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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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The interactions of AHLs with artificial membrane systems perturbs the membrane dipole potential.Fluorescence difference spectra obtained by subtracting di-8-ANEPPS excitation spectra (λem = 590 nm) of PC(100%) [A] or PC(70%)Cholesterol(30%) [B] membrane vesicles (400 µM) from those obtained after these membranes were exposed to the following QS molecules; 65 µM 3-oxo-C14-HSL (thick dashed line), 200 µM 3-oxo-C12-HSL (solid black line) and 200 µM 3-oxo-C10-HSL (thin dashed and dotted line). Before subtraction, each spectrum was normalized to the integrated areas so that the difference spectra would reflect only the spectral shifts. Each difference spectrum was then normalised to a DMSO control (grey line) and a three point moving average applied to reduce noise. In all experiments the dye concentration was 10 µM and temperature was maintained at 37°C. [C] A dual wavelength ratiometric measurement of the dipole potential variation in di-8-ANEPPS. Additions of 22 µM 3-oxo-C14-HSL or equivalent volumes of DMSO were made to 400 µM PC(100%). Samples were excited at 460 nm and 520 nm. Emission was read at 590 nm and the ratio R(460/520) was calculated (shown). All experiments n = 3.
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pone-0013522-g003: The interactions of AHLs with artificial membrane systems perturbs the membrane dipole potential.Fluorescence difference spectra obtained by subtracting di-8-ANEPPS excitation spectra (λem = 590 nm) of PC(100%) [A] or PC(70%)Cholesterol(30%) [B] membrane vesicles (400 µM) from those obtained after these membranes were exposed to the following QS molecules; 65 µM 3-oxo-C14-HSL (thick dashed line), 200 µM 3-oxo-C12-HSL (solid black line) and 200 µM 3-oxo-C10-HSL (thin dashed and dotted line). Before subtraction, each spectrum was normalized to the integrated areas so that the difference spectra would reflect only the spectral shifts. Each difference spectrum was then normalised to a DMSO control (grey line) and a three point moving average applied to reduce noise. In all experiments the dye concentration was 10 µM and temperature was maintained at 37°C. [C] A dual wavelength ratiometric measurement of the dipole potential variation in di-8-ANEPPS. Additions of 22 µM 3-oxo-C14-HSL or equivalent volumes of DMSO were made to 400 µM PC(100%). Samples were excited at 460 nm and 520 nm. Emission was read at 590 nm and the ratio R(460/520) was calculated (shown). All experiments n = 3.

Mentions: On this basis it is shown in Figure 3A & B that addition of 3-oxo-C14-HSL, 3-oxo-C12-HSL and 3-oxo-C10-HSL to Phosphotidylcholine100% and Phosphatidylcholine70% Cholesterol30% membranes vesicles led to a red shift in the excitation spectra of di-8-ANEPPS. A red shift (with a minimum of ∼440 nm and a maximum of ∼520 nm) is indicative of the ligands acting to decrease the membrane dipole potential upon insertion into the membrane. This finding is consistent with our previous work [29] reporting that addition of reagents known to decrease the membrane dipole potential, give rise to di-8-ANEPPS difference spectra of similar profiles to those reported here.


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)

The interactions of AHLs with artificial membrane systems perturbs the membrane dipole potential.Fluorescence difference spectra obtained by subtracting di-8-ANEPPS excitation spectra (λem = 590 nm) of PC(100%) [A] or PC(70%)Cholesterol(30%) [B] membrane vesicles (400 µM) from those obtained after these membranes were exposed to the following QS molecules; 65 µM 3-oxo-C14-HSL (thick dashed line), 200 µM 3-oxo-C12-HSL (solid black line) and 200 µM 3-oxo-C10-HSL (thin dashed and dotted line). Before subtraction, each spectrum was normalized to the integrated areas so that the difference spectra would reflect only the spectral shifts. Each difference spectrum was then normalised to a DMSO control (grey line) and a three point moving average applied to reduce noise. In all experiments the dye concentration was 10 µM and temperature was maintained at 37°C. [C] A dual wavelength ratiometric measurement of the dipole potential variation in di-8-ANEPPS. Additions of 22 µM 3-oxo-C14-HSL or equivalent volumes of DMSO were made to 400 µM PC(100%). Samples were excited at 460 nm and 520 nm. Emission was read at 590 nm and the ratio R(460/520) was calculated (shown). All experiments n = 3.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0013522-g003: The interactions of AHLs with artificial membrane systems perturbs the membrane dipole potential.Fluorescence difference spectra obtained by subtracting di-8-ANEPPS excitation spectra (λem = 590 nm) of PC(100%) [A] or PC(70%)Cholesterol(30%) [B] membrane vesicles (400 µM) from those obtained after these membranes were exposed to the following QS molecules; 65 µM 3-oxo-C14-HSL (thick dashed line), 200 µM 3-oxo-C12-HSL (solid black line) and 200 µM 3-oxo-C10-HSL (thin dashed and dotted line). Before subtraction, each spectrum was normalized to the integrated areas so that the difference spectra would reflect only the spectral shifts. Each difference spectrum was then normalised to a DMSO control (grey line) and a three point moving average applied to reduce noise. In all experiments the dye concentration was 10 µM and temperature was maintained at 37°C. [C] A dual wavelength ratiometric measurement of the dipole potential variation in di-8-ANEPPS. Additions of 22 µM 3-oxo-C14-HSL or equivalent volumes of DMSO were made to 400 µM PC(100%). Samples were excited at 460 nm and 520 nm. Emission was read at 590 nm and the ratio R(460/520) was calculated (shown). All experiments n = 3.
Mentions: On this basis it is shown in Figure 3A & B that addition of 3-oxo-C14-HSL, 3-oxo-C12-HSL and 3-oxo-C10-HSL to Phosphotidylcholine100% and Phosphatidylcholine70% Cholesterol30% membranes vesicles led to a red shift in the excitation spectra of di-8-ANEPPS. A red shift (with a minimum of ∼440 nm and a maximum of ∼520 nm) is indicative of the ligands acting to decrease the membrane dipole potential upon insertion into the membrane. This finding is consistent with our previous work [29] reporting that addition of reagents known to decrease the membrane dipole potential, give rise to di-8-ANEPPS difference spectra of similar profiles to those reported here.

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