Cardiolipin prevents membrane translocation and permeabilization by daptomycin

Abstract

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.

Keywords: Antibiotic Resistance; Antibiotics Action; Cardiolipin; Fluorescence; Isothermal Titration Calorimetry; Langmuir Monolayers; Lipopeptides; Liposomes; Phosphatidylglycerol.

FIGURE 1.

Structure of daptomycin. In NBD-daptomycin, the free amino group of ornithine is modified with NBD. In perylene-daptomycin, the N-terminally attached decanoyl residue is replaced by perylene-butanoic acid (13). Kynurenine has intrinsic fluorescence that can be used for FRET experiments in conjunction with NBD (15).

FIGURE 2.

Permeabilization by daptomycin of liposomes composed of PC and PG (A) and PC, PG, and CL (B). The pH-sensitive fluorophore pyranine was entrapped in liposomes. The increase in its fluorescence by the efflux of protons was mediated by carbonyl cyanide m-chlorophenylhydrazone (CCCP), in exchange for potassium influx mediated by valinomycin (green) or daptomycin (red). Valinomycin is active on both membranes (but acts faster on the ones containing CL), whereas daptomycin permeabilizes only the membranes devoid of CL. The fluorescence intensity is scaled to that observed after solubilization with Triton X-100, which equals 100 units.

FIGURE 3.

Fluorescence of perylene-daptomycin on liposomes containing PC alone or in various combinations with PG (mole fraction 30%) and CL (mole fraction 10%). A, on PC membranes as well as on PC/CL membranes, the spectrum is characteristic of the perylene monomer, indicating that oligomer formation does not occur. PC/PG membranes with or without 10% CL show a diminished monomer peak and a broad, overlapping excimer peak indicative of oligomer formation (13). The extent of excimer formation is slightly lower in the sample with CL. B, the ratio of emissions at 560 and 445 nm can be used to compare the extent of excimer formation. This ratio is shown here as a function of the CL mole fraction (0–70%). PG was constant at 30%, and the balance was made up by PC.

FIGURE 4.

Estimation of subunit stoichiometry of the daptomycin oligomer by FRET between native daptomycin (donor) and NBD-daptomycin (acceptor). A, native daptomycin (3 μm final) and NBD-daptomycin (0.5 μm final) were added to liposomes containing PC (40%), PG (50%), and CL (10%), either sequentially, with incubation for 5 min between additions, or premixed. Emission spectra were acquired 5 and 15 min after the final addition. The peak at 450 nm represents the kynurenine emission of native daptomycin, whereas the peak at 540 nm is due to NBD. The donor emission of the sequential sample is higher than that of the premixed sample, which is due to the formation of segregated, stable oligomers. It is lower than that of donor alone, which is due to FRET between those segregated oligomers. The emission of the sequential sample remains almost the same after 15 min, which shows that the oligomers are stable on the time scale of the experiment. AU, arbitrary units. B, the two species were mixed before application to liposomes containing PG (50%), CL (0–20%, as indicated), and PC (balance to 100%) to induce the formation of hybrid oligomers. The corrected relative donor fluorescence (F_r) was obtained as described in Ref. . The colored lines represent theoretical F_r functions for the indicated numbers of subunits; each data point represents the means ± S.D. of 3 or 4 repeated experiments.

FIGURE 5.

Surface pressure change of lipid monolayers consisting of CL (% mol/mol as indicated) and equal fractions of PC and PG, in response to injection of daptomycin into the subphase. A, time profiles; daptomycin was injected at t = 0 and at initial surface pressure of 20 micronewtons/m. B, changes in surface pressure derived from the data in panel A. Results were reproducible to within 5% (n = 3–5). Each data point represents the means ± S.D. of 3–5 repeated experiments. C, surface pressure change as a function of initial pressure on PC/PG membranes without CL. The surface pressure increment drops linearly with the starting pressure. The slope of the regression line is −0.885; at a slope of −1, the final surface pressure would be entirely independent of the starting pressure.

FIGURE 6.

ITC experiments on daptomycin binding to PC/PG membranes with or without CL. A, representative raw traces. Each peak is caused by the injection of 2.5 μl of liposome suspension (see “Experimental Procedures” for details). CP, chlorophenylhydrazone. B, integrals (data points) and single-site model fit curves for the traces shown in A. C, energies of interaction between daptomycin and liposome membranes (mole fraction of CL as indicated, balance of equal parts of PC and PG) obtained from a series of isothermal titrations (n = 2–3 at each lipid composition). Each data point represents the means ± S.D. of 3 or 4 repeated experiments.

FIGURE 7.

Hypothetical model of daptomycin membrane insertion, pore formation, and pore structure. A, a daptomycin tetramer forms in the outer membrane leaflet. B, its insertion into the head group layer is limited by the distension and creation of voids in the acyl chain layer; this is unfavorable in terms of enthalpy and increases disorder (entropy). If CL is present, it provides extra bulk in the acyl chain layer, which stabilizes this situation and thus allows for deeper penetration of daptomycin. C, if CL is not present, head group distension and voids in the acyl layer may alternatively cause the outer leaflet to cave in, forming a half-toroidal structure. D, a tetramer as shown in C may flip to the other leaflet and then combine with a second tetramer in the outer leaflet, which gives an octameric pore.

FIGURE 8.

Distribution of daptomycin across inner and outer leaflets, examined with NBD fluorescence quenching using dithionite. A, control (dithionite (10 mm)) reduces only 50% of NBD-PE (0.5% in PC/PG membranes), both in the absence and in the presence of daptomycin. Therefore, dithionite continues to be excluded from membranes in the presence of daptomycin pores. A. U., absorbance units. B, in PC/PG membranes containing 10 or 20% CL, most of the NBD-daptomycin (NBD-dap.) is reduced by dithionite instantly, indicating that it is confined to the outer leaflet. In contrast, in PC/PG membranes containing no CL, immediate reduction affects only 50% of NBD-daptomycin, whereas the remainder is reduced at a slower pace, presumably rate-limited by redistribution from the inner to the outer leaflet.