Cooperative partition model of nystatin interaction with phospholipid vesicles

Abstract

Nystatin is a membrane-active polyene antibiotic that is thought to kill fungal cells by forming ion-permeable channels. In this report we have investigated nystatin interaction with phosphatidylcholine liposomes of different sizes (large and small unilamellar vesicles) by time-resolved fluorescence measurements. Our data show that the fluorescence emission decay kinetics of the antibiotic interacting with gel-phase 1,2-dipalmitoyl-sn-glycero-3-phosphocholine vesicles is controlled by the mean number of membrane-bound antibiotic molecules per liposome, <A>. The transition from a monomeric to an oligomeric state of the antibiotic, which is associated with a sharp increase in nystatin mean fluorescence lifetime from approximately 7-10 to 35 ns, begins to occur at a critical concentration of 10 nystatin molecules per lipid vesicle. To gain further information about the transverse location (degree of penetration) of the membrane-bound antibiotic molecules, the spin-labeled fatty acids (5- and 16-doxyl stearic acids) were used in depth-dependent fluorescence quenching experiments. The results obtained show that monomeric nystatin is anchored at the phospholipid/water interface and suggest that nystatin oligomerization is accompanied by its insertion into the membrane. Globally, the experimental data was quantitatively described by a cooperative partition model which assumes that monomeric nystatin molecules partition into the lipid bilayer surface and reversibly assemble into aggregates of 6 +/- 2 antibiotic molecules.

FIGURE 1

Determination of the partition coefficient of nystatin from its fluorescence intensity increase upon incorporation into LUV of DPPC at 21°C. The nystatin concentrations used were (○) 3.2 and (•) 7.9 μM. The solid line is the best fit of Eq. 1 to the experimental data with K_p = (1.1 ± 0.3) × 10⁴.

FIGURE 2

Variation of (A) the steady-state fluorescence intensity, I_f (λ_exc = 304 nm; λ_em = 410 nm) and (B) light scattering intensity, I_d (λ_exc = λ_em = 450 nm), with nystatin concentration in (•) Tris and (○) HEPES buffers.

FIGURE 3

Relationship between the mean fluorescence lifetime of nystatin, 〈τ〉, and the phospholipid-to-antibiotic surface molar ratio, R_S, obtained with SUV of DPPC at 21°C. (A) The phospholipid concentration was kept constant (○) 1.1 mM, (•) 1.6 mM, (□) 2.3 mM, (▵) 2.4 mM, and (▴) 3.5 mM DPPC) while the antibiotic concentration was varied. (B) The nystatin concentration was kept constant (▾) 7.9 μM, (▪) 9.4 μM, and (▿) 11.0 μM antibiotic) while the phospholipid concentration was varied in each experiment. The dashed vertical lines define the range of R_S values (150 < R_S < 280) correspondent to the transition region between a long and short mean fluorescent lifetimes of nystatin.

FIGURE 4

Mean fluorescence lifetime of nystatin, 〈τ〉, at different (A) surface molar ratios of phospholipid-to-antibiotic, R_S, and (B) mean number of membrane bound antibiotic molecules per liposome, 〈A〉. The model membrane systems used were DPPC SUV (○) or LUV (•) at 21°C. In some experiments the phospholipid concentration was kept constant and the antibiotic concentration was varied and vice versa (see Fig. 3, legend). The solid line is the best fit of the cooperative partition model of the antibiotic to the experimental data (see the text and Fig. 8, legend, for details). (C, inset) Plot showing the relationship between the lowest value of the root-mean-square deviations (RMSD), and the aggregation number of nystatin, z. K′_ag was allowed to vary in each fitting while z was kept constant at different integer numbers (see Table 5).

FIGURE 5

Schematic representation of the expected transversal positions for the lipophilic probes 5- and 16-DS in a lipidic membrane. The dashed line represents the boundary between the polar and apolar regions of the membrane. Nystatin is represented perpendicular to the membrane surface just to illustrate that, upon internalization, its fluorophore group should get closer to both spin probes.

FIGURE 6

Stern-Volmer plots for the steady-state fluorescence quenching study of nystatin by 5- and 16-DS in 3 mM SUV of DPPC at 21°C. The nystatin concentrations used were (A) 3.0 and (B) 12.5 μM. Liposomes were prepared in Tris buffer. Lines are drawn just to guide the eye.

FIGURE 7

Schematic representation of the main stages of nystatin interaction with gel-phase lipid vesicles. (i) After nystatin addition to a liposome suspension, the monomeric antibiotic molecules partition into the membrane-water interface of the lipid vesicles. (ii) When the mean number of antibiotic molecules per liposome reaches a critical value (∼10 for gel-phase lipid vesicles), nystatin starts to self-associate. This process is accompanied by an increase in nystatin mean fluorescence lifetime from 7–10 ns to ∼35 ns. Concurrently, nystatin molecules translocate from the surface toward the interior of the lipid vesicles. The nystatin molecules that form an oligomer must shield their polar groups from contact with the acyl chains of the phospholipids.

FIGURE 8

Simulation of the effect of nystatin self-association upon the antibiotic binding to the lipid vesicles and its fluorescence emission decay kinetics. (A) Antibiotic mole fractions as a function of the bulk molar nystatin concentration added to the SUV suspension: (○) , (•) , and (▵) (calculated according to Eqs. 16–18). and are coincident. (B) Relationship between the partition coefficient of the antibiotic and its total concentration in solution. The dashed line depicts the value used for nystatin partition coefficient in this simulation ( = 9.2 × 10⁴), whereas (•) represent (calculated according to Eq. 20). (C) Mean fluorescence lifetime computed for nystatin, 〈τ〉_calc, at different mean number of membrane bound antibiotic molecules per liposome, 〈A〉. This last parameter was calculated according to Eq. 10 using either (○) or (•) in Eq. 9. The simulation of the cooperative partition model of nystatin was done considering that i), only monomeric antibiotic molecules partition into the liposomes ([L]_t = 1 mM; μ = 6,018; T = 21°C); and that ii), nystatin self-associates into hexamers (z = 6) with an association constant K_ag = 3.7 × 10¹¹¹. 〈τ〉_calc was computed according to Eq. 27 considering for monomeric nystatin α_M1 = 0.63; α_M2 = 0.36; α_M3 = 0.01; τ_M1 = 2.6 ns; τ_M2 = 8.1 ns e τ_M3 = 28 ns, and for the antibiotic molecules involved in aggregate formation α_Ag1 = 0.40; α_Ag2 = 0.10; α_Ag3 = 0.50; τ_Ag1 = 2.4 ns; τ_Ag2 = 14.0 ns e τ_Ag3 = 42 ns.