In situ assay for identifying inhibitors of bacterial transglycosylase.
An in situ transglycosylase assay has been developed using endogenously synthesized lipid II. The assay involves the preferential synthesis and accumulation of lipid II in a reaction mixture containing the cell wall membrane material isolated from Escherichia coli, exogenously supplied UDP-MurNAc-pentapeptide, and radiolabeled UDP-GlcNAc. In the presence of Triton X-100, the radiolabeled product formed is almost exclusively lipid II, while the subsequent formation of peptidoglycan is inhibited. Removal of the detergent resulted in the synthesis of peptidoglycan (25% incorporation of radiolabeled material) from the accumulated lipid II. This reaction was inhibited by moenomycin, a known transglycosylase inhibitor. In addition, tunicamycin, which affects an earlier step of the pathway by inhibiting MraY, had no effect on the formation of peptidoglycan in this assay, as expected. Similarly, ampicillin and bacitracin did not inhibit the formation of peptidoglycan under the conditions established.
Branstrom
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Advanced Medicine, Inc., 8 Clarke Drive, 08512, Cranbury, NJ, USA. abranstrom@advmedicine.com
Midha
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Goldman
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D000900:Anti-Bacterial Agents / Q000494:pharmacology; D001431:Bacteriological Techniques; D001455:Bambermycins / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D004926:Escherichia coli / Q000201:enzymology*; D016695:Glycosyltransferases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D017830:Octoxynol / Q000494:pharmacology; D010457:Peptidoglycan / Q000378:metabolism; D011104:Polyisoprenyl Phosphate Oligosaccharides / Q000378:metabolism; D014415:Tunicamycin / Q000494:pharmacology
D000900:Anti-Bacterial Agents; D004791:Enzyme Inhibitors; D010457:Peptidoglycan; D011104:Polyisoprenyl Phosphate Oligosaccharides; D001455:Bambermycins; D014415:Tunicamycin; C062770:undecaprenyl diphosphate-(N-acetylglucosaminyl)(1-4)-N-acetylmuramoyl pentapeptide; D017830:Octoxynol; D016695:Glycosyltransferases