title: Fluorescein-labeled beta-lactamase mutant for high-throughput screening of bacterial beta-lactamases against beta-lactam antibiotics.
abstract: The increasing emergence of new bacterial beta-lactamases that can efficiently hydrolyze beta-lactam antibiotics to clinically inactive carboxylic acids has created an intractable problem in the treatment of bacterial infections, and it is highly desirable to develop a useful tool that can rapidly screen bacteria for beta-lactamases against a variety of antibiotic candidates in a high-throughput manner. This paper describes the use of a fluorescein-labeled beta-lactamase mutant (E166Cf) as a convenient fluorescent tool to screen beta-lactamases, including the Bacillus cereus beta-lactamase I (PenPC), B. cereus beta-lactamase II, Bacillus licheniformis PenP, Escherichia coli TEM-1, and Enterobacter cloacae P99 against various beta-lactam antibiotics (penicillin G, penicillin V, ampicillin, cefuroxime, cefoxitin, moxalactam, cephaloridine), using a 96-well microplate reader. The E166Cf mutant was constructed by replacing Glu166 on the flexible Omega-loop, which is close to the enzyme's active site, with a cysteine residue on a class A beta-lactamase (B. cereus PenPC) and subsequently labeling the mutant with thiol-reactive fluorescein-5-maleimide. Such modifications significantly impaired the hydrolytic activity of the E166Cf mutant compared to that of the wild-type enzyme. The fluorescence intensity of the E166Cf mutant increases in the presence of beta-lactam antibiotics. For antibiotics that are resistant to hydrolysis by the E166Cf mutant (cefuroxime, cefoxitin, moxalactam), the fluorescence signal slowly increases until it reaches a plateau. For antibiotics that can be slowly hydrolyzed by the E166Cf mutant (penicillin G, penicillin V, ampicillin), the fluorescence signal rapidly increases to the plateau and then declines after a prolonged incubation. The E166Cf mutant retains its characteristic pattern of fluorescence signals in the presence of both bacterial beta-lactamases and beta-lactamase-resistant antibiotics. In contrast, in the presence of both bacterial beta-lactamases and beta-lactamase-sensitive antibiotics, the fluorescence signals of the E166Cf mutant were decreased. The fluorescence signals from the E166Cf mutant allow an unambiguous differentiation of beta-lactamase-resistant antibiotics from beta-lactamase-sensitive ones in the screening of bacterial beta-lactamases against a panel of antibiotic candidates. This simple method may provide an alternative tool in choosing potent beta-lactam antibiotics for treatment of bacterial infections.
authors.affiliation: Department of Applied Biology and Chemical Technology and Central Laboratory of the Institute of Molecular Technology for Drug Discovery and Synthesis, The Hong Kong Polytechnic University, Hung Hom, Kowloon, Hong Kong, China.
authors.forename: Pak-Ho
authors.initials: PH
authors.lastname: Chan
authors.forename: Kwok-Chu
authors.initials: KC
authors.lastname: Chan
authors.forename: Hong-Bing
authors.initials: HB
authors.lastname: Liu
authors.forename: Wai-Hong
authors.initials: WH
authors.lastname: Chung
authors.forename: Yun-Chung
authors.initials: YC
authors.lastname: Leung
authors.forename: Kwok-Yin
authors.initials: KY
authors.lastname: Wong
mesh_terms: D000818:Animals; D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D002384:Catalysis; D002511:Cephalosporins / Q000494:pharmacology; D019793:Fluorescein / Q000032:analysis* / Q000737:chemistry; D006868:Hydrolysis; D007700:Kinetics; D015394:Molecular Structure; D009154:Mutation / Q000235:genetics*; D010406:Penicillins / Q000494:pharmacology; D013552:Swine / Q000097:blood / Q000382:microbiology; D018440:beta-Lactam Resistance / Q000187:drug effects*; D001618:beta-Lactamases / Q000235:genetics* / Q000378:metabolism*; D047090:beta-Lactams / Q000378:metabolism*
chemical_list: D000900:Anti-Bacterial Agents; D002511:Cephalosporins; D010406:Penicillins; D047090:beta-Lactams; D001618:beta-Lactamases; D019793:Fluorescein