Characterization of natural product inhibitors of quorum sensing in Pseudomonas aeruginosa reveals competitive inhibition of RhlR by ortho-vanillin.

Quorum sensing (QS) is a cell-cell signaling system that enables bacteria to coordinate population density-dependent changes in behavior. This chemical communication pathway is mediated by diffusible N-acyl L-homoserine lactone signals and cytoplasmic signal-responsive LuxR-type receptors in Gram-negative bacteria. As many common pathogenic bacteria use QS to regulate virulence, there is significant interest in disrupting QS as a potential therapeutic strategy. Prior studies have implicated the natural products salicylic acid, cinnamaldehyde and other related benzaldehyde derivatives as inhibitors of QS in the opportunistic pathogen Pseudomonas aeruginosa, yet we lack an understanding of the mechanisms by which these compounds function. Herein, we evaluate the activity of a set of benzaldehyde derivatives using heterologous reporters of the P. aeruginosa LasR and RhlR QS signal receptors. We find that most tested benzaldehyde derivatives can antagonize LasR or RhlR reporter activation at micromolar concentrations, although certain molecules also caused mild growth defects and nonspecific reporter antagonism. Notably, several compounds showed promising RhlR or LasR specific inhibitory activities over a range of concentrations below that causing toxicity. Ortho-Vanillin, a previously untested compound, was the most promising within this set. Competition experiments against the native ligands for LasR and RhlR revealed that ortho-vanillin can interact competitively with RhlR but not with LasR. Overall, these studies expand our understanding of benzaldehyde activities in the LasR and RhlR receptors and reveal potentially promising effects of ortho-vanillin as a small molecule QS modulator against RhlR.

Abiotic Small Molecule Inhibitors and Activators of the LasR Quorum Sensing Receptor in Pseudomonas aeruginosa with Potencies Comparable or Surpassing N-Acyl Homoserine Lactones.

The opportunistic pathogen Pseudomonas aeruginosa controls almost 10% of its genome, including myriad virulence genes, via a cell-to-cell chemical communication system called quorum sensing (QS). Small molecules that either inhibit or activate QS in P. aeruginosa represent useful research tools to study the role of this signaling pathway in infection and interrogate its viability as an antivirulence target. However, despite active research in this area over the past 20+ years, there are relatively few synthetic compounds known to strongly inhibit or activate QS in P. aeruginosa. Most reported QS modulators in this pathogen are of low potency or have structural liabilities that limit their application in biologically relevant environments such as mimics of the native N-acyl l-homoserine lactone (AHL) signals. Here, we report the results of a high-throughput screen for abiotic small molecules that target LasR, a key QS regulator in P. aeruginosa. We screened a 25,000-compound library and discovered four new structural classes of abiotic LasR modulators. These compounds include antagonists that surpass the potency of all known AHL-type compounds and mimetics thereof, along with an agonist with potency approaching that of LasR's native ligand. The novel structures of this compound set, along with their anticipated robust physicochemical profiles, underscore their potential value as probe molecules to interrogate the roles of QS in this formidable pathogen.

Characterization of an Autoinducing Peptide Signal Reveals Highly Efficacious Synthetic Inhibitors and Activators of Quorum Sensing and Biofilm Formation in Listeria monocytogenes.

Bacteria can use chemical signals to assess their local population density in a process called quorum sensing (QS). Many of these bacteria are common pathogens, including Gram-positive bacteria that utilize agr QS systems regulated by macrocyclic autoinducing peptide (AIP) signals. Listeria monocytogenes, an important foodborne pathogen, uses an agr system to regulate a variety of virulence factors and biofilm formation, yet little is known about the specific roles of agr in Listeria infection and its persistence in various environments. Herein, we report synthetic peptide tools that will enable the study of QS in Listeria. We identified a 6-mer AIP signal in L. monocytogenes supernatants and selected it as a scaffold around which a collection of non-native AIP mimics was designed and synthesized. These peptides were evaluated in cell-based agr reporter assays to generate structure-activity relationships for AIP-based agonism and antagonism in L. monocytogenes. We discovered synthetic agonists with increased potency relative to native AIP and a synthetic antagonist capable of reducing agr activity to basal levels. Notably, the latter peptide was able to reduce biofilm formation by over 90%, a first for a synthetic QS modulator in wild-type L. monocytogenes. The lead agr agonist and antagonist in L. monocytogenes were also capable of antagonizing agr signaling in the related pathogen Staphylococcus aureus, further extending their utility and suggesting different mechanisms of agr activation in these two pathogens. This study represents an important first step in the application of chemical methods to modulate QS and concomitant virulence outcomes in L. monocytogenes.

