Abstract
The spread of β-lactamases that hydrolyze penicillins, cephalosporins and carbapenems among Gram-negative bacteria has limited options for treating bacterial infections. Initially, Klebsiella pneumoniae carbapenemase-2 (KPC-2) emerged as a widespread carbapenem hydrolyzing β-lactamase that also hydrolyzes penicillins and cephalosporins but not cephamycins and ceftazidime. In recent years, single and double amino acid substitution variants of KPC-2 have emerged among clinical isolates that show increased resistance to ceftazidime. Because it confers multi-drug resistance, KPC β-lactamase is a threat to public health. In this study, the evolution of KPC-2 function was determined in nine clinically isolated variants by examining the effects of the substitutions on enzyme kinetic parameters, protein stability and antibiotic resistance profile. The results indicate that the amino acid substitutions associated with KPC-2 natural variants lead to increased catalytic efficiency for ceftazidime hydrolysis and a consequent increase in ceftazidime resistance. Single substitutions lead to modest increases in catalytic activity while the double mutants exhibit significantly increased ceftazidime hydrolysis and resistance levels. The P104R, V240G and H274Y substitutions in single and double mutant combinations lead to the largest increases in ceftazidime hydrolysis and resistance. Molecular modeling suggests that the P104R and H274Y mutations could facilitate ceftazidime hydrolysis through increased hydrogen bonding interactions with the substrate while the V240G substitution may enhance backbone flexibility so that larger substrates might be accommodated in the active site. Additionally, we observed a strong correlation between gain of catalytic function for ceftazidime hydrolysis and loss of enzyme stability, which is in agreement with the ‘stability-function tradeoff’ phenomenon. The high Tm of KPC-2 (66.5°C) provides an evolutionary advantage as compared to other class A enzymes such as TEM (51.5°C) and CTX-M (51°C) in that it can acquire multiple destabilizing substitutions without losing the ability to fold into a functional enzyme.
Highlights
Because of their broad-spectrum activity, safety and favorable pharmacokinetic properties [1], β-lactam antibiotics have been the drugs of choice to treat bacterial infections
The absence of new antibiotics combined with the emergence of antibiotic-resistance enzymes like Klebsiella pneumoniae carbapenemase-2 (KPC-2) that can inactivate most β-lactam antibiotics has resulted in a longer duration of medical treatment, higher costs of medical care, and increased mortality
A number of amino acid sequence variants of KPC-2 have been identified in clinical isolates worldwide suggesting continued evolution of resistance in KPC-2
Summary
Because of their broad-spectrum activity, safety and favorable pharmacokinetic properties [1], β-lactam antibiotics have been the drugs of choice to treat bacterial infections. While antibiotics have helped save millions of lives, the extensive use of these drugs has resulted in the emergence of antibiotic resistant bacterial strains. This problem is compounded by the ability of these organisms to acquire mutations or obtain genes encoding antibiotic-inactivating enzymes from other bacteria, thereby reducing the efficacy of drugs. The penicillins and cephalosporins contain the β-lactam ring fused to a five or six-membered ring, respectively. The presence of a 6-α-hydroxyethyl side-chain at the C-6 position of the β-lactam nucleus is a feature that distinguishes the carbapenems from the penicillins and cephalosporins that have a 6-β- or 7-β-acylamino side-chain in the same position, respectively [5] (Fig 1). In addition to being a structural distinguishing factor for the carbapenems, the 6-α-hydroxyethyl side-chain is responsible for the broad-spectrum activity of the carbapenem antibiotics [6,7,8]
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