Development and optimization of fluorescent biosensors from various class A beta-lactamases

beta-Lactam antibiotics are the most commonly used and effective antibiotics for treatment of bacterial infections. Because of the effectiveness of beta-lactam antibiotics, they have been heavily used in the past several decades. However, the over and abuse use of beta-lactam antibiotics generated a strong selection pressure on bacteria and led to the emergence of several beta-lactam resistant mechanisms. The most dominant mechanism is the production of beta-lactamase by the bacteria to destroy the beta-lactam antibiotics. To reduce the emergence of beta-lactam resistant bacteria and to develop effective inhibitors against beta-lactamase, a fast and simple beta-lactam sensor is urgently needed for monitoring the improper use of beta-lactam antibiotics and for high-throughput beta-lactam inhibitors. In this project, fluorescent biosensors for beta-lactam antibiotics and beta-lactamase inhibitors were fabricated by covalently attaching an environment-sensitive fluorophore such as fluorescein onto a specific cysteine residue close to the active site of the beta-lactamase enzyme mutant.;Three class A beta-lactamases, namely PenP, PenPC and TEM, were chosen for this study. Different amino acid residues in these enzymes were mutated to cysteine, and these mutants were separately labeled with 3 different fluorophores namely tetramethylrhodamine-5-maleimide (TMRM), fluorescein-5-maleimide (FM) and 6-bromoacetyl-2-dimethylaminonaphthalene (badan). The badan-labeled PenP beta-lactamase with N170C mutation (PenP N170Cb) has significant improvement over the PenPC E166Cf and gives the largest signal change. The FM-labeled TEM-1 beta-lactamase with mutation V216C (TEM-1 V216Cf), with 2 folds fluorescence increase upon penicillin G binding was further improved by 3 additional mutations (E104K, M182T and G238S) which denoted as TEM-52 V216Cf, resulted in 3.6 folds increase in fluorescence intensity. E166 is the best residue for cysteine mutation and fluorophore labeling in the PenPC biosensor. The FM-labeled PenPC El 66Cf has 2 folds increase in fluorescence intensity upon addition of penicillin G and the fluorescence increase is raised to 3 folds by introducing the Y105W mutation. Labeling TMRM to PenPC Y105W/E166C (PenPC Y105W/E166Cr) produced an even better biosensor, with 4 folds increase in fluorescence intensity upon penicillin G addition.