IDENTIFICATION OF DAPTOMYCIN-BINDING PROTEINS IN THE MEMBRANE OF ENTEROCOCCUS-HIRAE

Daptomycin, a lipopeptide antibiotic active against gram-positive bact eria, was preliminarily shown to inhibit lipoteichoic acid (LTA) synth esis as a consequence of membrane binding in the presence of Ca2+ (P. Canepari, M. Boaretti, M. M. Lleo, and G. Satta, Antimicrob. Agents Ch emother. 34:1220-1224 1990). In the present study, it is shown that, a long with binding bacterial-membrane components, daptomycin binds the protein fraction with a noncovalent bond, as suggested by the instabil ity of the bond in the presence of ionic detergents such as sodium dod ecyl sulfate. Analysis of membrane proteins by isoelectric focusing el ectrophoresis reveals that five bands with isoelectric points ranging from 5.9 to 6.2 bind radioactive daptomycin. These proteins are theref ore called daptomycin-binding proteins. In an attempt to correlate the se proteins to the main inhibition observed during LTA synthesis, two- dimensional thin-layer chromatography of lipids synthesized during dap tomycin treatment was performed. A threefold increase in diglucosyl di acylglycerol is demonstrated, while the compounds phosphatidyl-alpha-k ojibiosyldiacylglycerol, ospho-phosphatidyl-alpha-kojibiosyldiacylglyc erol, and glycerophospho-kojibiosyldiacylglycerol, which follow digluc osyl diacylglycerol in LTA synthesis, decrease progressively with time during the course of daptomycin treatment.