Interaction Of Daptomycin Research Articles

Interaction of Daptomycin and Calcium with Lipid Bilayers

Daptomycin is a cyclic lipopeptide antibiotic recently approved for clinical treatment of gram positive bacterial infections. It displays strong bactericidal behavior in the presence of excess calcium ions and in biomembranes containing negatively charged PG headgroups. Yet, the molecular mechanism of daptomycin, beyond the suggestion of ion channel formation and cellular depolarization, remains unknown. Recent data has shown results inconsistent with ion channel formation, prompting a more detailed investigation of Daptomycin and calcium interactions with lipid bilayers at higher structural resolutions. In order to fully characterize the mechanism of Daptomycin, it is necessary to understand the role of individual components that are integral to Daptomycin's mode of action in the lipid bilayer. By using lamellar diffraction of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and 1,2-dioleoyl-sn-glycero-3-phosphoglycerol (DOPG) multilayers, we examined the individual and combined effects of Daptomycin and calcium ions on bilayer structure. We found that calcium ions alone result in a slight thickening effect of the bilayer, partially supporting molecular simulations of calcium ions and PG bilayers that propose a calcium ordering effect at physiological calcium concentrations. Inclusion of daptomycin alone produced a bilayer thinning effect, suggesting that Daptomycin is capable of inserting into the headgroup region of the lipid bilayer during binding. Lastly, samples that contained both Daptomycin and calcium ions showed a bilayer thickening effect. These results suggest that Daptomycin binds to the membrane in the absence of calcium ions but induces a structural thickening of the bilayer in the presence of calcium ions. The apparent thickening effect of Daptomycin is unlike other membrane-active antibiotics, such as melittin or LL-37, which induce bilayer thinning. These studies may form the basis for more detailed investigations to characterize the molecular mechanisms of Daptomycin and other cyclic lipopeptides.

Daptomycin Binds but does not Translocate across PC:PG Membranes

The lipopeptide daptomycin is a last-resort antimicrobial in multiresistant Staphylococcus aureus infections. Despite its importance, little is understood about the interaction of daptomycin with lipid membranes and the subsequent events leading to antibacterial effects. In this work we asked three fundamental questions: (1) Does daptomycin bind to giant unilamellar vesicles (GUVs) composed of phosphatidylcholine (PC) and phosphatidylglycerol (PG)? (2) Does it cause dye flux across the membrane? (3) Does the lipopeptide itself translocate across the membrane? Fluorescence confocal microscopy was used to visualize the binding of daptomycin to GUVs, dye flux into those vesicles, and peptide translocation across their membranes. First, we found that daptomycin binds to POPC:POPG GUVs in the presence of 0.5, 2.0, and 20 mM Ca2+. Second, we placed the vesicles in a solution containing carboxyfluorescein (CF) to test for influx. However, influx was never observed. Third, GUVs containing inner vesicles were observed to test for the translocation of daptomycin across the outer membrane of the GUVs, and onto the membrane of the inner vesicles. Again, translocation was never observed. However, in solutions containing 2 mM Ca2+ a significant amount (about 50%) of membranes that had bound daptomycin collapsed shortly after binding occurred, resulting in the destruction of the GUVs. Furthermore, at high Ca2+ concentrations (20 mM), the formation of daptomycin-rich clusters on the GUV membrane was observed. At lower concentrations of Ca2+ (2 mM), daptomycin was distributed evenly on the GUV membrane.Supported by NIH Grants GM072507 and AI088567.

Interaction of daptomycin with lipid bilayers: a lipid extracting effect.

Daptomycin is the first approved member of a new structural class of antibiotics, the cyclic lipopeptides. The peptide interacts with the lipid matrix of cell membranes, inducing permeability of the membrane to ions, but its molecular mechanism has been a puzzle. Unlike the ubiquitous membrane-acting host-defense antimicrobial peptides, daptomycin does not induce pores in the cell membranes. Thus, how it affects the permeability of a membrane to ions is not clear. We studied its interaction with giant unilamellar vesicles (GUVs) and discovered a lipid-extracting phenomenon that correlates with the direct action of daptomycin on bacterial membranes observed in a recent fluorescence microscopy study. Lipid extraction occurred only when the GUV lipid composition included phosphatidylglycerol and in the presence of Ca2+ ions, the same condition found to be necessary for daptomycin to be effective against bacteria. Furthermore, it occurred only when the peptide/lipid ratio exceeded a threshold value, which could be the basis of the minimal inhibitory concentration of daptomycin. In this first publication on the lipid extracting effect, we characterize its dependence on ions and lipid compositions. We also discuss possibilities for connecting the lipid extracting effect to the antibacterial activity of daptomycin.

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.