Genetic mapping of microbial and host traits reveals production of immunomodulatory lipids by Akkermansia muciniphila in the murine gut.

The molecular bases of how host genetic variation impacts the gut microbiome remain largely unknown. Here we used a genetically diverse mouse population and applied systems genetics strategies to identify interactions between host and microbe phenotypes including microbial functions, using faecal metagenomics, small intestinal transcripts and caecal lipids that influence microbe-host dynamics. Quantitative trait locus (QTL) mapping identified murine genomic regions associated with variations in bacterial taxa; bacterial functions including motility, sporulation and lipopolysaccharide production and levels of bacterial- and host-derived lipids. We found overlapping QTL for the abundance of Akkermansia muciniphila and caecal levels of ornithine lipids. Follow-up in vitro and in vivo studies revealed that A. muciniphila is a major source of these lipids in the gut, provided evidence that ornithine lipids have immunomodulatory effects and identified intestinal transcripts co-regulated with these traits including Atf3, which encodes for a transcription factor that plays vital roles in modulating metabolism and immunity. Collectively, these results suggest that ornithine lipids are potentially important for A. muciniphila-host interactions and support the role of host genetics as a determinant of responses to gut microbes.

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.

Potent pan-group quorum sensing inhibitors in Staphylococcus aureus revealed by N-terminal tailoring of peptidomimetics.

Staphylococcus aureus uses small peptides to assess its population densisty (i.e., quorum sensing) and regulate virulence at high cell number. Here, we report the design and synthesis of peptidomimetics based on these native signals that strongly block this communication pathway in all four specificity groups of S. aureus.

Chemical and biomolecular insights into the Staphylococcus aureus agr quorum sensing system: Current progress and ongoing challenges.

Staphylococcus aureus is a ubiquitous bacterium that has become a major threat to human health due to its extensive toxin production and tremendous capacity for antibiotic resistance (e.g., MRSA "superbug" infections). Amid a worsening antibiotic resistance crisis, new strategies to combat this deadly microbe that remove the selective pressure of traditional approaches are in high demand. S. aureus utilizes an accessory gene regulator (agr) quorum sensing network to monitor its local cellular population and trigger a devastating communal attack, like an invading horde, once a threshold cell density has been reached. The role of the agr system in a range of disease types is still being unraveled. Herein, we discuss the present-day biochemical understanding of agr along with unresolved details, describe its connection to the progression of infection, and review how chemical strategies have been implemented to study and intercept this signaling pathway. This research is illuminating the potential of agr as an anti-virulence target in S. aureus and should inform the study of similar, yet less studied, agr systems in related bacterial pathogens.

Small Molecules with Either Receptor-Selective or Pan-Receptor Activity in the Three LuxR-Type Receptors that Regulate Quorum Sensing in Pseudomonas aeruginosa.

Quorum sensing (QS) allows bacteria to assess their local cell density using chemical signals and plays a prominent role in the ability of common pathogens to infect a host. Non-native molecules capable of attenuating bacterial QS represent useful tools to explore the role of this pathway in virulence. As individual bacterial species can have multiple QS systems and/or reside in mixed communities with other bacteria capable of QS, chemical tools that are either selective for one QS system or "pan-active" and target all QS pathways are of significant interest. Herein we outline the analysis of a set of compounds reported to target one QS system in Pseudomonas aeruginosa for their activity in two other QS circuits in this pathogen and the discovery of molecules with novel activity profiles, including subsets that agonize all three QS systems, agonize one but antagonize the other two, or strongly antagonize just one.

Autoinducer-fluorophore conjugates enable FRET in LuxR proteins in vitro and in cells.

Cell-to-cell signaling, or quorum sensing (QS), in many Gram-negative bacteria is governed by small molecule signals (N-acyl-L-homoserine lactones, AHLs) and their cognate receptors (LuxR-type proteins). The mechanistic underpinnings of QS in these bacteria are severely limited due to the challenges of isolating and manipulating most LuxR-type proteins. Reports of quantitative direct-binding experiments on LuxR-type proteins are scarce, and robust and generalizable methods that provide such data are largely nonexistent. We report herein a Förster resonance energy transfer (FRET) assay that leverages (1) conserved tryptophans located in the LuxR-type protein ligand-binding site and synthetic fluorophore-AHL conjugates, and (2) isolation of the proteins bound to weak agonists. The FRET assay permits straightforward measurement of ligand-binding affinities with receptor-either in vitro or in cells-and was shown to be compatible with six LuxR-type proteins. These methods will advance fundamental investigations of LuxR-type protein mechanism and the development of small molecule QS modulators.

