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

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.

Keywords: KPC-2; avibactam; beta-lactam; beta-lactamase; carbapenemase; ceftazidime.

FIG 1

Ω-Loop hydrogen bond networking changes due to the aspartate (D)-to-asparagine (N) substitution at Ambler position 179 in KPC-2. (A) KPC-2. (B) Asp179Asn (D179N) variant.

FIG 2

Western blot of whole-cell preparations (A) and periplasmic extracts (B) of KPC-2 Asp179 variants in E. coli DH10B. vector, DH10B cells containing pBC SK vector; DH10B, unaltered cells. All variants are in the pBR322 vector except pBC SK-Ser, pBC SK-Ilu, and pBC SK-Glu.

FIG 3

Structures of β-lactams and β-lactamase inhibitors. The R1 groups are encompassed by boxes, and circles are used to surround the R2 groups. The dotted line indicates an R2 group that is observed intact when bound to D179N (Fig. S2).

FIG 4

(A) KPC-2 and Asp179Asn (D179N) (1 µM enzyme) hydrolysis of 25 µM ceftazidime (CAZ) at room temperature. (B) KPC-2 and Asp179Asn (0.5 µM enzyme) hydrolysis of 100 µM imipenem (IMI) at room temperature. (C) Examining pre-steady-state kinetics using a stopped-flow apparatus and hydrolysis of 25 µM ceftazidime by 2 µM KPC-2 or and Asp179Asn variant at 25°C for 1,000 s and (inset) 3 s. (D and E) Competitive inhibition curves determined with 50 µM nitrocefin and increasing concentrations of CAZ with 7 nM KPC-2 (apparent K_i, 3.5 mM) (D) and 425 nM Asp179Asn (apparent K_i, 0.13 mM) (E) at room temperature.

FIG 5

(A) Mass spectrometry of KPC-2 (8.7 µM) reacted with avibactam (AVI) ± ceftazidime (CAZ), imipenem (IMI), or aztreonam (AZT). The enzyme/substrate/inhibitor molar ratio was held constant at 1:1:1 at room temperature. Controls were incubated for 5 min. *, KPC-2 alone, mass of 28,720 Da. (B) Mass spectrometry of Asp179Asn (D179N) (8.7 µM) incubated with avibactam (AVI) ± ceftazidime (CAZ), imipenem (IMI), or aztreonam (AZT). The enzyme/substrate/inhibitor molar ratio was held constant at 1:1:1 at room temperature. Controls were incubated for 5 min. #, D179N alone, mass of 28,719 Da.

FIG 6

Conformational assessment of the molecular docking of ceftazidime in the acyl complex. (A) Root mean square deviations (RMSD) of Ω-loop residue data in Asp179Asn (D179N) and KPC-2. (B) Subangstrom movement (atomic RMS fluctuations) of each atom due to thermal energy during MDS. (C) Ω-Loop conformations of KPC-2 (C) and Asp179Asn (D). Molecular docking of KPC-2 (E) and Asp179Asn (F) with ceftazidime as acyl-enzyme during a 550-ps MDS analysis. Ω-Loops and deacylation waters are shown, but ceftazidime has been omitted from the image for clarity.