Effect of divalent cation removal on the structure of gram-negative bacterial outer membrane models

Abstract

The Gram-negative bacterial outer membrane (GNB-OM) is asymmetric in its lipid composition with a phospholipid-rich inner leaflet and an outer leaflet predominantly composed of lipopolysaccharides (LPS). LPS are polyanionic molecules, with numerous phosphate groups present in the lipid A and core oligosaccharide regions. The repulsive forces due to accumulation of the negative charges are screened and bridged by the divalent cations (Mg(2+) and Ca(2+)) that are known to be crucial for the integrity of the bacterial OM. Indeed, chelation of divalent cations is a well-established method to permeabilize Gram-negative bacteria such as Escherichia coli. Here, we use X-ray and neutron reflectivity (XRR and NR, respectively) techniques to examine the role of calcium ions in the stability of a model GNB-OM. Using XRR we show that Ca(2+) binds to the core region of the rough mutant LPS (RaLPS) films, producing more ordered structures in comparison to divalent cation free monolayers. Using recently developed solid-supported models of the GNB-OM, we study the effect of calcium removal on the asymmetry of DPPC:RaLPS bilayers. We show that without the charge screening effect of divalent cations, the LPS is forced to overcome the thermodynamically unfavorable energy barrier and flip across the hydrophobic bilayer to minimize the repulsive electrostatic forces, resulting in about 20% mixing of LPS and DPPC between the inner and outer bilayer leaflets. These results reveal for the first time the molecular details behind the well-known mechanism of outer membrane stabilization by divalent cations. This confirms the relevance of the asymmetric models for future studies of outer membrane stability and antibiotic penetration.

Figure 1

A comparison of the X-ray reflectometry profiles and model data fits (A) and the scattering length density profiles these fits describe (B) for air/liquid interface containing an RaLPS monolayer held at 35 mM m^–1 in the presence of 20 mM HEPES pH 7.2 H₂O buffer with either 5 mM CaCl₂ (red) or 3 mM EDTA (blue). The air/liquid interface was set to be between the tails and inner-core region of the LPS monolayer.

Figure 2

A GIXD contour plot obtained from an air/liquid interface containing an RaLPS monolayer held at 35 mM m^–1 in the presence of 20 mM HEPES pH 7.2 H₂O buffer with 5 mM CaCl₂ (A). A plot of this data integrated over Q_z is shown (B).

Figure 3

Neutron reflectometry profile and model data fits (A–C) and the scattering length density profiles these fits describe (D) for asymmetrically deposited DPPC (inner leaflet):RaLPS (outer leaflet) bilayer in the presence of 20 mM HEPES pH 7.2 buffer with 5 mM CaCl₂. The six simultaneously fitted isotopic contrasts shown are (A) d-DPPC/RaLPS in D₂O (red line), h-DPPC/RaLPS in D₂O (blue line); (B) d-DPPC/RaLPS in SMW (black line), h-DPPC/RaLPS in SMW (gray line); and (C) d-DPPC/RaLPS in H₂O (green line), h-DPPC/RaLPS in H₂O (purple line).

Figure 4

Neutron reflectometry profile and model data fits (A–C) and the scattering length density profiles these fits describe (D) for asymmetrically deposited DPPC (inner leaflet):RaLPS (outer leaflet) bilayer in the presence of 20 mM HEPES pH 7.2 buffer with 3 mM EDTA. The six simultaneously fitted isotopic contrasts shown are (A) d-DPPC/RaLPS in D₂O (red line), h-DPPC/RaLPS in D₂O (blue line); (B) d-DPPC/RaLPS in SMW (black line), h-DPPC/RaLPS in SMW (gray line); and (C) d-DPPC/RaLPS in H₂O (green line), h-DPPC/RaLPS in H₂O (purple line).

Figure 5

A comparison of the neutron reflectometry profile and model data fits (A) and the scattering length density profiles these fits describe (B) for asymmetrically deposited d-DPPC (inner leaflet):RaLPS (outer leaflet) bilayer in the presence of 20 mM HEPES pD 7.2 D₂O buffer with either 5 mM CaCl₂ (red) or 3 mM EDTA (blue). A pictorial representation of the bilayer structure before and following Ca²⁺ sequestration by EDTA is shown (C).