Identification of daptomycin-binding proteins in the membrane of Enterococcus hirae.
Daptomycin, a lipopeptide antibiotic active against gram-positive bacteria, was preliminarily shown to inhibit lipoteichoic acid (LTA) synthesis as a consequence of membrane binding in the presence of Ca2+ (P. Canepari, M. Boaretti, M. M. Lleó, and G. Satta, Antimicrob. Agents Chemother. 34:1220-1226, 1990). In the present study, it is shown that, along with binding bacterial-membrane components, daptomycin binds the protein fraction with a noncovalent bond, as suggested by the instability of the bond in the presence of ionic detergents such as sodium dodecyl sulfate. Analysis of membrane proteins by isoelectric focusing electrophoresis reveals that five bands with isoelectric points ranging from 5.9 to 6.2 bind radioactive daptomycin. These proteins are therefore called daptomycin-binding proteins. In an attempt to correlate these proteins to the main inhibition observed during LTA synthesis, two-dimensional thin-layer chromatography of lipids synthesized during daptomycin treatment was performed. A threefold increase in diglucosyl diacylglycerol is demonstrated, while the compounds phosphatidyl-alpha-kojibiosyldiacylglycerol, glycerophospho-phosphatidyl-alpha-kojibiosyldiacylglycerol, and glycerophospho-kojibiosyldiacylglycerol, which follow diglucosyl diacylglycerol in LTA synthesis, decrease progressively with time during the course of daptomycin treatment.
Boaretti
M
M
Istituto di Microbiologia, Università di Verona, Italy.
Canepari
P
P
D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D002352:Carrier Proteins / Q000032:analysis*; D002462:Cell Membrane / Q000737:chemistry / Q000187:drug effects; D002855:Chromatography, Thin Layer; D017576:Daptomycin / Q000494:pharmacology*; D016983:Enterococcus / Q000737:chemistry* / Q000187:drug effects; D007525:Isoelectric Focusing; D008055:Lipids / Q000096:biosynthesis; D008070:Lipopolysaccharides / Q000096:biosynthesis; D008565:Membrane Proteins / Q000378:metabolism; D011523:Protoplasts / Q000737:chemistry / Q000187:drug effects; D013682:Teichoic Acids / Q000096:biosynthesis
D000900:Anti-Bacterial Agents; D002352:Carrier Proteins; D008055:Lipids; D008070:Lipopolysaccharides; D008565:Membrane Proteins; D013682:Teichoic Acids; C009900:lipoteichoic acid; D017576:Daptomycin