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Receptor-independent interaction of bacterial lipopolysaccharide with lipid and lymphocyte membranes; the role of cholesterol.

Ciesielski F, Davis B, Rittig M, Bonev BB, O'Shea P - PLoS ONE (2012)

Bottom Line: LPS preparations from Klebsiella pneumoniae and Salmonella enterica were found to bind preferentially to mixed lipid membranes by comparison to pure PC bilayers.Insertion of LPS into model membranes confirmed the preference for sphingomyelin/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.

View Article: PubMed Central - PubMed

Affiliation: School of Biomedical Sciences, University of Nottingham, Nottingham, United Kingdom.

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 sphingomyelin/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.

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Related in: MedlinePlus

Excitation-emission fluorescence spectra of FPE-labelled phospholipid vesicles (top) before (dashed line) and after (solid line) LPS titration.Arrow indicates a small peak from residual, free FPE in solution; lower panel represents part of the LPS titration curve recorded over time for FPE-labelled PC55SM15Chol30 phospholipid vesicles – initial drop, A, is followed by signal re-equilibration, B. Binding curves (Figure 2) are obtained from measuring changes between the initial signal and the equilibrium state, C. Inset shows titration curve measured for pure PC100 vesicles, with significantly smaller difference between the initial binding and re-equilibration stages.
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pone-0038677-g001: Excitation-emission fluorescence spectra of FPE-labelled phospholipid vesicles (top) before (dashed line) and after (solid line) LPS titration.Arrow indicates a small peak from residual, free FPE in solution; lower panel represents part of the LPS titration curve recorded over time for FPE-labelled PC55SM15Chol30 phospholipid vesicles – initial drop, A, is followed by signal re-equilibration, B. Binding curves (Figure 2) are obtained from measuring changes between the initial signal and the equilibrium state, C. Inset shows titration curve measured for pure PC100 vesicles, with significantly smaller difference between the initial binding and re-equilibration stages.

Mentions: LPS has been shown to insert spontaneously into lipid bilayers and can lead to membrane breakdown at high concentrations [19]. 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. LPS was added from aqueous solution to large unilamellar vesicle suspensions and fluorescence spectra were recorded. The integrity of FPE-labelled vesicles was assessed by comparing excitation-emission spectra acquired before and after LPS addition (Figure 1a). No major changes in spectral line shape were observed, upon the LPS addition. Changes of the net fluorescence were observed to take place due to the molecular binding reactions and are in accordance with the established mode of action of the FPE reporting system [20], [23]. As the there are no concomitant or slower changes of the spectrum however this indicates that the molecular environment of the FPE is not changed and so the membrane structure is not modified by the interaction of FPE. This indicates that LPS does not disrupt the liposomal membranes over the concentration ranges employed in this study.


Receptor-independent interaction of bacterial lipopolysaccharide with lipid and lymphocyte membranes; the role of cholesterol.

Ciesielski F, Davis B, Rittig M, Bonev BB, O'Shea P - PLoS ONE (2012)

Excitation-emission fluorescence spectra of FPE-labelled phospholipid vesicles (top) before (dashed line) and after (solid line) LPS titration.Arrow indicates a small peak from residual, free FPE in solution; lower panel represents part of the LPS titration curve recorded over time for FPE-labelled PC55SM15Chol30 phospholipid vesicles – initial drop, A, is followed by signal re-equilibration, B. Binding curves (Figure 2) are obtained from measuring changes between the initial signal and the equilibrium state, C. Inset shows titration curve measured for pure PC100 vesicles, with significantly smaller difference between the initial binding and re-equilibration stages.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3369841&req=5

pone-0038677-g001: Excitation-emission fluorescence spectra of FPE-labelled phospholipid vesicles (top) before (dashed line) and after (solid line) LPS titration.Arrow indicates a small peak from residual, free FPE in solution; lower panel represents part of the LPS titration curve recorded over time for FPE-labelled PC55SM15Chol30 phospholipid vesicles – initial drop, A, is followed by signal re-equilibration, B. Binding curves (Figure 2) are obtained from measuring changes between the initial signal and the equilibrium state, C. Inset shows titration curve measured for pure PC100 vesicles, with significantly smaller difference between the initial binding and re-equilibration stages.
Mentions: LPS has been shown to insert spontaneously into lipid bilayers and can lead to membrane breakdown at high concentrations [19]. 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. LPS was added from aqueous solution to large unilamellar vesicle suspensions and fluorescence spectra were recorded. The integrity of FPE-labelled vesicles was assessed by comparing excitation-emission spectra acquired before and after LPS addition (Figure 1a). No major changes in spectral line shape were observed, upon the LPS addition. Changes of the net fluorescence were observed to take place due to the molecular binding reactions and are in accordance with the established mode of action of the FPE reporting system [20], [23]. As the there are no concomitant or slower changes of the spectrum however this indicates that the molecular environment of the FPE is not changed and so the membrane structure is not modified by the interaction of FPE. This indicates that LPS does not disrupt the liposomal membranes over the concentration ranges employed in this study.

Bottom Line: LPS preparations from Klebsiella pneumoniae and Salmonella enterica were found to bind preferentially to mixed lipid membranes by comparison to pure PC bilayers.Insertion of LPS into model membranes confirmed the preference for sphingomyelin/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.

View Article: PubMed Central - PubMed

Affiliation: School of Biomedical Sciences, University of Nottingham, Nottingham, United Kingdom.

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 sphingomyelin/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.

Show MeSH
Related in: MedlinePlus