Interactions of Bacterial Quorum Sensing Signals with Model Lipid Membranes: Influence of Membrane Composition on Membrane Remodeling.

We report the influence of membrane composition on the multiscale remodeling of multicomponent lipid bilayers initiated by contact with the amphiphilic bacterial quorum sensing signal N-(3-oxo)-dodecanoyl-l-homoserine lactone (3-oxo-C12-AHL) and its anionic headgroup hydrolysis product, 3-oxo-C12-HS. We used fluorescence microscopy and quartz crystal microbalance with dissipation (QCM-D) to characterize membrane reformation that occurs when these amphiphiles are placed in contact with supported lipid bilayers (SLBs) composed of (i) 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) containing varying amounts of cholesterol or (ii) mixtures of DOPC and either 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE, a conical zwitterionic lipid) or 1,2-dioleoyl-sn-glycero-3-phospho-l-serine (DOPS, a model anionic lipid). In general, we observe these mixed-lipid membranes to undergo remodeling events, including the formation and subsequent collapse of long tubules and the formation of hemispherical caps, upon introduction to biologically relevant concentrations of 3-oxo-C12-AHL and 3-oxo-C12-HS in ways that differ substantially from those observed in single-component DOPC membranes. These differences in bilayer reformation and their associated dynamics can be understood in terms of the influence of membrane composition on the time scales of molecular flip-flop, lipid packing defects, and lipid phase segregation in these materials. The lipid components investigated here are representative of classes of lipids that comprise both naturally occurring cell membranes and many useful synthetic soft materials. These studies thus represent a first step toward understanding the ways in which membrane composition can impact interactions with this important class of bacterial signaling molecules.

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

Many common bacteria use amphiphilic N-acyl-L-homoserine lactones (AHLs) as signaling molecules to coordinate group behaviors at high cell densities. Past studies demonstrate that AHLs can adsorb to and promote the remodeling of lipid membranes in ways that could underpin cell-cell or host-cell interactions. Here, we report that changes in AHL acyl tail group length and oxidation state (e.g., the presence or absence of a 3-oxo group) can lead to differences in the interactions of eight naturally occurring AHLs in solution and in contact with model lipid membranes. Our results reveal that the presence of a 3-oxo group impacts remodeling when AHLs are placed in contact with supported lipid bilayers (SLBs) of the phospholipid 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC). Whereas AHLs that have 3-oxo groups generally promote the formation of microtubules, AHLs that lack 3-oxo groups generally form hemispherical caps on the surfaces of SLBs. These results are interpreted in terms of the time scales on which AHLs translocate across bilayers to relieve asymmetrical bilayer stress. Quartz crystal microbalance with dissipation measurements also reveal that 3-oxo AHLs associate with DOPC bilayers to a greater extent than their non-3-oxo analogues. In contrast, we observed no monotonic relationship between AHL tail length and bilayer reformation. Finally, we observed that 3-oxo AHLs facilitate greater transport or leakage of molecular cargo across the membranes of DOPC vesicles relative to AHLs without 3-oxo groups, also suggesting increased bilayer disruption and destabilization. These fundamental studies hint at interactions and associated multiscale phenomena that may inform current interpretations of the behaviors of AHLs in biological contexts. These results could also provide guidance useful for the design of new classes of synthetic materials (e.g., sensor elements or drug delivery vehicles) that interact with or respond selectively to communities of bacteria that use 3-oxo AHLs for cell-cell communication.

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

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 large-scale remodeling in model lipid membranes. Characterization of supported lipid bilayers (SLBs) of the phospholipid 1,2-dioleoyl-sn-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-to-cell 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.

3D imaging of the early embryonic chicken heart with focused ion beam scanning electron microscopy.

Early embryonic heart development is a period of dynamic growth and remodeling, with rapid changes occurring at the tissue, cell, and subcellular levels. A detailed understanding of the events that establish the components of the heart wall has been hampered by a lack of methodologies for three-dimensional (3D), high-resolution imaging. Focused ion beam scanning electron microscopy (FIB-SEM) is a novel technology for imaging 3D tissue volumes at the subcellular level. FIB-SEM alternates between imaging the block face with a scanning electron beam and milling away thin sections of tissue with a FIB, allowing for collection and analysis of 3D data. FIB-SEM was used to image the three layers of the day 4 chicken embryo heart: myocardium, cardiac jelly, and endocardium. Individual images obtained with FIB-SEM were comparable in quality and resolution to those obtained with transmission electron microscopy. Up to 1,100 serial images were obtained in 4 nm increments at 4.88 nm resolution, and image stacks were aligned to create volumes 800-1,500 µm3 in size. Segmentation of organelles revealed their organization and distinct volume fractions between cardiac wall layers. We conclude that FIB-SEM is a powerful modality for 3D subcellular imaging of the embryonic heart wall.

