Structure of supported bilayers composed of lipopolysaccharides and bacterial phospholipids: raft formation and implications for bacterial resistance

Abstract

Lipopolysaccharide (LPS), the major lipid on the surface of Gram-negative bacteria, plays a key role in bacterial resistance to hydrophobic antibiotics and antimicrobial peptides. Using atomic force microscopy (AFM) we characterized supported bilayers composed of LPSs from two bacterial chemotypes with different sensitivities to such antibiotics and peptides. Rd LPS, from more sensitive "deep rough" mutants, contains only an inner saccharide core, whereas Ra LPS, from "rough" mutants, contains a longer polysaccharide region. A vesicle fusion technique was used to deposit LPS onto either freshly cleaved mica or polyethylenimine-coated mica substrates. The thickness of the supported bilayers measured with contact-mode AFM was 7 nm for Rd LPS and 9 nm for Ra LPS, consistent with previous x-ray diffraction measurements. In water the Ra LPS bilayer surface was more disordered than Rd LPS bilayers, likely due to the greater volume occupied by the longer Ra LPS polysaccharide region. Since deep rough mutants contain bacterial phospholipid (BPL) as well as LPS on their surfaces, we also investigated the organization of Rd LPS/BPL bilayers. Differential scanning calorimetry and x-ray diffraction indicated that incorporation of BPL reduced the phase transition temperature, enthalpy, and average bilayer thickness of Rd LPS. For Rd LPS/BPL mixtures, AFM showed irregularly shaped regions thinner than Rd LPS bilayers by 2 nm (the difference in thickness between Rd LPS and BPL bilayers), whose area increased with increasing BPL concentration. We argue that the increased permeability of deep rough mutants is due to structural modifications caused by BPL to the LPS membrane, in LPS hydrocarbon chain packing and in the formation of BPL-enriched microdomains.

FIGURE 1

Schematic representation of the chemical structures of (A) an LPS molecule, (B) a PEI monomer, and (C) an LPS bilayer on a PEI-coated mica substrate. In (A) the lipid acyl chains of the lipid A moiety are represented by jagged lines and the sugar residues of the LPS polysaccharide cores and the O-specific chain are represented by hexagons. Rd LPS contains only the inner saccharide core, whereas the larger Ra LPS contains both the inner and outer saccharide cores.

FIGURE 2

AFM images of Rd LPS bilayers on bare mica (A) scanned in water (fully hydrated condition) and (B) scanned in air (partially hydrated condition). Image size is 5 μm² and z-scale is 30 nm. In all images lower surfaces are darker. For each image, a section analysis along the horizontal line in the image is shown below the image, with the arrow on the left pointing to the bare mica surface and the arrow on the right pointing to a typical Rd LPS region. From the section analysis the thickness of the Rd LPS is measured to be ∼7 nm in each image. The Rd LPS bilayer coverage values are estimated to be 86% in water (A) and 36% in air (B).

FIGURE 3

AFM image of PEI-coated mica surface, image size is 5 μm² and z-scale is 20 nm. A central 1-μm² defect was created by the AFM tip at contact mode with a high force load and a high scan speed. A section analysis along the line in the image (shown below the image) gives the thickness of the PEI layer as ∼1.2 nm.

FIGURE 4

AFM images of Rd LPS bilayers formed on PEI-coated mica (A) scanned in water and (B) scanned in air. Image size, 5 μm²; z-scale, 30 nm. For each image, a section analysis along the horizontal line in the image is shown below the image, with the arrow on the left pointing to the PEI-coated mica surface and the arrow on the right pointing to a typical LPS bilayer region. From the section analysis the thickness of the bilayer is measured to be ∼7 nm in each image. The Rd LPS bilayer coverage values on PEI-coated mica are estimated to be 92% in water and 67% in air. The highest peaks in the section analysis of Rd LPS have a height of 14 nm and probably correspond to a double bilayer region.

FIGURE 5

AFM images of Ra LPS bilayers formed on PEI-coated mica by 4 h SUV deposition at 60°C: (A) scanned in water and (B) scanned in air. Image size: 5 × 5 μm, z-scale: 30 nm. For each image, a section analysis along the horizontal line in the image is shown below the image, with the lower arrow pointing to the PEI-coated mica surface and the upper arrow pointing to a typical LPS bilayer region. From the section analysis the thickness of the bilayer is measured to be ∼9 nm in each image.

FIGURE 6

DSC thermograms of Rd LPS (3 mg/ml) and equimolar Rd LPS/BPL (4 mg/ml) MLVs. Note that the two thermograms are shown on different vertical scales. The arrow points in the direction of an endothermic transition.

FIGURE 7

AFM images of (A) 1:1 Rd LPS/BPL and (B) 2:1 Rd LPS/BPL formed on PEI-coated mica scanned in water at 20°C. Image size, 2 μm²; z-scale, 20 nm. For each image, regions of two different thicknesses are observed. A section analysis along the horizontal line in the image is shown below the image, indicating that the difference in height of the two regions is ∼2 nm.

FIGURE 8

AFM images of (A) 1:1 Rd LPS/BPL and (B) 2:1 Rd LPS/BPL bilayers formed on PEI-coated mica, scanned in water at 40°C. Image size, 2 μm²; z-scale, 20 nm. For each image, a section analysis along the horizontal line in the image is shown below the image.

FIGURE 9

Relationship between the predicted and observed BPL area in Rd LPS/BPL supported bilayers at 20°C and 40°C. Error bars represent standard deviations for three to five different AFM scans, and the dotted line is a least-squares fit to the 20°C values (R² = 0.986).