Investigation of the Mechanism of Resistance to Third‐Generation Cephalosporins by Class C β‐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.

Directed evolution of O6-alkylguanine-DNA alkyltransferase for applications in protein labeling

“…This yields access to a much larger range of reaction conditions, such as variations in pH and temperature, and should also allow covering a larger dynamic range of the selections with respect to rate enhancements. Nevertheless, the here established yeast three-hybrid system is a valuable alternative to phage display for the directed evolution of AGT and another example of a growing list of yeast three-hybrid systems successfully used for the directed evolution of enzymatic activity (Firestine et al, 2000;Baker et al, 2002;Lin et al, 2004;Carter et al, 2005).…”

Design and Applications of Bifunctional Small Molecules: Why Two Heads Are Better Than One

“…In the presence of cephalosporinase, the molecule was cleaved, ablating the transcriptional response ( lacZ gene product) . Subsequently, they successfully used this system to screen a mutant library for β-lactamase mutants that could be involved in resistance to the cephalosporin cefotaxime . In a reverse application, they synthesized dexamethasone linked to cellobioside and MTX linked to lactosyl fluoride and screened for glycoside linkage formation by glycosynthases, a class of artificial glycosidases , .…”

Rose is a rose is a rose. Especially if you're a George

“…This initial work also laid the groundwork for much subsequent analysis of the role of omega (and other loops) in protein structure, function, and dynamics (see, for example, see Refs. 46–64). George has recently applied methods similar to those he used to classify and categorize proteins (what we call a “George‐like analysis”) to RNA structure and RNA structure formation 65–67.…”