Two successive calcium-dependent transitions mediate membrane binding and oligomerization of daptomycin and the related antibiotic A54145.

Daptomycin and A54145 are homologous lipopeptide antibiotics that permeabilize the cell membranes of Gram-positive bacteria. Membrane permeabilization depends on the presence of both phosphatidylglycerol (PG) and calcium, and it involves the formation of oligomeric transmembrane pores that consist of approximately 6-8 subunits. We here show that each lipopeptide molecule binds two calcium ions in separable, successive steps. The first calcium ion causes the lipopeptide molecule to bind to the target membrane, and likely to form a loosely associated oligomer. Higher calcium concentrations induce binding of a second ion, which produces the more tightly associated and more deeply membrane-inserted final, functional form of the oligomer. Both calcium-dependent steps are accompanied by fluorescence signals that indicate transition of specific amino acid residues into less polar environments, suggestive of insertion into the target membrane. Our findings agree with the earlier observation that two of the four acidic amino acid residues in the daptomycin molecule are essential for antibacterial activity.

Daptomycin forms cation- and size-selective pores in model membranes.

Daptomycin is a lipopeptide antibiotic that is used clinically to treat severe infections caused by Gram-positive bacteria. Its bactericidal action involves the calcium-dependent binding to membranes containing phosphatidylglycerol, followed by the formation of membrane-associated oligomers. Bacterial cells exposed to daptomycin undergo membrane depolarization, suggesting the formation of channels or pores in the target membranes. We here used a liposome model to detect and characterize the permeability properties of the daptomycin pores. The pores are selective for cations, with permeabilities being highest for Na(+), K(+), and other alkali metal ions. The permeability is approximately twice lower for Mg(++), and lower again for the organic cations choline and hexamethonium. Anions are excluded, as is the zwitterion cysteine. These observations account for the observed depolarization of bacterial cells by daptomycin and suggest that under typical in vivo conditions depolarization is mainly due to sodium influx.

Cardiolipin prevents membrane translocation and permeabilization by daptomycin.

Daptomycin is an acidic lipopeptide antibiotic that, in the presence of calcium, forms oligomeric pores on membranes containing phosphatidylglycerol. It is clinically used against various Gram-positive bacteria such as Staphylococcus aureus and Enterococcus species. Genetic studies have indicated that an increased content of cardiolipin in the bacterial membrane may contribute to bacterial resistance against the drug. Here, we used a liposome model to demonstrate that cardiolipin directly inhibits membrane permeabilization by daptomycin. When cardiolipin is added at molar fractions of 10 or 20% to membranes containing phosphatidylglycerol, daptomycin no longer forms pores or translocates to the inner membrane leaflet. Under the same conditions, daptomycin continues to form oligomers; however, these oligomers contain only close to four subunits, which is approximately half as many as observed on membranes without cardiolipin. The collective findings lead us to propose that a daptomycin pore consists of two aligned tetramers in opposite leaflets and that cardiolipin prevents the translocation of tetramers to the inner leaflet, thereby forestalling the formation of complete, octameric pores. Our findings suggest a possible mechanism by which cardiolipin may mediate resistance to daptomycin, and they provide new insights into the action mode of this important antibiotic.

Mutual inhibition through hybrid oligomer formation of daptomycin and the semisynthetic lipopeptide antibiotic CB-182,462.

Daptomycin is a clinically important lipopeptide antibiotic that kills Gram-positive bacteria through membrane depolarization. Its activity requires calcium and the presence of phosphatidylglycerol in the target membrane. Calcium and phosphatidylglycerol also promote the formation of daptomycin oligomers, which have been assumed but not proven to be required for the bactericidal effect. Daptomycin shares substantial structural similarity with another lipopeptide antibiotic, A54145; the two have identical amino acid residues in 5 out of 13 positions and similar ones in 4 more positions. We here examined whether these conserved residues are sufficient for oligomer formation. To this end, we used fluorescence energy transfer and excimer fluorescence to detect hybrid oligomers of daptomycin and CB-182,462, a semisynthetic derivative of A54145. Mixtures of the two compounds indeed produced hybrid oligomers, but at the same time displayed a significantly less than additive antibacterial activity against Bacillus subtilis. The existence of functionally impaired oligomers indicates that oligomer formation is indeed important for antibacterial function. However, it also shows that oligomerization is not sufficient; once formed, the oligomers must take another step in order to acquire antibacterial activity. Thus, the amino acid residues shared between daptomycin and CB-182,462 suffice for formation of the oligomer, but not for its subsequent activation.

Estimation of the subunit stoichiometry of the membrane-associated daptomycin oligomer by FRET.

Daptomycin is a lipopeptide antibiotic that kills Gram-positive bacteria by depolarizing their cell membranes. This antibacterial action of daptomycin is correlated with the formation of membrane-associated oligomers. We here examine the number of subunits contained in one oligomer using fluorescence resonance energy transfer (FRET). The results suggest that the oligomer contains approximately 6 to 7 subunits, or possibly twice this number if it spans both membrane monolayers.

Characterization of daptomycin oligomerization with perylene excimer fluorescence: stoichiometric binding of phosphatidylglycerol triggers oligomer formation.

Daptomycin is a lipopeptide antibiotic that binds to and depolarizes bacterial cell membranes. Its antibacterial activity requires calcium and correlates with the content of phosphatidylglycerol in the target membrane. Daptomycin has been shown to form oligomers on liposome membranes. We here use perylene excimer fluorescence to further characterize the membrane-associated oligomer. To this end, the N-terminal fatty acyl chain was replaced with perylene-butanoic acid. The perylene derivative retains one third of the antibacterial activity of native daptomycin. On liposomes containing phosphatidylcholine and phosphatidylglycerol, as well as on Bacillus subtilis cells, the perylene-labeled daptomycin forms excimers, which shows that the N-terminal acyl chains of neighboring oligomer subunits are in immediate contact with one another. In a lipid bicelle system, oligomer formation can be titrated with stoichiometric amounts of phosphatidylglycerol. Therefore, the interaction of daptomycin with a single molecule of phosphatidylglycerol is sufficient to trigger daptomycin oligomerization.

Oligomerization of daptomycin on membranes.

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.