Interkingdom signaling: integration, conformation, and orientation of N-acyl-L-homoserine lactones in supported lipid bilayers

Abstract

N-Acyl-L-homoserine lactones (AHLs) are small cell-to-cell signaling molecules involved in the regulation of population density and local gene expression in microbial communities. Recent evidence shows that contact of this signaling system, usually referred to as quorum sensing, to living eukaryotes results in interactions of AHL with host cells in a process termed "interkingdom signaling". So far details of this process and the binding site of the AHLs remain unknown; both an intracellular and a membrane-bound receptor seem possible, the first of which requires passage through the cell membrane. Here, we used sum-frequency-generation (SFG) spectroscopy to investigate the integration, conformation, orientation, and translocation of deuterated N-acyl-L-homoserine lactones (AHL-d(n)) with varying chain length (8, 12, and 14 C atoms) in lipid bilayers consisting of a 1:1 mixture of POPC:POPG supported on SiO(2) substrates (prepared by vesicle fusion). We found that all AHL-d(n) derivatives are well-ordered within the supported lipid bilayer (SLB) in a preferentially all-trans conformation of the deuterated alkyl chain and integrated into the upper leaflet of the SLB with the methyl terminal groups pointing downward. For the bilayer system described above, no flip-flop of AHL-d(n) from the upper leaflet to the lower one could be observed. Spectral assignments and interpretations were further supported by Fourier transform infrared and Raman spectroscopy.

Figure 1

The structure of the deuterated AHLs (AHL-d_n): N-(3-oxooctanoyl-d₉)-L-homoserine lactone (AHL-d₉; 1a), N-(3-oxododecanoyl-d₁₇)-L-homoserine lactone (AHL-d₁₇; 1b), and N-(3-oxotetradecanoyl-d₂₁)-L-homoserine lactone (AHL-d₂₁; 1c)

Figure 2

Background-suppressed SFG spectra in the spectral region of deuterated methyl and methylene groups for AHL-d₉ in solution and for the three AHL derivatives AHL-d₉, AHL-d₁₇, and AHL-d₂₁ in the presence of the SLB. Only in the presence of AHL-d_n and the SLB can an SFG signal be detected. This increases in intensity with increasing chain length. Prominent peak positions are marked as dotted vertical lines.

Figure 3

IR (bottom 3) and Raman (top 3) spectra of incorporated AHL-d_n derivatives in its solid state. The bands that get more prominent with decreasing chain length can be associated to CD₃ vibrations (dotted vertical lines) and coincide in peak positions to the ones detected in the SFG spectra of incorporated AHL-d_n (Fig. 2).

Figure 4

Non background-suppressed SFG spectra of incorporated AHL-d₉, AHL-d₁₇, and AHL-d₂₁. The envelope reflects the non-resonant signal of the Au layer with a Gaussian-like IR intensity profile. The r⁻ band located at 2220 cm⁻¹ has a constructive relative phase to the non-resonant background resulting in a peak in the spectrum. The appearance of a peak in the spectrum is an indicator for a deuterated methyl group pointing towards the surface. Fits of the spectra are plotted as dotted lines.

Figure 5

Integrated SFG intensities of the background-suppressed spectra for AHL-d₂₁ (triangles), AHL-d₁₇ (circles) and AHL-d₉ (squares). Values are normalized to their respective initial intensities. Full lines are exponential fits as described in the text.