Bacterial Quorum Sensing Signals Promote Large-Scale Remodeling of Lipid Membranes

Abstract

We report that N-acyl-L-homoserine lactones (AHLs), a class of nonionic amphiphiles that common bacteria use as signals to coordinate group behaviors, can promote largescale remodeling in model lipid membranes. Characterization of supported lipid bilayers (SLBs) of the phospholipid 1,2-dioleoylsn-glycero-3-phosphocholine (DOPC) by fluorescence microscopy and quartz crystal microbalance with dissipation (QCM-D) reveals the well-studied AHL signal 3-oxo-C12-AHL and its anionic head group hydrolysis product (3-oxo-C12-HS) to promote the formation of long microtubules that can retract into hemispherical caps on the surface of the bilayer. These transformations are dynamic, reversible, and dependent upon the head group structure. Additional experiments demonstrate that 3-oxo-C12-AHL can promote remodeling to form microtubules in lipid vesicles and promote molecular transport across bilayers. Molecular dynamics (MD) simulations predict differences in thermodynamic barriers to translocation of these amphiphiles across a bilayer that are reflected in both the type and extent of reformation and associated dynamics. Our experimental observations can thus be interpreted in terms of accumulation and relief of asymmetric stresses in the inner and outer leaflets of a bilayer upon intercalation and translocation of these amphiphiles. Finally, experiments on Pseudomonas aeruginosa, a pathogen that uses 3-oxo-C12-AHL for cell-tocell signaling, demonstrate that 3-oxo-C12-AHL and 3-oxo-C12-HS can promote membrane remodeling at biologically relevant concentrations and in the absence of other biosurfactants, such as rhamnolipids, that are produced at high population densities. Overall, these results have implications for the roles that 3-oxo-C12-AHL and its hydrolysis product may play in not only mediating intraspecies bacterial communication but also processes such as interspecies signaling and bacterial control of host-cell response. Our findings also provide guidance that could prove useful for the design of synthetic self-assembled materials that respond to bacteria in ways that are useful in the context of sensing, drug delivery, and in other fundamental and applied areas.

Interactions of Bacterial Quorum Sensing Signals with Model Lipid Membranes: Influence of Acyl Tail Structure on Multiscale Response

“…Daniels et al also demonstrated that some long-chained AHLs can self-assemble and undergo phase changes when compressed at air–water interfaces . Finally, recent reports have demonstrated that AHLs can intercalate into lipid membranes, , allowing them to translocate across lipid bilayers, promote membrane depolarization, dissolve lipid phases, and, of particular interest to the work reported here, promote large-scale membrane remodeling . These studies reveal that both head-group and aliphatic tail structure can have substantial impacts on the behaviors of AHLs, including self-association in solution , and adsorption , and subsequent multiscale phenomena ,, that occur when they come in contact with lipid bilayers.…”

“…Calcein-loaded LUVs were prepared using a previously described freeze–thaw and vesicle extrusion procedure. , In brief, DOPC dissolved in chloroform was aliquoted into vials, and the chloroform was subsequently removed under a stream of N 2 and further dried under vacuum for at least 1 h. A 70 mM calcein solution in buffer was then added to the dried lipids and vortexed vigorously to form a turbid 5 mg/mL DOPC solution. The calcein-containing solution was prepared by massing an appropriate amount of calcein, to which buffer was added and the pH of the solution was adjusted to 7.4 with 10 M NaOH.…”

“…Statistical significance against the buffer control or other AHL-containing solutions was determined using a one-way ANOVA with all nine normalized replicates for each condition used that was corrected for multiple comparisons using the Tukey method. The fluorescence intensity for each reading was normalized using a procedure adapted from our past study and summarized in eq , where FI is the fluorescence intensity of the test solution, FI Buffer is the fluorescence intensity of the calcein vesicles in buffer as the negative control, and FI TX‑100 is the fluorescence intensity of a vesicle solution containing 1% Triton X-100 (previously demonstrated to completely lyse vesicles at high concentrations) as the positive control. …”

“…We recently reported that 3-oxo-C12-AHL (Figure ), a naturally occurring long-tailed AHL produced by P. aeruginosa and other related bacteria, can promote the formation of extended microtubules and large hemispherical caps on the surfaces of model lipid bilayers formed using the phospholipid 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) . That study also showed that 3-oxo-C12-HSthe anionic head-group hydrolysis product of 3-oxo-C12-AHL that arises naturally in P.…”

“…The work reported here was motivated by a growing body of work demonstrating that the amphiphilic nature of AHLs can lead to self-assembly in solution and at interfaces. − The HL “head” group and the acyl “tail” group of varying length and structure (e.g., ranging from 4–20 carbons, oxidation state in the 3-position, etc.) provide polar and hydrophobic motifs, respectively (Figure ).…”