Lipopolysaccharide-induced dynamic lipid membrane reorganization: tubules, perforations, and stacks

Abstract

Lipopolysaccharide (LPS) is a unique lipoglycan, with two major physiological roles: 1), as a major structural component of the outer membrane of Gram-negative bacteria and 2), as a highly potent mammalian toxin when released from cells into solution (endotoxin). LPS is an amphiphile that spontaneously inserts into the outer leaflet of lipid bilayers to bury its hydrophobic lipidic domain, leaving the hydrophilic polysaccharide chain exposed to the exterior polar solvent. Divalent cations have long been known to neutralize and stabilize LPS in the outer membrane, whereas LPS in the presence of monovalent cations forms highly mobile negatively-charged aggregates. Yet, much of our understanding of LPS and its interactions with the cell membrane does not take into account its amphiphilic biochemistry and charge polarization. Herein, we report fluorescence microscopy and atomic force microscopy analysis of the interaction between LPS and fluid-phase supported lipid bilayer assemblies (sLBAs), as model membranes. Depending on cation availability, LPS induces three remarkably different effects on simple sLBAs. Net-negative LPS-Na(+) leads to the formation of 100-μm-long flexible lipid tubules from surface-associated lipid vesicles and the destabilization of the sLBA resulting in micron-size hole formation. Neutral LPS-Ca(2+) gives rise to 100-μm-wide single- or multilamellar planar sheets of lipid and LPS formed from surface-associated lipid vesicles. Our findings have important implications about the physical interactions between LPS and lipids and demonstrate that sLBAs can be useful platforms to study the interactions of amphiphilic virulence factors with cell membranes. Additionally, our study supports the general phenomenon that lipids with highly charged or bulky headgroups can promote highly curved membrane architectures due to electrostatic and/or steric repulsions.

Figure 1

Comparison of the chemical structure of lipopolysaccharide from E. coli serotype O111 and the phospholipid DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine). Partial covalent modifications, which may result in additional phosphate groups, are shown (dotted lines) and are dependent on growth conditions and other factors. Number of repeat units in the O-chain, n, ranges from 1 to 18. COL, colitose (3,6-dideoxy-L-xylo-hexose); GAL, galactopyranose; GLC, glucopyranose; GLCN, 2-amino-2-deoxyglucopyranose; HEP, L-glycero-D-manno-heptopyranose; KDO, 3-deoxy-D-manno-oct-2-ulopyranosonic acid; P, phosphate.

Figure 2

Lipid tubule formation induced by LPS in PBS. (A) AFM topographs showing a DOPC sLBA in PBS at low and high magnification. A height profile across the lower image (dashed white line) shows a relatively flat surface. (B) LSCM of the DOPC sLBA (doped with 0.5% green fluorescent lipids) in PBS before and after addition of 100 μg/mL LPS. Sequential images are shown at selected time periods after addition of the LPS. (C) Representative epifluorescence microscopy image of lipid tubules. (D) Representative TIRFM image showing long tubules. The background of green fluorescence suggested a homogenous lipid bilayer (note: 45° periodic noise is an optical artifact that should be ignored). To see this figure in color, go online.

Figure 3

Holes in sLBAs after LPS treatment and washing. (A) LSCM fluorescence images showing DOPC sLBAs after treatment with 100 μg/mL LPS in PBS followed by washing the surface (PBS, 10 changes). (B) Higher-magnification LSCM image. A profile of the fluorescence intensity (below) shows the SPC counts along a line drawn across the image (white dashed line). (C) A representative TIRFM image of a similar sample. (D) FRAP experiment from the sample in panel A. A circular region was photobleached and then sequential images acquired to show the lateral diffusion of fluorescent lipids. (E) AFM topograph showing accurate width and depth of holes induced by LPS, similar sample to panel A. Height profiles (below, red lines) show the height data (across white dashed lines) in the image, chosen to show the depth of holes in the lipid bilayer. (F) Higher magnification topograph from the field of holes in panel E. To see this figure in color, go online.

Figure 4

LPS concentration and time dependence on hole formation. LSCM fluorescence images of a DOPC sLBA treated for 20 min with LPS at varying concentrations of LPS in PBS: (A) 500 μg/mL, (B) 100 μg/mL, or (C) 20 μg/mL, and then washed with PBS, showing decreasing numbers of holes with LPS concentration. Parallel samples were treated with 5 μg/mL LPS in PBS for (D) 20 min, (E) 60 min, or (F) 180 min, and then washed and imaged immediately. More holes are observed with increased incubation time. These images were acquired at higher pixel density (4096 × 4096) to resolve small holes. (D–F, inset) Digitally magnified areas of these images showing small holes more clearly. To see this figure in color, go online.

Figure 5

LPS in Ca²⁺ buffer causes formation and growth of multilamellar stacks. (A) LSCM of the DOPC sLBA in Ca²⁺ buffer before and after addition of 100 μg/mL LPS in Ca²⁺ buffer. Sequential images are shown from selected time points after addition of the LPS. Fluorescent patches are observed in images immediately after LPS addition (10 s) and continue to grow in size over the following minutes (1–23 min). (B) Image at high magnification showing a patch of contiguous fluorescence of approximately double the intensity of the DOPC bilayer. (C) Image at a high magnification showing a fluorescent patch with multiple distinct step-changes in the intensity. Numbers (2), (4), and (6) indicate expected stacked bilayers with multiples of intensity of a single bilayer (1). (D) Fluorescence image after washing the LPS-Ca²⁺-treated surface with 10 changes of Ca²⁺ buffer. (Note: images in panel C acquired with lower exposure settings than in panels B and D, hence, lower fluorescence intensity.) To see this figure in color, go online.

Figure 6

Schematic of the mechanism of LPS-induced lipid bilayer deformation. See text for description. LPS is represented by a simplified molecular structure showing the hydrophobic domain with six fatty acid tails linked to core sugar units and the extended O-chain. Sugar units are represented by their cyclic rings. Note that, for clarity, side groups are not displayed and the polysaccharide chain is greatly shortened, represented by (…). Normal phospholipids are represented by their two fatty acid tails linked to a headgroup (green boxes represent BODIPY dye). Negatively-charged groups of LPS are represented by their charge symbols (blue). Cations that associate with LPS are represented by their elemental symbol and single or double charge (in red). Electrostatic repulsion is shown (Cyan lines). To see this figure in color, go online.