Slippery Antifouling Polymer Coatings Fabricated Entirely from Biodegradable and Biocompatible Components.

We report the design of slippery liquid-infused porous surfaces (SLIPS) fabricated from building blocks that are biodegradable, edible, or generally regarded to be biocompatible. Our approach involves infusion of lubricating oils, including food oils, into nanofiber-based mats fabricated by electrospinning or blow spinning of poly(ε-caprolactone), a hydrophobic biodegradable polymer used widely in medical implants and drug delivery devices. This approach leads to durable and biodegradable SLIPS that prevent fouling by liquids and other materials, including microbial pathogens, on objects of arbitrary shape, size, and topography. This degradable polymer approach also provides practical means to design "controlled-release" SLIPS that release molecular cargo at rates that can be manipulated by the properties of the infused oils (e.g., viscosity or chemical structure). Together, our results provide new designs and introduce useful properties and behaviors to antifouling SLIPS, address important issues related to biocompatibility and environmental persistence, and thus advance new potential applications, including the use of slippery materials for food packaging, industrial and marine coatings, and biomedical implants.

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.

Fabrication of Slippery Liquid-Infused Coatings in Flexible Narrow-Bore Tubing.

We report a layer-by-layer suction-and-flow approach that enables the fabrication of polymer-based "slippery" liquid-infused porous surfaces (SLIPS) in the confined luminal spaces of flexible, narrow-bore tubing. These SLIPS-coated tubes can prevent or strongly reduce surface fouling after prolonged contact, storage, or flow of a broad range of complex fluids and viscoelastic materials, including many that are relevant in the contexts of medical devices (e.g., blood and urine), food processing (beverages and fluids), and other commercial and industrial applications. The robust and mechanically compliant nature of the nanoporous coating used to host the lubricating oil phase allows these coated tubes to be bent, flexed, and coiled repeatedly without affecting their inherent slippery and antifouling behaviors. Our results also show that SLIPS-coated tubes can prevent the formation of bacterial biofilms after prolonged and repeated flow-based exposure to the human pathogen Staphylococcus aureus and that the anti-biofouling properties of these coated tubes can be further improved or prolonged by coupling this approach with strategies that permit the sustained release of broad-spectrum antimicrobial agents. The suction-and-flow approach used here enables the application of slippery coatings in the confined luminal spaces of narrow-bore tubing that are difficult to access using several other methods for the fabrication of liquid-infused coatings and can be applied to tubing of arbitrary length and diameter. We anticipate that the materials and approaches reported here will prove useful for reducing or preventing biofouling, process fouling, and the clogging or occlusion of tubing in a wide range of consumer, industrial, and healthcare-oriented applications.

Slippery nanoemulsion-infused porous surfaces (SNIPS): anti-fouling coatings that can host and sustain the release of water-soluble agents.

We report the design of 'slippery' nanoemulsion-infused porous surfaces (SNIPS). These materials are strongly anti-fouling to a broad range of substances, including microorganisms. Infusion with water-in-oil nanoemulsions also endows these slippery coatings with the ability to host and control or sustain the release of water-soluble agents, including polymers, peptides, and nucleic acids, opening the door to new applications of liquid-infused materials.

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.

Liquid Crystal-Infused Porous Polymer Surfaces: A "Slippery" Soft Material Platform for the Naked-Eye Detection and Discrimination of Amphiphilic Species.

