title: Investigation of the mechanism of resistance to third-generation cephalosporins by class C beta-lactamases by using chemical complementation.
abstract: The widespread use of antibiotics to treat bacterial infections has led to the continuing challenge of antibiotic resistance. For beta-lactam antibiotics, the most common form of resistance is the expression of beta-lactamase enzymes, which inactivate the antibiotics by cleavage of the beta-lactam core. In this study, chemical complementation, which is a general method to link the formation or cleavage of a chemical bond to the transcription of a reporter gene in vivo, was employed in combination with combinatorial mutagenesis to study the mechanism by which the class C beta-lactamase P99 might evolve resistance to the commonly administered third-generation cephalosporin cefotaxime. The chemical complementation system was first shown to be able to distinguish between the wild-type (wt) class C beta-lactamase P99 and the clinically isolated extended-spectrum class C beta-lactamase GC1 in the presence of cefotaxime. The system was then employed to evaluate the activity of mutants of wt P99 towards cefotaxime. A number of single-point mutations at position 221 (Tyr in wt P99) were identified that conferred resistance towards inhibition by cefotaxime, with as much as a 2000-fold increase in k(cat) and a 100-fold increase in k(cat)/K(M) (k(cat)=the rate of catalysis; K(M)=the Michaelis constant), as compared to those of the wt enzyme. Finally, the chemical complementation system was employed in a high-throughput screen to identify a number of mutants of P99 that have multiple mutations around the substrate-binding pocket that increase resistance towards cefotaxime inhibition. The catalytic turnover of cefotaxime by the most active mutant identified was 5500 times higher than that of the wt P99. The resistant mutants suggest a mechanism by which a number of mutations can confer resistance by increasing the flexibility of the Omega loop and altering the positioning of residue 221. Thus, as illustrated in this study, chemical complementation has the potential to be used as a high-throughput screen to study a wide range of enzyme-drug interactions.
authors.affiliation: Department of Chemistry, Columbia University, New York, NY 10027, USA.
authors.forename: Brian T
authors.initials: BT
authors.lastname: Carter
authors.forename: Hening
authors.initials: H
authors.lastname: Lin
authors.forename: Shalom D
authors.initials: SD
authors.lastname: Goldberg
authors.forename: Eric A
authors.initials: EA
authors.lastname: Althoff
authors.forename: Jessica
authors.initials: J
authors.lastname: Raushel
authors.forename: Virginia W
authors.initials: VW
authors.lastname: Cornish
mesh_terms: D000900:Anti-Bacterial Agents / Q000037:antagonists & inhibitors / Q000145:classification / Q000494:pharmacology; D002439:Cefotaxime / Q000145:classification / Q000494:pharmacology; D002511:Cephalosporins / Q000037:antagonists & inhibitors / Q000145:classification* / Q000494:pharmacology*; D018360:Crystallography, X-Ray; D019020:Directed Molecular Evolution; D015195:Drug Design; D024881:Drug Resistance, Bacterial / Q000235:genetics / Q000502:physiology*; D008826:Microbial Sensitivity Tests; D008958:Models, Molecular; D015394:Molecular Structure; D009154:Mutation; D001618:beta-Lactamases / Q000145:classification / Q000494:pharmacology / Q000502:physiology*
chemical_list: D000900:Anti-Bacterial Agents; D002511:Cephalosporins; C415153:oxyiminocephalosporinase type II; D001618:beta-Lactamases; D002439:Cefotaxime