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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 Lymphocyte membranes.Binding profiles of 3-oxo-C14-HSL (Δ, hyperbolic), 3-oxo-C12-HSL (•, sigmoidal) and 3-oxo-C10-HSL (×, neither) on titration to di-8-ANEPPS labeled T-Lymphocytes (40,000 cells/ml) at 37°C normalised to DMSO controls. Profiles were fitted to simple hyperbolic and sigmoidal binding models (equations 1 and 2) and F-Tests were used to determine the best fitting model. In each experiment n = 3, ±SEM.
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pone-0013522-g007: Binding profiles of the interactions of AHLs with Lymphocyte membranes.Binding profiles of 3-oxo-C14-HSL (Δ, hyperbolic), 3-oxo-C12-HSL (•, sigmoidal) and 3-oxo-C10-HSL (×, neither) on titration to di-8-ANEPPS labeled T-Lymphocytes (40,000 cells/ml) at 37°C normalised to DMSO controls. Profiles were fitted to simple hyperbolic and sigmoidal binding models (equations 1 and 2) and F-Tests were used to determine the best fitting model. In each experiment n = 3, ±SEM.

Mentions: Figure 6B indicates that upon titration of di-8-ANEPPS labeled Jurkat T-lymphocytes with AHLs, a similar decrease in the R(460/520) parameter was observed as for titration with the artificial membrane systems. These data were plotted and fitted as before and are shown in Figure 7. It was found that 3-oxo-C14-HSL exhibited a strong interaction with the cell membrane with both a greater binding capacity (1.40±0.11 compared to 0.86±0.08, p = 0.02) and dissociation constant significantly less than 3-oxo-C12-HSL (39 µM±6 µM compared to 153 µM±38 µM, p = 0.04) as determined by two tailed T test. The latter exhibited a more complex binding reaction that was poorly described by Eq. 1. Eq. 2 however was found to be able to describe the binding isotherm and this model indicates that cooperativity appears to be occurring. The cooperativity index (sometimes referred to as the Hill coefficient) for these studies was found to be 1.87±0.27. There are several possible interpretations of this finding, such as that two 3-oxo-C12-HSL molecules come together on/in the membrane to promote their respective interaction. There are, however other explanations that accommodate this behavior of which the most likely is that the membrane is modified by the presence of the AHLs and this affects the subsequent binding of further molecules (see below).


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 Lymphocyte membranes.Binding profiles of 3-oxo-C14-HSL (Δ, hyperbolic), 3-oxo-C12-HSL (•, sigmoidal) and 3-oxo-C10-HSL (×, neither) on titration to di-8-ANEPPS labeled T-Lymphocytes (40,000 cells/ml) at 37°C normalised to DMSO controls. Profiles were fitted to simple hyperbolic and sigmoidal binding models (equations 1 and 2) and F-Tests were used to determine the best fitting model. In each experiment n = 3, ±SEM.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2958149&req=5

pone-0013522-g007: Binding profiles of the interactions of AHLs with Lymphocyte membranes.Binding profiles of 3-oxo-C14-HSL (Δ, hyperbolic), 3-oxo-C12-HSL (•, sigmoidal) and 3-oxo-C10-HSL (×, neither) on titration to di-8-ANEPPS labeled T-Lymphocytes (40,000 cells/ml) at 37°C normalised to DMSO controls. Profiles were fitted to simple hyperbolic and sigmoidal binding models (equations 1 and 2) and F-Tests were used to determine the best fitting model. In each experiment n = 3, ±SEM.
Mentions: Figure 6B indicates that upon titration of di-8-ANEPPS labeled Jurkat T-lymphocytes with AHLs, a similar decrease in the R(460/520) parameter was observed as for titration with the artificial membrane systems. These data were plotted and fitted as before and are shown in Figure 7. It was found that 3-oxo-C14-HSL exhibited a strong interaction with the cell membrane with both a greater binding capacity (1.40±0.11 compared to 0.86±0.08, p = 0.02) and dissociation constant significantly less than 3-oxo-C12-HSL (39 µM±6 µM compared to 153 µM±38 µM, p = 0.04) as determined by two tailed T test. The latter exhibited a more complex binding reaction that was poorly described by Eq. 1. Eq. 2 however was found to be able to describe the binding isotherm and this model indicates that cooperativity appears to be occurring. The cooperativity index (sometimes referred to as the Hill coefficient) for these studies was found to be 1.87±0.27. There are several possible interpretations of this finding, such as that two 3-oxo-C12-HSL molecules come together on/in the membrane to promote their respective interaction. There are, however other explanations that accommodate this behavior of which the most likely is that the membrane is modified by the presence of the AHLs and this affects the subsequent binding of further molecules (see below).

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