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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

Difference fluorescence spectraof di-8-ANEPPS-labelled PC100 and PC55SM15Chol30 phospholipid vesicles before and after titration of LPS from S. enterica (A).The addition of endotoxin to both types of vesicles results in red shift. Changes in R460/520 ratio were plotted in (B) and fitted to Equation 1. The graphs are normalized to Bmax of the PC100 and to the starting fluorescence intensity. The Bmax and Kd values were estimated from the fits and are presented in (C) and (D), respectively.
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pone-0038677-g004: Difference fluorescence spectraof di-8-ANEPPS-labelled PC100 and PC55SM15Chol30 phospholipid vesicles before and after titration of LPS from S. enterica (A).The addition of endotoxin to both types of vesicles results in red shift. Changes in R460/520 ratio were plotted in (B) and fitted to Equation 1. The graphs are normalized to Bmax of the PC100 and to the starting fluorescence intensity. The Bmax and Kd values were estimated from the fits and are presented in (C) and (D), respectively.

Mentions: We investigated the role of membrane composition on insertion of LPS into membranes using the fluorescent probe di-8-ANEPPS (4-[2-[6-(Dioctylamino)-2-naphthalenyl] ethenyl]-1-(3-sulfopropyl)-pyridinium, inner salt), one advantage of this probe is that it can yield information about the penetration of macromolecules into the membrane interior [22]. Thus by measuring the emission at different excitations, the ratio (R460/520) has been shown to be a good approximation of the level of the membrane dipole potential and any concomitant changes due to molecular interactions. Smooth LPS from S. enterica was titrated into di-8-ANEPPS-containing phospholipid vesicles made up of PC100 and PC55SM15Chol30. Excitation was measured before and after endotoxin addition and subtracted to obtain from the difference spectra a red shift in fluorescence (Figure 4A). The magnitude of such shifts has been shown to be dependent on the concentration of the interacting species ([28]), thus as more LPS became bound to the membrane this led to greater changes of the membrane dipole potential with one interpretation being that the LPS penetrates the membrane interior. R460/520 ratios were measured before and after LPS addition and differences were plotted against LPS concentration (Figure 4B). The experiments were carried out with pure lipid, PC100, and mixed lipid membranes, PC55SM15Chol30, to investigate the role of lateral phase separation and the presence of cholesterol. Comparison between the membrane binding of smooth LPS from opportunistic pathogens S. enterica and K. pneumoniae showed very similar kinetics thus we report only our studies of membrane insertion of S. enterica LPS.


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)

Difference fluorescence spectraof di-8-ANEPPS-labelled PC100 and PC55SM15Chol30 phospholipid vesicles before and after titration of LPS from S. enterica (A).The addition of endotoxin to both types of vesicles results in red shift. Changes in R460/520 ratio were plotted in (B) and fitted to Equation 1. The graphs are normalized to Bmax of the PC100 and to the starting fluorescence intensity. The Bmax and Kd values were estimated from the fits and are presented in (C) and (D), respectively.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0038677-g004: Difference fluorescence spectraof di-8-ANEPPS-labelled PC100 and PC55SM15Chol30 phospholipid vesicles before and after titration of LPS from S. enterica (A).The addition of endotoxin to both types of vesicles results in red shift. Changes in R460/520 ratio were plotted in (B) and fitted to Equation 1. The graphs are normalized to Bmax of the PC100 and to the starting fluorescence intensity. The Bmax and Kd values were estimated from the fits and are presented in (C) and (D), respectively.
Mentions: We investigated the role of membrane composition on insertion of LPS into membranes using the fluorescent probe di-8-ANEPPS (4-[2-[6-(Dioctylamino)-2-naphthalenyl] ethenyl]-1-(3-sulfopropyl)-pyridinium, inner salt), one advantage of this probe is that it can yield information about the penetration of macromolecules into the membrane interior [22]. Thus by measuring the emission at different excitations, the ratio (R460/520) has been shown to be a good approximation of the level of the membrane dipole potential and any concomitant changes due to molecular interactions. Smooth LPS from S. enterica was titrated into di-8-ANEPPS-containing phospholipid vesicles made up of PC100 and PC55SM15Chol30. Excitation was measured before and after endotoxin addition and subtracted to obtain from the difference spectra a red shift in fluorescence (Figure 4A). The magnitude of such shifts has been shown to be dependent on the concentration of the interacting species ([28]), thus as more LPS became bound to the membrane this led to greater changes of the membrane dipole potential with one interpretation being that the LPS penetrates the membrane interior. R460/520 ratios were measured before and after LPS addition and differences were plotted against LPS concentration (Figure 4B). The experiments were carried out with pure lipid, PC100, and mixed lipid membranes, PC55SM15Chol30, to investigate the role of lateral phase separation and the presence of cholesterol. Comparison between the membrane binding of smooth LPS from opportunistic pathogens S. enterica and K. pneumoniae showed very similar kinetics thus we report only our studies of membrane insertion of S. enterica LPS.

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