Synthetic Peptides Capable of Potent Multigroup Staphylococcal Quorum Sensing Activation and Inhibition in Both Cultures and Biofilm Communities.

The pathogen Staphylococcus epidermidis uses a chemical signaling process, i.e., quorum sensing (QS), to form robust biofilms and cause human infection. Many questions remain about QS in S. epidermidis, as it uses this intercellular communication pathway to both negatively and positively regulate virulence traits. Herein, we report synthetic multigroup agonists and antagonists of the S. epidermidis accessory gene regulator (agr) QS system capable of potent superactivation and complete inhibition, respectively. These macrocyclic peptides maintain full efficacy across the three major agr specificity groups, and their activity can be "mode-switched" from agonist to antagonist via subtle residue-specific structural changes. We describe the design and synthesis of these non-native peptides and demonstrate that they can appreciably decrease biofilm formation on abiotic surfaces, underscoring the potential for agr agonism as a route to block S. epidermidis virulence. Additionally, we show that both the S. epidermidis agonists and antagonists are active in S. aureus, another common pathogen with a related agr system, yet only as antagonists. This result not only revealed one of the most potent agr inhibitors known in S. aureus but also highlighted differences in the mechanisms of agr agonism and antagonism between these related bacteria. Finally, our investigations reveal unexpected inhibitory behavior for certain S. epidermidis agr agonists at sub-activating concentrations, an observation that can be leveraged for the design of future probes with enhanced potencies. Together, these peptides provide a powerful tool set to interrogate the role of QS in S. epidermidis infections and in Staphylococcal pathogenicity in general.

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.

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.

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.

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.

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.

Conformational Switch to a ß-Turn in a Staphylococcal Quorum Sensing Signal Peptide Causes a Dramatic Increase in Potency.

We report the solution-phase structures of native signal peptides and related analogs capable of either strongly agonizing or antagonizing the AgrC quorum sensing (QS) receptor in the emerging pathogen Staphylococcus epidermidis. Chronic S. epidermidis infections are often recalcitrant to traditional therapies due to antibiotic resistance and formation of robust biofilms. The accessory gene regulator (agr) QS system plays an important role in biofilm formation in this opportunistic pathogen, and the binding of an autoinducing peptide (AIP) signal to its cognate transmembrane receptor (AgrC) is responsible for controlling agr. Small molecules or peptides capable of modulating this binding event are of significant interest as probes to investigate both the agr system and QS as a potential antivirulence target. We used NMR spectroscopy to characterize the structures of the three native S. epidermidis AIP signals and five non-native analogs with distinct activity profiles in the AgrC-I receptor from S. epidermidis. These studies revealed a suite of structural motifs critical for ligand activity. Interestingly, a unique ß-turn was present in the macrocycles of the two most potent AgrC-I modulators, in both an agonist and an antagonist, which was distinct from the macrocycle conformation in the less-potent AgrC-I modulators and in the native AIP-I itself. This previously unknown ß-turn provides a structural rationale for these ligands' respective biological activity profiles. Development of analogs to reinforce the ß-turn resulted in our first antagonist with subnanomolar potency in AgrC-I, while analogs designed to contain a disrupted ß-turn were dramatically less potent relative to their parent compounds. Collectively, these studies provide new insights into the AIP:AgrC interactions crucial for QS activation in S. epidermidis and advance the understanding of QS at the molecular level.

Mechanism of agonism and antagonism of the Pseudomonas aeruginosa quorum sensing regulator QscR with non-native ligands.

Pseudomonas aeruginosa is an opportunistic pathogen that uses the process of quorum sensing (QS) to coordinate the expression of many virulence genes. During quorum sensing, N-acyl-homoserine lactone (AHL) signaling molecules regulate the activity of three LuxR-type transcription factors, LasR, RhlR and QscR. To better understand P. aeruginosa QS signal reception, we examined the mechanism underlying the response of QscR to synthetic agonists and antagonists using biophysical and structural approaches. The structure of QscR bound to a synthetic agonist reveals a novel mode of ligand binding supporting a general mechanism for agonist activity. In turn, antagonists of QscR with partial agonist activity were found to destabilize and greatly impair QscR dimerization and DNA binding. These results highlight the diversity of LuxR-type receptor responses to small molecule agonists and antagonists and demonstrate the potential for chemical strategies for the selective targeting of individual QS systems.

The State of the Union Is Strong: a Review of ASM's 6th Conference on Cell-Cell Communication in Bacteria.

The 6th American Society for Microbiology Conference on Cell-Cell Communication in Bacteria convened from 16 to 19 October 2017 in Athens, GA. In this minireview, we highlight some of the research presented at that meeting that addresses central questions emerging in the field, including the following questions. How are cell-cell communication circuits designed to generate responses? Where are bacteria communicating? Finally, why are bacteria engaging in such behaviors?

