Lipopolysaccharide confinement in the bacterial outer membrane is governed by interactions within the conserved Lipid A anchor

TL;DR

This study reveals how lipopolysaccharide (LPS) is confined in the bacterial outer membrane through specific interactions in the Lipid A anchor, which is crucial for the membrane's function.

Contribution

The study identifies the biophysical mechanisms behind LPS confinement in Gram-negative bacteria, including the role of divalent cations and hydrophobic interactions.

Findings

LPS confinement is independent of the oligosaccharide domain structure and is conserved across multiple bacterial species.

Divalent cations, especially Mg2+, significantly influence LPS lateral mobility more than Ca2+.

Disruption of LPS-LPS interactions leads to a new sub-population of freely diffusing LPS molecules.

Abstract

The outer membrane of Gram-negative bacteria is an asymmetric bilayer in which lipopolysaccharide (LPS), the principal component of the outer leaflet, promotes tight packing and ordering of the membrane components that are essential for the barrier and load-bearing functions of this membrane. Lipopolysaccharide mobility is known to be restricted in the outer membrane, but this confinement and the underlying biophysical interactions responsible remain to be fully characterized. Here, we apply a bio-orthogonal strategy for in situ site-specific fluorescent labeling of LPS. Using fluorescence microscopy, we quantify LPS lateral confinement in the outer membrane of Escherichia coli and demonstrate that this confinement is independent of oligosaccharide domain structure. We show that lipopolysaccharide assembles into discrete supramolecular structures, and that restricted lateral mobility arises from a combination of divalent cation-mediated electrostatic interactions in the anionic Lipid A headgroup, and hydrophobic interactions between acyl chains within the lipid milieu. Magnesium cations exert a greater influence than calcium cations on lipopolysaccharide lateral mobility. These traits are conserved across multiple pathogenic bacterial species, irrespective of O-antigen serotype, showing that lipopolysaccharide confinement is a ubiquitous feature of Gram-negative bacteria.

Lipopolysaccharide (LPS) mobility is known to be restricted in the bacterial outer membrane, but this confinement and the governing biophysical interactions have not been fully characterized. This study uses in vivo super-resolution imaging, single-particle tracking and FRAP microscopy to quantify LPS lateral confinement, delineating its dependence on lipopolysaccharide structure, membrane asymmetry, and divalent cations.

LPS lateral confinement is a ubiquitous trait of Gram-negative bacteria and is independent of differences in oligosaccharide domain length and capsular serotype.LPS-rich regions exist in the native outer membrane and are similar in size and frequency to previously described outer membrane protein-rich regions.Loss of outer membrane asymmetry and targeted disruption of intermolecular LPS-LPS interactions by chelator treatments reduce LPS confinement, producing a new sub-population of freely diffusing LPS molecules not observed in the native outer membrane.Mg2+ generates an outer membrane with a more significant degree of LPS confinement compared to Ca2+ irrespective of oligosaccharide domain length.

LPS lateral confinement is a ubiquitous trait of Gram-negative bacteria and is independent of differences in oligosaccharide domain length and capsular serotype.

LPS-rich regions exist in the native outer membrane and are similar in size and frequency to previously described outer membrane protein-rich regions.

Loss of outer membrane asymmetry and targeted disruption of intermolecular LPS-LPS interactions by chelator treatments reduce LPS confinement, producing a new sub-population of freely diffusing LPS molecules not observed in the native outer membrane.

Mg2+ generates an outer membrane with a more significant degree of LPS confinement compared to Ca2+ irrespective of oligosaccharide domain length.

LPS mobility in the outer membrane of Gram-negative bacteria depends on intermolecular Lipid A-mediated interactions, membrane asymmetry, and divalent cations.

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