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 (Mg2+ and Ca2+) 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 Ca2+ 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.

Understanding the electrostatic interactions between bacterial membranes and exogenous proteins is crucial to designing effective antimicrobial agents against Gram-negative bacteria. Here we study, using neutron reflecometry under multiple isotopic contrast conditions, the role of the uncharged sugar groups in the outer core region of lipopolysaccharide (LPS) in protecting the phosphate-rich inner core region from electrostatic interactions with antimicrobial proteins. Models of the asymmetric Gram negative outer membrane on silicon were prepared with phopshatidylcholine (PC) in the inner leaflet (closest to the silicon), whereas rough LPS was used to form the outer leaflet (facing the bulk solution). We show how salt concentration can be used to reversibly alter the binding affinity of a protein antibiotic colicin N (ColN) to the anionic LPS confirming that the interaction is electrostatic in nature. By examining the interaction of ColN with two rough LPS types with different-sized core oligosaccharide regions we demonstrate the role of uncharged sugars in blocking short-range electrostatic interactions between the cationic antibiotics and the vulnerable anionic phosphate groups.

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Lipopolysaccharide (LPS) is a major component of the external leaflet of bacterial outer membranes, key pro-inflammatory factor and an important mediator of host-pathogen interactions. In host cells it activates the complement along with a pro-inflammatory response via a TLR4-mediated signalling cascade and shows preference for cholesterol-containing membranes. Here, we use solid state (13)C and (31)P MAS NMR to investigate the interactions of LPS from three bacterial species, Brucella melitensis, Klebsiella pneumoniae and Escherichia coli, with mixed lipid membranes, raft models. All endotoxin types are found to be pyrophosphorylated and Klebsiellar LPS is phosphonylated, as well. Carbon-13 MAS NMR indicates an increase in lipid order in the presence of LPS. Longitudinal (31)P relaxation, providing a direct probe of LPS molecular and segmental mobility, reveals a significant reduction in (31)P T1 times and lower molecular mobility in the presence of ternary lipid mixtures. Along with the ordering effect on membrane lipid, this suggests a preferential partitioning of LPS into ordered bilayer sphingomyelin/cholesterol-rich domains. We hypothesise that this is an important evolutionary drive for the selection of GPI-anchored raft-associated LPS-binding proteins as a first line of response to membrane-associated LPS.

Lipopolysaccharide (LPS) is a major constituent of bacterial outer membranes where it makes up the bulk of the outer leaflet and plays a key role as determinant of bacterial interactions with the host. Membrane-free LPS is known to activate T-lymphocytes through interactions with Toll-like receptor 4 via multiprotein complexes. In the present study, we investigate the role of cholesterol and membrane heterogeneities as facilitators of receptor-independent LPS binding and insertion, which underpin bacterial interactions with the host in symbiosis, pathogenesis and cell invasion. We use fluorescence spectroscopy to investigate the interactions of membrane-free LPS from intestinal Gram-negative organisms with cholesterol-containing model membranes and with T-lymphocytes. LPS preparations from Klebsiella pneumoniae and Salmonella enterica were found to bind preferentially to mixed lipid membranes by comparison to pure PC bilayers. The same was observed for LPS from the symbiote Escherichia coli but with an order of magnitude higher dissociation constant. Insertion of LPS into model membranes confirmed the preference for sphimgomyelin/cholesterol-containing systems. LPS insertion into Jurkat T-lymphocyte membranes reveals that they have a significantly greater LPS-binding capacity by comparison to methyl-β-cyclodextrin cholesterol-depleted lymphocyte membranes, albeit at slightly lower binding rates.

Direct observation of J-couplings remains a challenge in high-resolution solid-state NMR. In some cases, it is possible to use Lee-Goldburg (LG) homonuclear decoupling during rare spin observation in MAS NMR correlation spectroscopy of lipid membranes to obtain J-resolved spectra in the direct dimension. In one simple implementation, a wide line separation-type (13)C-(1)H HETCOR can provide high-resolution (1)H/(13)C spectra, which are J-resolved in both dimensions. Coupling constants, (1)J(HC), obtained from (1)H doublets, can be compared with scaled (1)J(θ)(CH)-values obtained from the (13)C multiplets to assess the LG efficiency and scaling factor. The use of homonuclear decoupling during proton evolution, LG-HETCOR-LG, can provide J-values, at least in the rare spin dimension, and allows measurements in less mobile membrane environments. The LG-decoupled spectroscopic approach is demonstrated on pure dioleoylphosphatidylcholine (DOPC) membranes and used to investigate lipid mixtures of DOPC/cholesterol and DOPC/cholesterol/sphingomyelin.

The molecular action of polyene macrolides with antifungal activity, amphotericin B and natamycin, involves recognition of sterols in membranes. Physicochemical and functional studies have contributed details to understanding the interactions between amphotericin B and ergosterol and, to a lesser extent, with cholesterol. Fewer molecular details are available on interactions between natamycin with sterols. We use solid state 13C MAS NMR to characterize the impact of amphotericin B and natamycin on mixed lipid membranes of DOPC/cholesterol or DOPC/ergosterol. In cholesterol-containing membranes, amphotericin B addition resulted in marked increase in both DOPC and cholesterol 13C MAS NMR linewidth, reflecting membrane insertion and cooperative perturbation of the bilayer. By contrast, natamycin affects little either DOPC or cholesterol linewidth but attenuates cholesterol resonance intensity preferentially for sterol core with lesser impact on the chain. Ergosterol resonances, attenuated by amphotericin B, reveal specific interactions in the sterol core and chain base. Natamycin addition selectively augmented ergosterol resonances from sterol core ring one and, at the same time, from the end of the chain. This puts forward an interaction model similar to the head-to-tail model for amphotericin B/ergosterol pairing but with docking on opposite sterol faces. Low toxicity of natamycin is attributed to selective, non-cooperative sterol engagement compared to cooperative membrane perturbation by amphotericin B.