We report the design and characterization of liquid crystal (LC)-infused porous polymer membranes that can detect and report on the presence of natural and synthetic amphiphiles in aqueous solution. We demonstrate that thermotropic LCs can be infused into nanoporous polymer membranes to yield LC-infused surfaces that exhibit slippery behaviors in contact with a range of aqueous fluids. In contrast to conventional liquid-infused surfaces (LIS) or slippery liquid-infused porous surfaces (SLIPS) prepared using isotropic oils, aqueous solutions slide over the surfaces of these LC-infused materials at speeds that depend strongly upon the composition of the fluid, including the presence, concentration, or structure of a dissolved surfactant. In general, the sliding times of aqueous droplets on these LC-infused surfaces increase significantly (e.g., from times on the order of seconds to times on the order of minutes) with increasing amphiphile concentration, allowing sliding times to be used to estimate the concentration of the amphiphile. Additional experiments revealed other intrinsic and extrinsic variables or parameters that can be used to further manipulate droplet sliding times and discriminate among amphiphiles of similar structure. Our results are consistent with a physical picture that involves reversible changes in the interfacial orientation of anisotropic LCs mediated by the interfacial adsorption of amphiphiles. These materials thus permit facile "naked-eye" detection and discrimination of amphiphiles in aqueous samples using equipment no more sophisticated than a stopwatch. We demonstrate the potential utility of these LC-infused surfaces for the unaided, naked-eye detection and monitoring of amphiphilic biotoxins in small droplets of fluid extracted directly from cultures of two common bacterial pathogens (Pseudomonas aeruginosa and Staphylococcus aureus). The ability to translate molecular interactions at aqueous/LC interfaces into large and readily observed changes in the sliding times of small aqueous droplets on surfaces could open the door to new applications for antifouling, liquid-infused materials in the context of environmental sensing and other fundamental and applied areas.

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.

Non-Native Peptides Capable of Pan-Activating the agr Quorum Sensing System across Multiple Specificity Groups of Staphylococcus epidermidis.

Staphylococcus epidermidis is a leading cause of hospital-acquired infections. Traditional antibiotics have significantly reduced efficacy against this pathogen due to its ability to form biofilms on abiotic surfaces and drug resistance. The accessory gene regulator (agr) quorum sensing system is directly involved in S. epidermidis pathogenesis. Activation of agr is achieved via binding of the autoinducing peptide (AIP) signal to the extracellular sensor domain of its cognate receptor, AgrC. Divergent evolution has given rise to four agr specificity groups in S. epidermidis defined by the unique AIP sequence used by each group (AIPs-I-IV) with observed cross-group activities. As agr agonism has been shown to reduce biofilm growth in S. epidermidis, the development of pan-group activators of the agr system is of interest as a potential antivirulence strategy. To date, no synthetic compounds have been identified that are capable of appreciably activating the agr system of more than one specificity group of S. epidermidis or, to our knowledge, of any of the other Staphylococci. Here, we report the characterization of the structure-activity relationships for agr agonism by S. epidermidis AIP-II and AIP-III and the application of these new SAR data and those previously reported for AIP-I for the design and synthesis of the first multigroup agr agonists. These non-native peptides were capable of inducing the expression of critical biofilm dispersal agents (i.e., phenol-soluble modulins) in cell culture and represent new tools to study the role of quorum sensing in S. epidermidis infections.

Chemical Control of Quorum Sensing in E. coli: Identification of Small Molecule Modulators of SdiA and Mechanistic Characterization of a Covalent Inhibitor.

Enterohemorrhagic Escherichia coli (EHEC) is the causative agent of severe diarrheal disease in humans. Cattle are the natural reservoir of EHEC, and approximately 75% of EHEC infections in humans stem from bovine products. Many common bacterial pathogens, including EHEC, rely on chemical communication systems, such as quorum sensing (QS), to regulate virulence and facilitate host colonization. EHEC uses SdiA from E. coli (SdiAEC), an orphan LuxR-type receptor, to sense N-acyl l-homoserine lactone (AHL) QS signals produced by other members of the bovine enteric microbiome. SdiAEC regulates two phenotypes critical for colonizing cattle: acid resistance and the formation of attaching and effacing lesions. Despite the importance of SdiAEC, there is very little known about its selectivity for different AHL signals, and no chemical inhibitors that act specifically on SdiAEC have been reported. Such compounds would represent valuable tools to study the roles of QS in EHEC virulence. To identify chemical modulators of SdiAEC and delineate the structure-activity relationships (SARs) for AHL activity in this receptor, we report herein the screening of a focused library composed largely of AHLs and AHL analogues in an SdiAEC reporter assay. We describe the identity and SARs of potent modulators of SdiAEC activity, examine the promiscuity of SdiAEC, characterize the mechanism of a covalent inhibitor, and provide phenotypic assay data to support that these compounds can control SdiAEC-dependent acid resistance in E. coli. These SdiAEC modulators could be used to advance the study of LuxR-type receptor/ligand interactions, the biological roles of orphan LuxR-type receptors, and potential QS-based therapeutic approaches.