Oligomerization of daptomycin on membranes

Abstract

Daptomycin is a lipopeptide antibiotic that kills Gram-positive bacteria by membrane depolarization. While it has long been assumed that the mode of action of daptomycin involves the formation of membrane-associated oligomers, this has so far not been experimentally demonstrated. We here use FRET between native daptomycin and an NBD-labeled daptomycin derivative to show that such oligomerization indeed occurs. The oligomers are observed in the presence of calcium ions on membrane vesicles isolated from Bacillus subtilis, as well as on model membranes containing the negatively charged phospholipid phosphatidylglycerol. In contrast, oligomerization does not occur on membranes containing phosphatidylcholine only, nor in solution at micromolar daptomycin concentrations. The requirements for oligomerization of daptomycin resemble those previously reported for antibacterial activity, suggesting that oligomerization is necessary for the activity.

The action mechanism of daptomycin

“…Fluorescence experiments using micromolar concentrations of two derivatives of daptomycin labelled with Alexa Fluor 350 and 7-nitro-2,1,3-benzoxadiazol (NBD-Dap; Figure 3) detected no FRET between both, with or without Ca 2+ present, 45 suggesting that aggregation does not occur at concentrations near the MIC. It cannot be ruled out, however, that the covalently attached fluorescent labels may have affected the extent of aggregation.…”

“…Accordingly, the proposal by Silverman et al 73 that daptomycin also forms oligomeric membrane pores or channels was quite readily accepted, although at the time no experimental evidence of oligomer formation had been obtained. More recently, Muraih et al 45 demonstrated oligomer formation on liposomes and on bacterial membrane vesicles by FRET, using the Kyn (13) residue of unlabelled daptomycin as the donor and an extrinsic NBD moiety attached to Orn (6) as the acceptor (NBDdaptomycin, Figure 3). When NBD-daptomycin was used alone, oligomer formation was also evident by self-quenching of the dye.…”

“…Emission blue-shifts and increased quantum yields are also observed with the extrinsic labels NBD and acrylodan attached to Orn (6) in daptomycin and to Lys (8) in A54145, indicating that these residues, too, assume membrane-inserted positions. 45,63,85 When the fluorescence of all four residues-1, 6, 8, and 13-are monitored at different calcium concentrations, it turns out that the spectral changes that signal membrane insertion occur at lower calcium concentrations with residues 6 and 8 than with residues 1 and 13, 85 suggesting that residues 6 and 8 insert in a partially Ca 2+ -saturated state of daptomycin, whereas residues 1 and 13 insert only at full Ca 2+ saturation. This agrees with the observation by ITC that, in the presence of PC/PG liposomes in molar excess, each daptomycin molecule binds two Ca 2+ ions.…”

Antimicrobial Properties of MX-2401, an Expanded-Spectrum Lipopeptide Active in the Presence of Lung Surfactant

“…Daptomycin, however, exhibits a different mechanism of action, as Ca 2ϩ helps daptomycin to interact with and aggregate in the membrane (2,15,19,24), leading to membrane depolarization and eventual bacterial cell death (28). Nevertheless, the presence of Ca 2ϩ had less of an effect on the activity and secondary structure/membrane interaction of MX-2401 than on those of daptomycin, consistent with its different mechanism of action, inhibiting the biosynthesis of the cell wall and teichoic acid precursors (25).…”

“…The addition of one or more equivalents of Ca 2ϩ completely inverted the positive ellipticity at 230 nm in the absence of calcium. This effect has been interpreted as being due to a Ca 2ϩ -dependent change in structure (15,19) or daptomycin-mediated fusion of POPC-POPG liposomes in the presence of calcium (16) without a structural change (12).…”