Abstract
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.
Highlights
Gram-negative bacteria, co-evolving alongside human hosts, have adapted to occupying available ecological niches as extracellular symbiotes, facultative or true intracellular pathogens
LPS is released as endotoxin in oligomeric and monomeric form during outer membrane renewal in Gramnegatives and plays an important role in pathogen-host signalling in activating immune response to bacterial presence through Tolllike receptor 4, TLR4 [9,10]
To investigate the lipid specificity of LPS/membrane interactions and obtain quantitative measurements of the binding capacity of membranes for LPS, membranes of different composition were prepared with fluorescein phosphatidylethanolamine (FPE) at levels known not to have any influence on membrane interactions
Summary
Gram-negative bacteria, co-evolving alongside human hosts, have adapted to occupying available ecological niches as extracellular symbiotes, facultative or true intracellular pathogens. The host response to environmental stimuli, associated with bacterial presence, is governed by cell surface receptor-activated cascades [3,4]. An important role in signalling has been attributed to phase heterogeneities in the host cell membranes, such as lipid microdomains or rafts [5,6]. LPS is released as endotoxin in oligomeric and monomeric form during outer membrane renewal in Gramnegatives and plays an important role in pathogen-host signalling in activating immune response to bacterial presence through Tolllike receptor 4, TLR4 [9,10]. LPB facilitates LPS binding to the GPI-anchored receptor CD14 [12], which, in turn, stimulates TLR4 dimerisation and initiation of the cellular signalling cascade [13]. S. typhimurium, for example, produces LPS with an altered membrane-associated domain, lipid A, which reduces TNF-a expression by monocytes [15]
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