Lipid-specific binding of the calcium-dependent antibiotic daptomycin leads to changes in lipid polymorphism of model membranes

Daptomycin is a cyclic anionic lipopeptide with an antibiotic activity that is completely dependent on the presence of calcium (as Ca 2+). In a previous study [Jung et al., 2004. Chem. Biol. 11, 949–957], it was concluded that daptomycin underwent two Ca 2+-dependent structural transitions, whereby the first transition was solely dependent on Ca 2+, while the second transition was dependent on both Ca 2+ and the presence of negatively charged lipids that allowed daptomycin to insert into and perturb bilayer membranes with acidic character. Differences in the interaction of daptomycin with acidic and neutral membranes were further investigated by spectroscopic means. The lack of quenching of intrinsic fluorescence by the water-soluble quencher, KI, confirmed the insertion of the daptomycin Trp residue into the membrane bilayer, while the kynurenine residue was inaccessible even in an aqueous environment. Differential scanning calorimetry (DSC) indicated that the binding of daptomycin to neutral bilayers occurred through a combination of electrostatic and hydrophobic interactions, while the binding of daptomycin to bilayers containing acidic lipids primarily involved electrostatic interactions. The binding of daptomycin to acidic membranes led to the induction of non-lamellar lipid phases and membrane fusion.

Interaction of daptomycin with two recombinant thromboplastin reagents leads to falsely prolonged patient prothrombin time/International Normalized Ratio results

A cluster of patients experiencing elevations of International Normalized Ratio without clinical bleeding in temporal association with daptomycin therapy was identified during postmarketing safety surveillance. A common element was the thromboplastin reagent used for the laboratory assay. The present study evaluated the effect of daptomycin on measured prothrombin time using commercially available thromboplastin reagent kits commonly used in the United States. Thirty reagent kits were obtained. Daptomycin was added to pooled normal human plasma samples to achieve final concentrations of 0-200 microg/ml. Quality control ranges were established for each reagent kit using normal and abnormal control plasmas. Triplicate assays of the prothrombin time were performed on the daptomycin-spiked plasma samples using each of the 30 kits. The activated partial thromboplastin time and thrombin time were also assessed. Statistical comparisons of interest were performed using analysis of variance with the Bonferroni t-test for multiple comparisons; alpha = 0.05 was used. Addition of daptomycin to human plasma samples dose-dependently prolonged measured prothrombin times when two recombinant thromboplastin reagents were utilized. The findings were statistically and clinically significant. No clinically meaningful effect was observed with the other reagents. The activated partial thromboplastin time and thrombin time were not affected. Prolonged International Normalized Ratio patient values were an artifact caused by the interaction of daptomycin with the in-vitro prothrombin time test reagent; an in-vivo anticoagulant effect was not observed. Healthcare providers should consider a possible drug-laboratory test interaction if prolonged prothrombin time or elevated International Normalized Ratio values are observed in patients receiving daptomycin.

Evaluation of In Vitro Interaction of Daptomycin with Gentamicin or Beta-lactam Antibiotics Against taphylococcus aureus and Enterococci by FIC Index and Timed-Kill Curves

The interactions of daptomycin with gentamicin and 5 β-lactam agents were determined by checkerboard and timed-kill studies. Eighty isolates were tested by checkerboard: 20 each of methicillin-resistant Staphylococcus aureus (MRSA), methicillin-susceptible Staphylococcus aureus (MSSA), vancomycin-susceptible Enterococcus faecalis (VSEF) and vancomycin-resistant enterococci (VRE). Time kill curves were performed on 8 selected isolates: 2 each of MRSA, MSSA, VSEF and VRE. Checkerboard results showed highest frequency of synergistic effects against VSEF (35-75%) with daptomycin combined with ceftriaxone, cefepime or imipenem; a modest effect with most combinations against VRE and MRSA (5-20%); and indifference with daptomycin and most agents against MSSA (0-5%); except with daptomycin with oxacillin where 10-20% synergy was observed.Synergistic interaction was confirmed by time kill studies in seven of ten isolates where checkerboard suggested synergy. A bactericidal effect was exerted in 5/7 synergistic combinations.The in vitro data suggest that daptomycin combined with other antibiotics may be microbiologically beneficial and not antagonistic.

Structural transitions as determinants of the action of the calcium-dependent antibiotic daptomycin.

Daptomycin is a cyclic anionic lipopeptide antibiotic recently approved for the treatment of complicated skin infections (Cubicin). Its function is dependent on calcium (as Ca2+). Circular dichroism spectroscopy indicated that daptomycin experienced two structural transitions: a transition upon interaction of daptomycin with Ca2+, and a further transition upon interaction with Ca2+ and the bacterial acidic phospholipid, phosphatidyl glycerol. The Ca2+-dependent insertion of daptomycin into model membranes promoted mild and more pronounced perturbations as assessed by the increase of lipid flip-flop and membrane leakage, respectively. The NMR structure of daptomycin indicated that Ca2+ induced a conformational change in daptomycin that increased its amphipathicity. These results are consistent with the hypothesis that the association of Ca2+ with daptomycin permits it to interact with bacterial membranes with effects that are similar to those of the cationic antimicrobial peptides.