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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

Time dependent changes in fluorescence from di-8-ANEPPS-labelled phospholipid vesicles and from Jurkat cells exposed to a single concentration of S. enterica LPS.Panel (A) shows changes in di-8-ANEPPS fluorescence recorded over time from phospholipid vesicles and panel (B), form Jurkat cells; orange lines show membranes without lipid domains (either PC100 or MβCD-treated Jurkat cells). The PC55SM15Chol30 curve is normalised to PC100 and Jurkats fluorescence is normalised to MβCD-treated cells.
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pone-0038677-g006: Time dependent changes in fluorescence from di-8-ANEPPS-labelled phospholipid vesicles and from Jurkat cells exposed to a single concentration of S. enterica LPS.Panel (A) shows changes in di-8-ANEPPS fluorescence recorded over time from phospholipid vesicles and panel (B), form Jurkat cells; orange lines show membranes without lipid domains (either PC100 or MβCD-treated Jurkat cells). The PC55SM15Chol30 curve is normalised to PC100 and Jurkats fluorescence is normalised to MβCD-treated cells.

Mentions: The time-dependent insertion of LPS from S. enterica into the Jurkat lymphocyte cell membranes and into the cholesterol-depleted membranes (MβCD-treated) was investigated following changes in the fluorescence of membrane embedded di-8-ANEPPS and was compared to model systems PC55SM15Chol30 and PC100, respectively (Figure 6). Following addition of LPS, significantly longer equilibration times on the order of 30 minutes were required to achieve a fluorescence steady state in the live system by comparison to the model membrane system. Such observations may arise due to structures absent in a model system compared to that of the cellular system. This strategy has been employed previously in other systems and allows some discrimination between purely lipid-based interactions and those that may involve receptor systems (see e.g. Asawakarn et al. [28].


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)

Time dependent changes in fluorescence from di-8-ANEPPS-labelled phospholipid vesicles and from Jurkat cells exposed to a single concentration of S. enterica LPS.Panel (A) shows changes in di-8-ANEPPS fluorescence recorded over time from phospholipid vesicles and panel (B), form Jurkat cells; orange lines show membranes without lipid domains (either PC100 or MβCD-treated Jurkat cells). The PC55SM15Chol30 curve is normalised to PC100 and Jurkats fluorescence is normalised to MβCD-treated cells.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0038677-g006: Time dependent changes in fluorescence from di-8-ANEPPS-labelled phospholipid vesicles and from Jurkat cells exposed to a single concentration of S. enterica LPS.Panel (A) shows changes in di-8-ANEPPS fluorescence recorded over time from phospholipid vesicles and panel (B), form Jurkat cells; orange lines show membranes without lipid domains (either PC100 or MβCD-treated Jurkat cells). The PC55SM15Chol30 curve is normalised to PC100 and Jurkats fluorescence is normalised to MβCD-treated cells.
Mentions: The time-dependent insertion of LPS from S. enterica into the Jurkat lymphocyte cell membranes and into the cholesterol-depleted membranes (MβCD-treated) was investigated following changes in the fluorescence of membrane embedded di-8-ANEPPS and was compared to model systems PC55SM15Chol30 and PC100, respectively (Figure 6). Following addition of LPS, significantly longer equilibration times on the order of 30 minutes were required to achieve a fluorescence steady state in the live system by comparison to the model membrane system. Such observations may arise due to structures absent in a model system compared to that of the cellular system. This strategy has been employed previously in other systems and allows some discrimination between purely lipid-based interactions and those that may involve receptor systems (see e.g. Asawakarn et al. [28].

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