Lipid Lateral Organization on Giant Unilamellar Vesicles Containing Lipopolysaccharides

Abstract

We developed a new (to our knowledge) protocol to generate giant unilamellar vesicles (GUVs) composed of mixtures of single lipopolysaccharide (LPS) species and Escherichia coli polar lipid extracts. Four different LPSs that differed in the size of the polar headgroup (i.e., LPS smooth > LPS-Ra > LPS-Rc > LPS-Rd) were selected to generate GUVs composed of different LPS/E. coli polar lipid mixtures. Our procedure consists of two main steps: 1), generation and purification of oligolamellar liposomes containing LPSs; and 2), electroformation of GUVs using the LPS-containing oligolamellar vesicles at physiological salt and pH conditions. Analysis of LPS incorporation into the membrane models (both oligolamellar vesicles and GUVs) shows that the final concentration of LPS is lower than that expected from the initial E. coli lipids/LPS mixture. In particular, our protocol allows incorporation of no more than 15 mol % for LPS-smooth and LPS-Ra, and up to 25 mol % for LPS-Rc and LPS-Rd (with respect to total lipids). We used the GUVs to evaluate the impact of different LPS species on the lateral structure of the host membrane (i.e., E. coli polar lipid extract). Rhodamine-DPPE-labeled GUVs show the presence of elongated micrometer-sized lipid domains for GUVs containing either LPS-Rc or LPS-Rd above 10 mol %. Laurdan GP images confirm this finding and show that this particular lateral scenario corresponds to the coexistence of fluid disordered and gel (LPS-enriched)-like micron-sized domains, in similarity to what is observed when LPS is replaced with lipid A. For LPSs containing the more bulky polar headgroup (i.e., LPS-smooth and LPS-Ra), an absence of micrometer-sized domains is observed for all LPS concentrations explored in the GUVs (up to ∼15 mol %). However, fluorescence correlation spectroscopy (using fluorescently labeled LPS) and Laurdan GP experiments in these microscopically homogeneous membranes suggests the presence of LPS clusters with dimensions below our microscope's resolution (∼380 nm radial). Our results indicate that LPSs can cluster into gel-like domains in these bacterial model membranes, and that the size of these domains depends on the chemical structure and concentration of the LPSs.

Blood-Borne Lipopolysaccharide Is Rapidly Eliminated by Liver Sinusoidal Endothelial Cells via High-Density Lipoprotein

“…After sonication, LPS was double oxidized with galactose oxidase (57) followed by NaIO4 (58). The oxidized LPS was dialyzed using 2 kDa dialysis cassette against PBS (pH 7.4) buffer overnight with a minimum of 4 volume exchanges.…”

Impact of bacterial endotoxin on the structure of DMPC membranes

“…Multiple earlier studies have focused on the specific binding of LPS by ordered sphingomyelin/cholesterol-rich domains, so called rafts [11,12,30]. It was shown that in ternary systems which offer classical phospholipid bilayers next to rafts, LPS will partition with preference for the raft structures [11,12,30].…”

“…It was shown that in ternary systems which offer classical phospholipid bilayers next to rafts, LPS will partition with preference for the raft structures [11,12,30]. This selectivity may be induced by the highly saturated alkyl chains of the membrane-integrated part of the LPS leading to a preferred association with the more ordered rafts [11].…”

“…It has been reported to insert spontaneously into giant unilamellar vesicles generating shape changes and vesicle fission [11]. Further, Kubiak et al have shown that, in vesicles of Escherichia coli lipid extract, LPS causes an expansion of gel-like areas which strongly supports their association with LPS [12]. In general, TLR-independent interactions between LPS and lipid membranes as well as rafts are of high interest in the pathophysiology and genesis of sepsis and septic shock and may explain LPS-toxicity for cells missing TLR.…”

Asymmetric phospholipid: lipopolysaccharide bilayers; a Gram-negative bacterial outer membrane mimic

“…Studies have ranged from examining the formation, structure and physiochemical properties of LPS-containing vesicles in solution [8,9] and in dry and hydrated powders [10]. Neutron and X-ray diffraction studies have been used to examine suspensions and stacked bilayers of both smooth and rough LPS types [11,12].…”