Development of a whole-cell assay for peptidoglycan biosynthesis inhibitors.
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.
Barbosa
Maria D F S
MD
Department of Antimicrobial Research, Bristol-Myers Squibb Pharma, Wilmington, Delaware 19880, USA. barbodamdf@aol.com
Yang
Gaoyun
G
Fang
Jie
J
Kurilla
Michael G
MG
Pompliano
David L
DL
D019883:Alkyl and Aryl Transferases / Q000037:antagonists & inhibitors; D001681:Biological Assay; D002384:Catalysis; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000235:genetics / Q000302:isolation & purification; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D004926:Escherichia coli / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D007202:Indicators and Reagents; D008826:Microbial Sensitivity Tests; D010457:Peptidoglycan / Q000096:biosynthesis*; D010957:Plasmids; D014537:Uridine Diphosphate N-Acetylglucosamine / Q000037:antagonists & inhibitors / Q000096:biosynthesis
D004269:DNA, Bacterial; D004791:Enzyme Inhibitors; D007202:Indicators and Reagents; D010457:Peptidoglycan; D014537:Uridine Diphosphate N-Acetylglucosamine; D019883:Alkyl and Aryl Transferases; C022566:UDP-N-acetylglucosamine 1-carboxyvinyltransferase