The potent effect of mycolactone on lipid membranes

Abstract

Mycolactone is a lipid-like endotoxin synthesized by an environmental human pathogen, Mycobacterium ulcerans, the causal agent of Buruli ulcer disease. Mycolactone has pleiotropic effects on fundamental cellular processes (cell adhesion, cell death and inflammation). Various cellular targets of mycolactone have been identified and a literature survey revealed that most of these targets are membrane receptors residing in ordered plasma membrane nanodomains, within which their functionalities can be modulated. We investigated the capacity of mycolactone to interact with membranes, to evaluate its effects on membrane lipid organization following its diffusion across the cell membrane. We used Langmuir monolayers as a cell membrane model. Experiments were carried out with a lipid composition chosen to be as similar as possible to that of the plasma membrane. Mycolactone, which has surfactant properties, with an apparent saturation concentration of 1 μM, interacted with the membrane at very low concentrations (60 nM). The interaction of mycolactone with the membrane was mediated by the presence of cholesterol and, like detergents, mycolactone reshaped the membrane. In its monomeric form, this toxin modifies lipid segregation in the monolayer, strongly affecting the formation of ordered microdomains. These findings suggest that mycolactone disturbs lipid organization in the biological membranes it crosses, with potential effects on cell functions and signaling pathways. Microdomain remodeling may therefore underlie molecular events, accounting for the ability of mycolactone to attack multiple targets and providing new insight into a single unifying mechanism underlying the pleiotropic effects of this molecule. This membrane remodeling may act in synergy with the other known effects of mycolactone on its intracellular targets, potentiating these effects.

Fig 1. Adsorption of mycolactone at the air/buffer interface.

Equilibrium surface pressures (π_e) reached at the end of the adsorption kinetics for different concentrations of mycolactone injected into the subphase (PBS pH 7.4). Each point corresponds to the mean value of three kinetic experiments. The surface saturation concentration (1 μM) was determined at the start of the plateau. Inset: BAM images at equilibrium surface pressure (π_e), after the injection of mycolactone at a final concentration of 0.6 μM (a), 1.2 μM (b) or 6 μM (c). Image scale: 483 × 383 μm². All measurements were repeated at least three times for each concentration, with a satisfactory reproducibility, and the mean values are presented here.

Fig 2. Surface pressure (π)–molecular area (A) isotherms and corresponding BAM images of monolayers with and without cholesterol.

(A) π-A isotherms of mixture 1 (39% POPC, 33% SM, 9% POPE, 19% Chol given in mol%) recorded at 20°C (solid line) or 25°C (dashed line). (B) π-A isotherms of mixture 2 (48% POPC, 41% SM, 11% POPE given in mol%) recorded at 20°C (dashed-dotted line) or 25°C (dotted line). (C) Comparison of isotherms of the above-mentioned monolayers. Isotherms were recorded on PBS subphase (pH 7.4). Each isotherm corresponds to the mean of three experiments. BAM images were recorded during compression of the monolayer at a constant rate of 0.045 nm².molecule^-1.min^-1. Images A and B were recorded at 20°C. The images obtained for C were identical for 20 and 25°C. The estimated error for monolayers is ±0.05 mN/m for π and ≤ 0.01 nm² for (A). Image scale: 483 × 383 μm².

Fig 3. Effect of mycolactone on the lipid organization of mixed monolayers in the presence of cholesterol.

BAM images of 39% POPC, 33% SM, 9% POPE, 19% Chol (mixture 1) monolayers after the injection (4.45 μL) of ethanol (row a) or mycolactone (row b) into the PBS subphase (pH 7.4) beneath the interfacial film compressed at an initial surface pressure of 30 mN/m at 20° (A) or 25°C (B). The injection was performed after a relaxation time of one hour, with the surface area kept constant (mobile barriers were stopped). The final concentration of mycolactone was 60 nM. Image scale: 483 × 383 μm².

Fig 4. Effect of mycolactone on the lipid organization of mixed monolayers without cholesterol.

