Development of a whole-cell assay for peptidoglycan biosynthesis inhibitors

Abstract

Osmotically stabilized Escherichia coli cells subjected to freezing and thawing were utilized as the source of enzymes for a peptidoglycan pathway assay that can be used to simultaneously test all targets of the committed steps of cell wall biosynthesis. The use of (14)C-labeled UDP-N-acetylglucosamine (UDP-GlcNAc) as a substrate allows the direct detection of cross-linked peptidoglycan formed. The assay was validated with known antibiotics. Fosfomycin was the strongest inhibitor of the pathway assay, with a 50% inhibitory concentration of 1 microM. Flavomycin, bacitracin, vancomycin, D-cycloserine, penicillin G, and ampicillin also inhibited formation of radiolabeled peptidoglycan by the E. coli cells. Screening of compounds identified two inhibitors of the pathway, Cpd1 and Cpd2. Subsequent tests with a biochemical assay utilizing purified enzyme implicated UDP-GlcNAc enolpyruvyl transferase (MurA) as the target of Cpd1. This compound inhibits the first enzyme of the pathway in a time-dependent manner. Moreover, enzyme inactivation is dependent on preincubation in the presence of UDP-GlcNAc, which forms a complex with MurA, exposing its active site. Cpd1 also displayed antimicrobial activity against a panel of microorganisms. The pathway assay used in conjunction with assays for individual enzymes provides an efficient means of detecting and characterizing novel antimicrobial agents.

FIG. 1.

Cell lysis as measured by optical density decrease (600 nm). Open circles, mix 1; closed circles, mix 1 supplemented with 50 mM NH₄Cl. E. coli cells were subjected to one cycle of freezing and thawing.

FIG. 2.

Inhibition of the peptidoglycan pathway assay by fosfomycin (•), vancomycin (○), flavomycin (□), and bacitracin (▪).

FIG. 3.

Chemical structures of peptidoglycan biosynthesis inhibitors screened with a pathway assay utilizing whole E. coli cells as a source of enzymes.

FIG. 4.

MurA inactivation by Cpd1 following preincubation in the presence (closed circles) or absence (open circles) of 1 mM UDP-GlcNAc.

FIG. 5.

Time-dependent inactivation of MurA during preincubation with Cpd1 at 2.5 (▴), 5 (▵), 10 (○), 20 (•), or 30 (□) μM or with DMSO (▪). The compound was dissolved in 100% DMSO.