11/2/entrez PY - 2017/11/2/pubmed PY - 2018/6/15/medline KW - KPC-2 KW - avibactam KW - beta-lactam KW - beta-lactamase KW - carbapenemase KW - ceftazidime JF - mBio JO - MBio VL - 8 IS - 5 N2 - 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. SN - 2150-7511 UR - https://www.unboundmedicine.com/medline/citation/29089425/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_ L2 - http://mbio.asm.org/cgi/pmidlookup?view=long&pmid=29089425 DB - PRIME DP - Unbound Medicine ER -