Abstract
Directed evolution can be a powerful tool for revealing the mutational pathways that lead to more resistant bacterial strains. In this study, we focused on the bacterium Mycobacterium tuberculosis, which is resistant to members of the β-lactam class of antibiotics and thus continues to pose a major public health threat. Resistance of this organism is the result of a chromosomally encoded, extended spectrum class A β-lactamase, BlaC, that is constitutively produced. Here, combinatorial enzyme libraries were selected on ampicillin to identify mutations that increased resistance of bacteria to β-lactams. After just a single round of mutagenesis and selection, BlaC mutants were evolved that conferred 5-fold greater antibiotic resistance to cells and enhanced the catalytic efficiency of BlaC by 3-fold compared to the wild-type enzyme. All isolated mutants carried a mutation at position 105 (e.g., I105F) that appears to widen access to the active site by 3.6 Å while also stabilizing the reorganized topology. In light of these findings, we propose that I105 is a ‘gatekeeper’ residue of the active site that regulates substrate hydrolysis by BlaC. Moreover, our results suggest that directed evolution can provide insight into the development of highly drug resistant microorganisms.
Highlights
Ever since the discovery of penicillin G, b-lactams have emerged as one of the most clinically important classes of antibacterial chemotherapeutics [1]
Antibiotic resistance to blactams primarily arises through the horizontal transfer of blactamase genes contained on plasmids [3], intrinsic mechanisms not specified by mobile elements are recognized as key contributors to antibiotic resistance in bacteria [4]
A key aspect of our studies was the functional transfer of BlaCmediated drug resistance to E. coli, which enabled an evolutionary strategy for revealing mutational pathways that lead to enhanced antibiotic resistance associated with BlaC
Summary
Ever since the discovery of penicillin G, b-lactams have emerged as one of the most clinically important classes of antibacterial chemotherapeutics [1]. Despite the wide-ranging clinical utility of blactam antibiotics, a significant number of b-lactam-resistant strains have emerged in recent years, compromising our ability to effectively treat bacterial infections. The production of b-lactamase was proposed to be the most significant reason for the intrinsic resistance of M. tuberculosis to these antibiotics [6]. When blaC was deleted from M. tuberculosis, the resulting cells exhibited increased susceptibility to b-lactam antibiotics by 8- to 256-fold [7,8], thereby linking BlaC with the intrinsic resistance to b-lactam chemotherapy. The efficiency with which BlaC thwarts b-lactam chemotherapy stems from its ability to hydrolyze penicillin, cephalosporin, and carbapenem classes of b-lactams [9,10]. One promising approach to combat the inherent b-lactam resistance of M. tuberculosis is to use b-lactam antibiotics in combination with blactamase inhibitors such as clavulanate [9,11]
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