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The interaction of N-acylhomoserine lactone quorum sensing signaling molecules with biological membranes: implications for inter-kingdom signaling.

Davis BM, Jensen R, Williams P, O'Shea P - PLoS ONE (2010)

Bottom Line: The long chain N-acylhomoserine lactone (AHL) quorum sensing signal molecules released by Pseudomonas aeruginosa have long been known to elicit immunomodulatory effects through a process termed inter-kingdom signaling.Our observations support previous findings that increasing AHL lipophilicity increases the immunomodulatory activity of these quorum compounds, while providing evidence to suggest membrane interaction plays an important role in quorum sensing and implies a role for membrane microdomains in this process.Finally, our results suggest the existence of a eukaryotic membrane-located system that acts as an AHL receptor.

View Article: PubMed Central - PubMed

Affiliation: Cell Biophysics Group, Institute of Biophysics, Imaging and Optical Science, University of Nottingham, Nottingham, United Kingdom.

ABSTRACT

Background: The long chain N-acylhomoserine lactone (AHL) quorum sensing signal molecules released by Pseudomonas aeruginosa have long been known to elicit immunomodulatory effects through a process termed inter-kingdom signaling. However, to date very little is known regarding the exact mechanism of action of these compounds on their eukaryotic targets.

Methodology/principal findings: The use of the membrane dipole fluorescent sensor di-8-ANEPPS to characterise the interactions of AHL quorum sensing signal molecules, N-(3-oxotetradecanoyl)-L-homoserine lactone (3-oxo-C14-HSL), N-(3-oxododecanoyl)homoserine-L-lactone (3-oxo-C12-HSL) and N-(3-oxodecanoyl) homoserine-L-lactone (3-oxo-C10 HSL) produced by Pseudomonas aeruginosa with model and cellular membranes is reported. The interactions of these AHLs with artificial membranes reveal that each of the compounds is capable of membrane interaction in the micromolar concentration range causing significant modulation of the membrane dipole potential. These interactions fit simple hyperbolic binding models with membrane affinity increasing with acyl chain length. Similar results were obtained with T-lymphocytes providing the evidence that AHLs are capable of direct interaction with the plasma membrane. 3-oxo-C12-HSL interacts with lymphocytes via a cooperative binding model therefore implying the existence of an AHL membrane receptor. The role of cholesterol in the interactions of AHLs with membranes, the significance of modulating cellular dipole potential for receptor conformation and the implications for immune modulation are discussed.

Conclusions/ significance: Our observations support previous findings that increasing AHL lipophilicity increases the immunomodulatory activity of these quorum compounds, while providing evidence to suggest membrane interaction plays an important role in quorum sensing and implies a role for membrane microdomains in this process. Finally, our results suggest the existence of a eukaryotic membrane-located system that acts as an AHL receptor.

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Binding profiles of the interactions of AHLs with artificial membrane systems.Binding profiles of [A] 3-oxo-C14-HSL, [B] 3-oxo-C12-HSL and [C] 3-oxo-C10-HSL on titration to PC100% (•) or PC70%Cholesterol30% (Δ) di-8-ANEPPS labeled liposomes (400 µM) at 37°C (n = 5) normalised to DMSO controls. Profiles were fitted to simple hyperbolic and sigmoidal binding models (equations 1 and 2) and extra sum of squares F-Tests were used to determine the best fitting in each case (all hyperbolic). [D] Average saturation points and [E] Average dissociation constant of the best fitting models±SEM.
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pone-0013522-g004: Binding profiles of the interactions of AHLs with artificial membrane systems.Binding profiles of [A] 3-oxo-C14-HSL, [B] 3-oxo-C12-HSL and [C] 3-oxo-C10-HSL on titration to PC100% (•) or PC70%Cholesterol30% (Δ) di-8-ANEPPS labeled liposomes (400 µM) at 37°C (n = 5) normalised to DMSO controls. Profiles were fitted to simple hyperbolic and sigmoidal binding models (equations 1 and 2) and extra sum of squares F-Tests were used to determine the best fitting in each case (all hyperbolic). [D] Average saturation points and [E] Average dissociation constant of the best fitting models±SEM.

