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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 of LPS from S. enterica to FPE-labelled Jurkat cells and to Jurkat cells, from which cholesterol has been removed with MβCD prior to addition of LPS (A).Both curves are normalized to initial fluorescence intensity of untreated cells. Values for Bmax and Kd values are show in panel (B).
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pone-0038677-g005: Binding isotherms of LPS from S. enterica to FPE-labelled Jurkat cells and to Jurkat cells, from which cholesterol has been removed with MβCD prior to addition of LPS (A).Both curves are normalized to initial fluorescence intensity of untreated cells. Values for Bmax and Kd values are show in panel (B).

Mentions: LPS from S. enterica was titrated into a suspension of T lymphocytes and the membrane interactions monitored as changes in FPE fluorescence. Studies were also performed with Lymphocytes which had been pre-treated with MβCD to deplete the cell membrane cholesterol. Fluorescence intensity is shown in Figure 5 along with Bmax and Kd. Experimental scatter in the LPS titration curves hindered differentiation between binding and charge re-arrangement and so only the composite total signal profiles can be reported (i.e. comprising both the binding and rearrangement phases of the LPS interaction – defined in Figure 1). The observed LPS binding capacity Bmax values are higher in MβCD-treated Jurkat cells, suggesting a greater LPS binding capacity to cholesterol-depleted membranes. By contrast, the corresponding dissociation constant is also higher, which shows a lower affinity for LPS after cholesterol depletion by comparison to untreated cells. The apparent increased LPS binding affinity observed for the MβCD treated cells is interesting and may be the result of combining the 2 interaction phases we define in Fig. 1. Alternatively (or in addition) it may reside in the possibility that MβCD is only known to remove cholesterol whereas in cells microdomains may be stabilised by several other factors in addition to cholesterol. These factors may include cytoskeletal elements, ECM and other lipid components unaffected by MβCD treatment or modulations of the levels of the fluid membrane free volume by cholesterol [29]. The lower Kd values observed in untreated cells is consistent with our FPE fluorescence results from mixed lipid PC55SM15Chol30 membranes (see earlier section) and supports the hypothesis of a greater LPS affinity for phase separated/lipid raft-containing membranes.


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 of LPS from S. enterica to FPE-labelled Jurkat cells and to Jurkat cells, from which cholesterol has been removed with MβCD prior to addition of LPS (A).Both curves are normalized to initial fluorescence intensity of untreated cells. Values for Bmax and Kd values are show in panel (B).
© Copyright Policy
Related In: Results  -  Collection

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

pone-0038677-g005: Binding isotherms of LPS from S. enterica to FPE-labelled Jurkat cells and to Jurkat cells, from which cholesterol has been removed with MβCD prior to addition of LPS (A).Both curves are normalized to initial fluorescence intensity of untreated cells. Values for Bmax and Kd values are show in panel (B).
Mentions: LPS from S. enterica was titrated into a suspension of T lymphocytes and the membrane interactions monitored as changes in FPE fluorescence. Studies were also performed with Lymphocytes which had been pre-treated with MβCD to deplete the cell membrane cholesterol. Fluorescence intensity is shown in Figure 5 along with Bmax and Kd. Experimental scatter in the LPS titration curves hindered differentiation between binding and charge re-arrangement and so only the composite total signal profiles can be reported (i.e. comprising both the binding and rearrangement phases of the LPS interaction – defined in Figure 1). The observed LPS binding capacity Bmax values are higher in MβCD-treated Jurkat cells, suggesting a greater LPS binding capacity to cholesterol-depleted membranes. By contrast, the corresponding dissociation constant is also higher, which shows a lower affinity for LPS after cholesterol depletion by comparison to untreated cells. The apparent increased LPS binding affinity observed for the MβCD treated cells is interesting and may be the result of combining the 2 interaction phases we define in Fig. 1. Alternatively (or in addition) it may reside in the possibility that MβCD is only known to remove cholesterol whereas in cells microdomains may be stabilised by several other factors in addition to cholesterol. These factors may include cytoskeletal elements, ECM and other lipid components unaffected by MβCD treatment or modulations of the levels of the fluid membrane free volume by cholesterol [29]. The lower Kd values observed in untreated cells is consistent with our FPE fluorescence results from mixed lipid PC55SM15Chol30 membranes (see earlier section) and supports the hypothesis of a greater LPS affinity for phase separated/lipid raft-containing membranes.

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