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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

Binding isotherms showing changes in initial fluorescence intensity (cf.Figure 1) for three types of smooth LPS from S. enterica, (squares) K. pneumoniae (triangles) and E. coli (circles) upon binding to FPE-labelled PC100 (dashed lines) or PC55SM15Chol30 (solid lines) phospholipid vesicles.(A). In each case, the average of three repeats was used and the values are shown in comparisons to the data obtained for studies with PC100. Histograms of Bmax and Kd values, corresponding to (A) but obtained from non-normalized data, are shown in panels (B) and (C), respectively and summarised in Table 1. The values of Kd and Bmax for E. coli LPS are approximated from the fits, as Kd is greater than the concentration range investigated experimentally. Tolerances in Kd and Bmax arise from fitting the data to Equation 1, while error bars in (A) show variance between runs.
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pone-0038677-g002: Binding isotherms showing changes in initial fluorescence intensity (cf.Figure 1) for three types of smooth LPS from S. enterica, (squares) K. pneumoniae (triangles) and E. coli (circles) upon binding to FPE-labelled PC100 (dashed lines) or PC55SM15Chol30 (solid lines) phospholipid vesicles.(A). In each case, the average of three repeats was used and the values are shown in comparisons to the data obtained for studies with PC100. Histograms of Bmax and Kd values, corresponding to (A) but obtained from non-normalized data, are shown in panels (B) and (C), respectively and summarised in Table 1. The values of Kd and Bmax for E. coli LPS are approximated from the fits, as Kd is greater than the concentration range investigated experimentally. Tolerances in Kd and Bmax arise from fitting the data to Equation 1, while error bars in (A) show variance between runs.

Mentions: 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.


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)

Binding isotherms showing changes in initial fluorescence intensity (cf.Figure 1) for three types of smooth LPS from S. enterica, (squares) K. pneumoniae (triangles) and E. coli (circles) upon binding to FPE-labelled PC100 (dashed lines) or PC55SM15Chol30 (solid lines) phospholipid vesicles.(A). In each case, the average of three repeats was used and the values are shown in comparisons to the data obtained for studies with PC100. Histograms of Bmax and Kd values, corresponding to (A) but obtained from non-normalized data, are shown in panels (B) and (C), respectively and summarised in Table 1. The values of Kd and Bmax for E. coli LPS are approximated from the fits, as Kd is greater than the concentration range investigated experimentally. Tolerances in Kd and Bmax arise from fitting the data to Equation 1, while error bars in (A) show variance between runs.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0038677-g002: Binding isotherms showing changes in initial fluorescence intensity (cf.Figure 1) for three types of smooth LPS from S. enterica, (squares) K. pneumoniae (triangles) and E. coli (circles) upon binding to FPE-labelled PC100 (dashed lines) or PC55SM15Chol30 (solid lines) phospholipid vesicles.(A). In each case, the average of three repeats was used and the values are shown in comparisons to the data obtained for studies with PC100. Histograms of Bmax and Kd values, corresponding to (A) but obtained from non-normalized data, are shown in panels (B) and (C), respectively and summarised in Table 1. The values of Kd and Bmax for E. coli LPS are approximated from the fits, as Kd is greater than the concentration range investigated experimentally. Tolerances in Kd and Bmax arise from fitting the data to Equation 1, while error bars in (A) show variance between runs.
Mentions: 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.

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