Mentions: Changes in the membrane dipole potential can be tracked over time using the ratio of di-8-ANEPPS fluorescence at 460 nm and 520 nm excitation and a fixed emission (termed R(460/520)), which is sensitive solely to variations of the local electric field due to dipolar molecular properties. Upon titration of the AHLs into these artificial membranes a concentration dependent decrease in membrane dipole potential was observed as shown in Figure 3C. These data were plotted as the incremental change of di-8-ANEPPS fluorescence versus the concentration of AHL added and fitted to various ‘binding’ models (eq. 1 & 2) as shown in Figure 4A–C. Such observations are consistent with the interaction and insertion of AHL molecules with the membrane vesicles. All ligands were found to interact with both Phosphotidylcholine100% and Phosphatidylcholine70%Cholesterol30% membranes via a simple hyperbolic (i.e. non-cooperative) binding mechanism, Figure 4D & E compare the dissociation constant (Kd) and saturation point (Bmax) obtained in each case. Overall these figures depict that as acyl chain length increases it leads to a decrease of the observed the Kd, suggesting that longer chain AHLs have a higher membrane affinity than the shorter chain variants. This was found to be the case for both Phosphotidylcholine100% and Phosphatidylcholine70%Cholesterol30% membranes. Titration of the AHLs into membranes containing 30% cholesterol did not result in a significant change in Kd (Figure 4D), however the binding capacity of 3-oxo-C12 HSL and 3-oxo-C14 HSL (i.e. saturation in fluorescence units) of Phosphatidylcholine70% Cholesterol30% membranes was significantly greater than for Phosphatidylcholine100% membranes (p<0.05), suggesting that QS compounds may be accumulating in membrane microdomains present in Phosphatidylcholine70%Cholesterol30% membranes.


The interaction of N-acylhomoserine lactone quorum sensing signaling molecules with biological membranes: implications for inter-kingdom signaling.

Davis BM, Jensen R, Williams P, O'Shea P - PLoS ONE (2010)

Binding profiles of the interactions of AHLs with artificial membrane systems.Binding profiles of [A] 3-oxo-C14-HSL, [B] 3-oxo-C12-HSL and [C] 3-oxo-C10-HSL on titration to PC100% (•) or PC70%Cholesterol30% (Δ) di-8-ANEPPS labeled liposomes (400 µM) at 37°C (n = 5) normalised to DMSO controls. Profiles were fitted to simple hyperbolic and sigmoidal binding models (equations 1 and 2) and extra sum of squares F-Tests were used to determine the best fitting in each case (all hyperbolic). [D] Average saturation points and [E] Average dissociation constant of the best fitting models±SEM.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2958149&req=5