BAM images of 48% POPC, 41% SM, 11% POPE (mixture 2) monolayers after the injection (4.45 μL) of ethanol (row a) or mycolactone (row b) in the PBS subphase (pH 7.4) beneath the interfacial film compressed at an initial surface pressure of 30 mN/m at 20°C (A) or 25°C (B). The injection was performed after a relaxation time of one hour, with the surface area kept constant (mobile barriers were stopped). The final concentration of mycolactone was 60 nM. Image scale: 483 × 383 μm².

Fig 5. Interaction of mycolactone with mixed monolayers in the presence or absence of cholesterol.

Adsorption kinetics (π-t) curves of mycolactone on monolayers composed of (A) mixture 1 (39% POPC, 33% SM, 9% POPE, 19% Chol) or (B) mixture 2 (48% POPC, 41% SM, 11% POPE) at 20°C (solid line) or 25°C (dashed line). Mycolactone was injected (4.45 μL) into the PBS subphase (pH 7.4) beneath the monolayer compressed at an initial surface pressure π_i of 30 mN/m after a relaxation time of one hour (arrows). Surface area was kept constant during the run. The final concentration of mycolactone was 60 nM. Each measurement was performed at least three times for each condition, and a representative curve is presented here.

Fig 6. Influence of lipid packing on the membrane-binding properties of mycolactone.

Change in surface pressure (Δπ, mN/m) when mycolactone interacts with mixed monolayers at different initial surface pressures (π_i, mN/m). The nature of the lipid membrane was as follows: (A) and (B) mixture 1 consisting of 39% POPC, 33% SM, 9% POPE, 19% Chol. (C) and (D) mixture 2 consisting of 48% POPC, 41% SM, 11% POPE (given in mol%). Experiments were performed at 20°C (A) and (C), or 25°C (B) and (D). Each point corresponds to an independent measurement with a new lipid monolayer formed on PBS subphase (pH 7.4). The final concentration of mycolactone was 60 nM. Representative data from two or three independent assays are shown.

Fig 7. Influence of mycolactone interaction on the distribution of the liquid-ordered (L_o) phase in the membrane.

Fluorescence images of monolayers consisting of 39% POPC, 33% SM, 9% POPE, 19% Chol and including 0.5% BODIPY-cholesterol (TopFluor Cholesterol), after the injection (4.45 μL) of ethanol (row a) or mycolactone (row b) into the PBS subphase (pH 7.4) beneath the interfacial film compressed at an initial surface pressure of 30 mN/m at 20°C. The injection was performed after a relaxation time of one hour, and surface area was kept constant (mobile barriers were stopped). The final concentration of mycolactone was 60 nM. Scale bar: 50 μm.

Fig 8. Detergent effect on mixed monolayers in the presence of cholesterol.

Adsorption kinetics (π-t) curves of detergent on monolayers consisting of 39% POPC, 33% SM, 9% POPE, 19% Chol (mixture 1) at 20°C, and the corresponding BAM images. (A) Tween 20 or Triton X-100 was injected into the PBS subphase (pH 7.4) beneath the monolayer at a final concentration of 60 nM. (B) Tween 20 or Triton X-100 was injected into the PBS subphase (pH 7.4) beneath the monolayer at a constant final “Active concentration/CMC ratio” of 0.06. (C) BAM images corresponding to the adsorption kinetics (π-t) curves after the injection of Tween 20 (b) and (d), or Triton X-100 (c) and (e), at a constant final concentration of 60 nM (b) and (c), or an “Active concentration/CMC ratio” of 0.06 (d) and (e). (a) Images recorded after the injection of a 60nM mycolactone (final concentration). In all experiments, the monolayer was compressed at an initial surface pressure π_i of 30 mN/m and detergents were injected after a relaxation time of one hour (arrows). Surface area was kept constant during the run. Each measurement was performed at least three times for each condition, and a representative curve is presented here. Image scale: 483 × 383 μm².