Lipopolysaccharide lateral mobility in the Gram-negative bacterial outer membrane is confined and governed by interactions within the conserved Lipid A anchor

The Gram-negative bacterial cell envelope is defined by an asymmetric outer membrane where the outer leaflet adopts a highly ordered structure composed principally of lipopolysaccharide molecules. The organisation and dynamics of these glycolipids are key to the ability of the outer membrane to act as an innate barrier against chemical and antibiotic challenges, and as a load bearing element for the cell. Strong intermolecular forces are thought to govern the lateral diffusion of lipopolysaccharide in the outer membrane, but the extent and molecular basis of this diffusion has remained a controversial topic for over 50 years. Here we use a bio-orthogonal labelling strategy and in vivo fluorescence microscopy to unequivocally demonstrate extreme lateral confinement of lipopolysaccharide in the outer membrane of Escherichia coli , regardless of carbohydrate domain size and structure. We specifically identify magnesium cation-mediated interactions at the base of the carbohydrate and hydrophobic interactions within the lipid milieu as critical for lipopolysaccharide confinement. Importantly, these traits are conserved across multiple pathogenic species irrespective of O-antigen and capsular serotype. Together, these findings establish lipopolysaccharide endotoxin lateral confinement as a ubiquitous feature of the outer membrane and highlight potential universal vulnerabilities of the bacterial cell envelope.