pone-0013522-g004: Binding profiles of the interactions of AHLs with artificial membrane systems.Binding profiles of [A] 3-oxo-C14-HSL, [B] 3-oxo-C12-HSL and [C] 3-oxo-C10-HSL on titration to PC100% (•) or PC70%Cholesterol30% (Δ) di-8-ANEPPS labeled liposomes (400 µM) at 37°C (n = 5) normalised to DMSO controls. Profiles were fitted to simple hyperbolic and sigmoidal binding models (equations 1 and 2) and extra sum of squares F-Tests were used to determine the best fitting in each case (all hyperbolic). [D] Average saturation points and [E] Average dissociation constant of the best fitting models±SEM.
Mentions: Changes in the membrane dipole potential can be tracked over time using the ratio of di-8-ANEPPS fluorescence at 460 nm and 520 nm excitation and a fixed emission (termed R(460/520)), which is sensitive solely to variations of the local electric field due to dipolar molecular properties. Upon titration of the AHLs into these artificial membranes a concentration dependent decrease in membrane dipole potential was observed as shown in Figure 3C. These data were plotted as the incremental change of di-8-ANEPPS fluorescence versus the concentration of AHL added and fitted to various ‘binding’ models (eq. 1 & 2) as shown in Figure 4A–C. Such observations are consistent with the interaction and insertion of AHL molecules with the membrane vesicles. All ligands were found to interact with both Phosphotidylcholine100% and Phosphatidylcholine70%Cholesterol30% membranes via a simple hyperbolic (i.e. non-cooperative) binding mechanism, Figure 4D & E compare the dissociation constant (Kd) and saturation point (Bmax) obtained in each case. Overall these figures depict that as acyl chain length increases it leads to a decrease of the observed the Kd, suggesting that longer chain AHLs have a higher membrane affinity than the shorter chain variants. This was found to be the case for both Phosphotidylcholine100% and Phosphatidylcholine70%Cholesterol30% membranes. Titration of the AHLs into membranes containing 30% cholesterol did not result in a significant change in Kd (Figure 4D), however the binding capacity of 3-oxo-C12 HSL and 3-oxo-C14 HSL (i.e. saturation in fluorescence units) of Phosphatidylcholine70% Cholesterol30% membranes was significantly greater than for Phosphatidylcholine100% membranes (p<0.05), suggesting that QS compounds may be accumulating in membrane microdomains present in Phosphatidylcholine70%Cholesterol30% membranes.

Bottom Line: The long chain N-acylhomoserine lactone (AHL) quorum sensing signal molecules released by Pseudomonas aeruginosa have long been known to elicit immunomodulatory effects through a process termed inter-kingdom signaling.Our observations support previous findings that increasing AHL lipophilicity increases the immunomodulatory activity of these quorum compounds, while providing evidence to suggest membrane interaction plays an important role in quorum sensing and implies a role for membrane microdomains in this process.Finally, our results suggest the existence of a eukaryotic membrane-located system that acts as an AHL receptor.

View Article: PubMed Central - PubMed

Affiliation: Cell Biophysics Group, Institute of Biophysics, Imaging and Optical Science, University of Nottingham, Nottingham, United Kingdom.

ABSTRACT

Background: The long chain N-acylhomoserine lactone (AHL) quorum sensing signal molecules released by Pseudomonas aeruginosa have long been known to elicit immunomodulatory effects through a process termed inter-kingdom signaling. However, to date very little is known regarding the exact mechanism of action of these compounds on their eukaryotic targets.

Methodology/principal findings: The use of the membrane dipole fluorescent sensor di-8-ANEPPS to characterise the interactions of AHL quorum sensing signal molecules, N-(3-oxotetradecanoyl)-L-homoserine lactone (3-oxo-C14-HSL), N-(3-oxododecanoyl)homoserine-L-lactone (3-oxo-C12-HSL) and N-(3-oxodecanoyl) homoserine-L-lactone (3-oxo-C10 HSL) produced by Pseudomonas aeruginosa with model and cellular membranes is reported. The interactions of these AHLs with artificial membranes reveal that each of the compounds is capable of membrane interaction in the micromolar concentration range causing significant modulation of the membrane dipole potential. These interactions fit simple hyperbolic binding models with membrane affinity increasing with acyl chain length. Similar results were obtained with T-lymphocytes providing the evidence that AHLs are capable of direct interaction with the plasma membrane. 3-oxo-C12-HSL interacts with lymphocytes via a cooperative binding model therefore implying the existence of an AHL membrane receptor. The role of cholesterol in the interactions of AHLs with membranes, the significance of modulating cellular dipole potential for receptor conformation and the implications for immune modulation are discussed.

Conclusions/ significance: Our observations support previous findings that increasing AHL lipophilicity increases the immunomodulatory activity of these quorum compounds, while providing evidence to suggest membrane interaction plays an important role in quorum sensing and implies a role for membrane microdomains in this process. Finally, our results suggest the existence of a eukaryotic membrane-located system that acts as an AHL receptor.

Show MeSH
Related in: MedlinePlus