Effect of fengycin, a lipopeptide produced by Bacillus subtilis, on model biomembranes

Abstract

Fengycin is a biologically active lipopeptide produced by several Bacillus subtilis strains. The lipopeptide is known to develop antifungal activity against filamentous fungi and to have hemolytic activity 40-fold lower than that of surfactin, another lipopeptide produced by B. subtilis. The aim of this work is to use complementary biophysical techniques to reveal the mechanism of membrane perturbation by fengycin. These include: 1), the Langmuir trough technique in combination with Brewster angle microscopy to study the lipopeptide penetration into monolayers; 2), ellipsometry to investigate the adsorption of fengycin onto supported lipid bilayers; 3), differential scanning calorimetry to determine the thermotropic properties of lipid bilayers in the presence of fengycin; and 4), cryogenic transmission electron microscopy, which provides information on the structural organization of the lipid/lipopeptide system. From these experiments, the mechanism of fengycin action appears to be based on a two-state transition controlled by the lipopeptide concentration. One state is the monomeric, not deeply anchored and nonperturbing lipopeptide, and the other state is a buried, aggregated form, which is responsible for membrane leakage and bioactivity. The mechanism, thus, appears to be driven mainly by the physicochemical properties of the lipopeptide, i.e., its amphiphilic character and affinity for lipid bilayers.

FIGURE 1

Primary structure of fengycin. R varies between 11 and 15.

FIGURE 2

(A) Surface pressure increase after injection of fengycin (final concentration: 27.5 × 10⁻⁷ mM) under a DPPC monolayer previously compressed at a defined initial surface pressure. Numbers in the figure correspond to defined steps discussed in the text. (B) BAM images of the interface (a) before injection of fengycin and (b) 5 min (corresponding to step 1 in A), (c) 10 min (corresponding to step 2 in A), and (d) 35 min (corresponding to step 4 in A) after injection of fengycin under the DPPC monolayer previously compressed at Π_i = 21 mN/m. Subphase is a 10 mM Tris, 150 mM NaCl buffer at pH 7.4. Temperature is 30°C.

FIGURE 3

Surface pressure increase after injection of fengycin under a DPPC monolayer previously compressed at a defined initial surface pressure. Subphase is a 10 mM Tris, 150 mM NaCl at pH 7.4. Temperature is 30°C. Fengycin is injected into the subphase under the DPPC monolayer at a defined position in one step to a final concentration of 27.5 × 10⁻⁷ M. The line represents the linear regression that best fits the experimental data. The intersection with the abscissa estimates the exclusion pressure (Π_ex).

FIGURE 4

Cryo-TEM image of DPPC vesicles after the heating-cooling cycle performed in the DSC experiments.

FIGURE 5

Second heating scan of fengycin and/or DPPC vesicles in a 10 mM Tris, 150 mM NaCl buffer at pH 7.4. (a) DPPC vesicles 1 mM; (b) DPPC/fengycin (300:1), [fengycin] = 3.3 μM; (c) DPPC/fengycin (37.5:1), [fengycin] = 25.8 μM; (d) DPPC/fengycin (10:1), [fengycin] = 96 μM; (e) DPPC/fengycin (2:1), [fengycin] = 0.48 mM; (f) fengycin at a concentration of 0.48 mM.

FIGURE 6

Upper graph: Adsorbed amount (open circles) and layer thickness (crosses) as a function of time for the DPPC/fengycin system. Bottom graphs: magnification of the interesting part of the experiment. (Left) Formation and stabilization of the bilayer. Arrows indicate the point at which the composition of the solution was changed. At 0 min, adsorption from 0.114 g/l of DDM/DPPC, (2) rinsing with Tris-NaCl, (3) adsorption from 0.0114 g/l of DDM/DPPC, (4) rinsing with Tris-NaCl, (5) adsorption from 0.00114 g/l of DDM/DPPC, (6) rinsing with Tris-NaCl. (Right) Effect of the increasing fengycin concentration. Arrows indicate the point at which fengycin is injected in the cuvette. Final concentration of fengycin in the cuvette is (7) 0.2 μM, (8) 2 μM, (9) 10 μM, (10) 20 μM, (11) 100 μM, and (12) 180 μM.

FIGURE 7

(Upper graph) Adsorbed amount (open circles) and layer thickness (crosses) as a function of time for the DOPC/fengycin system. (Lower graphs) Magnification of the interesting part of the experiment. (Left) Formation and stabilization of the bilayer. Arrows indicate the point at which the composition of the solution was changed. At 0 min, adsorption from 0.114 g/l of DDM/DOPC, (2) rinsing with Tris-NaCl, (3) adsorption from 0.0114 g/l of DDM/DOPC, (4) rinsing with Tris-NaCl, (5) adsorption from 0.00114 g/l of DDM/DOPC, (6) rinsing with Tris-NaCl. (Right) Effect of the increasing fengycin concentration. Arrows indicate the point at which fengycin is injected in the cuvette. Final concentration of fengycin in the cuvette is (7) 0.2 μM, (8) 2 μM, (9) 10 μM, (10) 20 μM, (11) 100 μM, and (12) 180 μM.

FIGURE 8

Cryo-TEM images (a) DPPC vesicles 5 mM; (b) fengycin 133 μM, the arrow points out, as an example, a grid border where fengycin micelles are adsorbed; (c) fengycin 2.425 mM; (d) DPPC/fengycin (500:1), [fengycin] = 10 μM; (e) DPPC/fengycin (37.5:1), [fengycin] = 133 μM; (f) DPPC/fengycin (2:1), [fengycin] = 2.425 mM. For (d–f), fengycin is incubated with DPPC vesicles at 50°C for 30 min. After this time the sample temperature is brought back to room temperature and the vitrification is operated from 25°C. (g) DPPC/fengycin (37.5:1), [fengycin] = 133 μM, fengycin is incubated with DPPC vesicles at room temperature for 30 min and the vitrification is operated from 25°C. (h) DPPC/fengycin (37.5:1), [fengycin] = 133 μM, fengycin is incubated with DPPC vesicles at 50°C for 30 min, and the vitrification is operated from 45°C.

FIGURE 9

Proposed model for the concentration-dependent mechanism of membrane perturbation by fengycin. (i), (ii), and (iii) and arrows: see text for explanation.