Identification of small molecules that strongly inhibit bacterial quorum sensing using a high-throughput lipid vesicle lysis assay.

Strategies to both monitor and block bacterial quorum sensing (QS), and thus associated infections, are of significant interest. We developed a straightforward assay to monitor biosurfactants and lytic agents produced by bacteria under the control of QS. The method is based on the lysis of synthetic lipid vesicles containing the environmentally sensitive fluorescent dye calcein. This assay allows for the in situ screening of compounds capable of altering biosurfactant production by bacteria, and thereby the identification of molecules that could potentially modulate QS pathways, and avoids the constraints of many of the cell-based assays in use today. Application of this assay in a high-throughput format revealed five molecules capable of blocking vesicle lysis by S. aureus. Two of these compounds were found to almost completely inhibit agr-based QS in S. aureus and represent the most potent small-molecule-derived QS inhibitors reported in this formidable pathogen.

Membrane Remodeling and Stimulation of Aggregation Following α-Synuclein Adsorption to Phosphotidylserine Vesicles.

α-Synuclein is an intrinsically disordered protein abundant in presynaptic terminals in neurons and in synaptic vesicles. α-Synuclein's interaction with lipid bilayers is important not only for its normal physiological function but also in its pathological aggregation and deposition as Lewy bodies in Parkinson's disease. α-Synuclein binds preferentially to lipids with acidic head groups and to high-curvature vesicles and can modulate membrane curvature. The relationship between the protein's role as a membrane curvature sensor and generator and the role of membranes in facilitating its aggregation remains unknown. We investigated the interaction of α-synuclein with vesicles of 1,2-dioleoyl-sn-glycero-3-phospho-l-serine (DOPS) or 1,2-dilauroyl-sn-glycero-3-phospho-l-serine (DLPS). Using nanoparticle tracking along with electron microscopy, we demonstrate that α-synuclein induces extensive vesicle disruption and membrane remodeling into discoids, tubules, and ribbons with DLPS vesicles but not DOPS. Coarse-grained molecular dynamics simulations revealed that adsorption of α-synuclein to DLPS but not DOPS vesicles induced vesicle elongation and redistribution of protein to regions of higher curvature, a process that could drive protein aggregation. In agreement with this hypothesis, DLPS but not DOPS strongly stimulated α-synuclein aggregation. Our results provide new insights into the critical contribution of bilayer stability in the membrane response to α-synuclein adsorption and in stimulation of aggregation.

Bacterial Quorum Sensing Signals Self-Assemble in Aqueous Media to Form Micelles and Vesicles: An Integrated Experimental and Molecular Dynamics Study.

Many species of common bacteria communicate and coordinate group behaviors, including toxin production and surface fouling, through a process known as quorum sensing (QS). In Gram-negative bacteria, QS is regulated by N-acyl-l-homoserine lactones (AHLs) that possess a polar homoserine lactone headgroup and a nonpolar aliphatic tail. Past studies demonstrate that AHLs can aggregate in water or adsorb at interfaces, suggesting that molecular self-assembly could play a role in processes that govern bacterial communication. We used a combination of biophysical characterization and atomistic molecular dynamics (MD) simulations to characterize the self-assembly behaviors of 12 structurally related AHLs. We used static light scattering and measurements of surface tension to characterize the assembly of four naturally occurring AHLs (3-oxo-C8-AHL, 3-oxo-C12-AHL, C12-AHL, and C16-AHL) in aqueous media and determine their critical aggregation concentrations (CACs). MD simulations and alchemical free energy calculations were used to predict thermodynamically preferred aggregate structures for each AHL. Those calculations predicted that AHLs with 10 or 12 tail carbon atoms should form spherical micelles and that AHLs with 14 or 16 tail carbon atoms should form vesicles in solution. Characterization of solutions of AHLs using negative stain transmission electron microscopy (TEM) and dynamic light scattering (DLS) revealed aggregates with sizes consistent with spherical micelles or small unilamellar vesicles for 3-oxo-C12-AHL and C12-AHL and the formation of large vesicles (∼250 nm) in solutions of C16-AHL. These experimental findings are in general agreement with our simulation predictions. Overall, our results provide insight into processes of self-assembly that can occur in this class of bacterial amphiphiles and, more broadly, provide a potential basis for understanding how AHL structure could influence processes that bacteria use to drive important group behaviors.