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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

Two stages of S. enterica LPS interaction with mixed PC55SM15Chol30 membranes.(A): initial binding (hexagons) and conformational re-equilibration (diamonds). Both datasets were normalized to the starting fluorescent intensities for LPS binding to PC100 and the values of Bmax are normalized to one for PC100 (B) and values for Kd are shown in (C). The average of three repeats is shown and fitted to Equation 1. Similar binding curves were obtained from K. pneumoniae LPS binding. The Bmax and Kd values for both types of LPS are shown in (B) and (C) and summarised in Table 2.
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pone-0038677-g003: Two stages of S. enterica LPS interaction with mixed PC55SM15Chol30 membranes.(A): initial binding (hexagons) and conformational re-equilibration (diamonds). Both datasets were normalized to the starting fluorescent intensities for LPS binding to PC100 and the values of Bmax are normalized to one for PC100 (B) and values for Kd are shown in (C). The average of three repeats is shown and fitted to Equation 1. Similar binding curves were obtained from K. pneumoniae LPS binding. The Bmax and Kd values for both types of LPS are shown in (B) and (C) and summarised in Table 2.

Mentions: Receptor-independent binding of LPS to membranes is important to opportunistic pathogens as a route to host invasion. To gain a better insight into this process, the equilibrium properties of initial binding and fluorescence re-equilibration following LPS/membrane interactions were analysed for smooth type LPS from S. enterica and K. pneumoniae. The binding/equilibration curves are shown in Figure 3A and the corresponding Bmax and Kd values are compared in Figure 3B,C and summarised in Table 2. LPS from E. coli showed similar kinetics of binding to both types of membranes and is not included in the following analysis. The values of Bmax for the binding step are slightly higher than the re-equilibration step in both types of LPS. This suggests that only a fraction of the charges that bind the lipid bilayer is then rearranged in the second step. The Kd values obtained from initial binding curves are significantly higher than from the re-equilibration step, which suggests that hydrophobic interactions play a significant role in LPS redistribution within the membrane. Differences in kinetic constants between S. enterica and K. pneumoniae LPS are minimal, which points to a common mechanism of host target engagement.


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)

Two stages of S. enterica LPS interaction with mixed PC55SM15Chol30 membranes.(A): initial binding (hexagons) and conformational re-equilibration (diamonds). Both datasets were normalized to the starting fluorescent intensities for LPS binding to PC100 and the values of Bmax are normalized to one for PC100 (B) and values for Kd are shown in (C). The average of three repeats is shown and fitted to Equation 1. Similar binding curves were obtained from K. pneumoniae LPS binding. The Bmax and Kd values for both types of LPS are shown in (B) and (C) and summarised in Table 2.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0038677-g003: Two stages of S. enterica LPS interaction with mixed PC55SM15Chol30 membranes.(A): initial binding (hexagons) and conformational re-equilibration (diamonds). Both datasets were normalized to the starting fluorescent intensities for LPS binding to PC100 and the values of Bmax are normalized to one for PC100 (B) and values for Kd are shown in (C). The average of three repeats is shown and fitted to Equation 1. Similar binding curves were obtained from K. pneumoniae LPS binding. The Bmax and Kd values for both types of LPS are shown in (B) and (C) and summarised in Table 2.
Mentions: Receptor-independent binding of LPS to membranes is important to opportunistic pathogens as a route to host invasion. To gain a better insight into this process, the equilibrium properties of initial binding and fluorescence re-equilibration following LPS/membrane interactions were analysed for smooth type LPS from S. enterica and K. pneumoniae. The binding/equilibration curves are shown in Figure 3A and the corresponding Bmax and Kd values are compared in Figure 3B,C and summarised in Table 2. LPS from E. coli showed similar kinetics of binding to both types of membranes and is not included in the following analysis. The values of Bmax for the binding step are slightly higher than the re-equilibration step in both types of LPS. This suggests that only a fraction of the charges that bind the lipid bilayer is then rearranged in the second step. The Kd values obtained from initial binding curves are significantly higher than from the re-equilibration step, which suggests that hydrophobic interactions play a significant role in LPS redistribution within the membrane. Differences in kinetic constants between S. enterica and K. pneumoniae LPS are minimal, which points to a common mechanism of host target engagement.

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