A comparative study of non-native N-acyl l-homoserine lactone analogs in two Pseudomonas aeruginosa quorum sensing receptors that share a common native ligand yet inversely regulate virulence.

Certain bacteria can coordinate group behaviors via a chemical communication system known as quorum sensing (QS). Gram-negative bacteria typically use N-acyl l-homoserine lactone (AHL) signals and their cognate intracellular LuxR-type receptors for QS. The opportunistic pathogen Pseudomonas aeruginosa has a relatively complex QS circuit in which two of its LuxR-type receptors, LasR and QscR, are activated by the same natural signal, N-(3-oxo)-dodecanoyl l-homoserine lactone. Intriguingly, once active, LasR activates virulence pathways in P. aeruginosa, while activated QscR can inactivate LasR and thus repress virulence. We have a limited understanding of the structural features of AHLs that engender either agonistic activity in both receptors or receptor-selective activity. Compounds with the latter activity profile could prove especially useful tools to tease out the roles of these two receptors in virulence regulation. A small collection of AHL analogs was assembled and screened in cell-based reporter assays for activity in both LasR and QscR. We identified several structural motifs that bias ligand activation towards each of the two receptors. These findings will inform the development of new synthetic ligands for LasR and QscR with improved potencies and selectivities.

Non-native autoinducer analogs capable of modulating the SdiA quorum sensing receptor in Salmonella enterica serovar Typhimurium.

Quorum sensing (QS) allows many common bacterial pathogens to coordinate group behaviors such as virulence factor production, host colonization, and biofilm formation at high population densities. This cell-cell signaling process is regulated by N -acyl L-homoserine lactone (AHL) signals, or autoinducers, and LuxR-type receptors in Gram-negative bacteria. SdiA is an orphan LuxR-type receptor found in Escherichia, Salmonella, Klebsiella, and Enterobacter genera that responds to AHL signals produced by other species and regulates genes involved in several aspects of host colonization. The inhibition of QS using non-native small molecules that target LuxR-type receptors offers a non-biocidal approach for studying, and potentially controlling, virulence in these bacteria. To date, few studies have characterized the features of AHLs and other small molecules capable of SdiA agonism, and no SdiA antagonists have been reported. Herein, we report the screening of a set of AHL analogs to both uncover agonists and antagonists of SdiA and to start to delineate structure-activity relationships (SARs) for SdiA:AHL interactions. Using a cell-based reporter of SdiA in Salmonella enterica serovar Typhimurium, several non-natural SdiA agonists and the first set of SdiA antagonists were identified and characterized. These compounds represent new chemical probes for exploring the mechanisms by which SdiA functions during infection and its role in interspecies interactions. Moreover, as SdiA is highly stable when produced in vitro, these compounds could advance fundamental studies of LuxR-type receptor:ligand interactions that engender both agonism and antagonism.

Simplified AIP-II Peptidomimetics Are Potent Inhibitors of Staphylococcus aureus AgrC Quorum Sensing Receptors.

The bacterial pathogen Staphylococcus aureus controls many aspects of virulence by using the accessory gene regulator (agr) quorum sensing (QS) system. The agr system is activated by a macrocyclic peptide signal known as an autoinducing peptide (AIP). We sought to develop structurally simplified mimetics of AIPs for use as chemical tools to study QS in S. aureus. Herein, we report new peptidomimetic AgrC receptor inhibitors based on a tail-truncated AIP-II peptide that have almost analogous inhibitory activities to the parent peptide. Structural comparison of one of these peptidomimetics to the parent peptide and a highly potent, all-peptide-derived, S. aureus agr inhibitor (AIP-III D4A) revealed a conserved hydrophobic motif and overall amphipathic nature. Our results suggest that the AIP scaffold is amenable to structural mimicry and minimization for the development of synthetic agr inhibitors.

Slippery Liquid-Infused Porous Surfaces that Prevent Microbial Surface Fouling and Kill Non-Adherent Pathogens in Surrounding Media: A Controlled Release Approach.

Many types of slippery liquid-infused porous surfaces (or 'SLIPS') can resist adhesion and colonization by microorganisms. These 'slippery' materials thus offer new approaches to prevent fouling on a range of commercial and industrial surfaces, including biomedical devices. However, while SLIPS can prevent fouling on surfaces to which they are applied, they can currently do little to prevent the proliferation of non-adherent (planktonic) organisms, stop them from colonizing other surfaces, or prevent them from engaging in other behaviors that could lead to infection and associated burdens. Here, we report an approach to the design of multi-functional SLIPS that addresses these issues and expands the potential utility of slippery surfaces in antimicrobial contexts. Our approach is based on the incorporation and controlled release of small-molecule antimicrobial agents from the porous matrices used to host infused slippery oil phases. We demonstrate that SLIPS fabricated using nanoporous polymer multilayers can prevent short- and longer-term colonization and biofilm formation by four common fungal and bacterial pathogens (Candida albicans, Pseudomonas aeruginosa, Escherichia coli, and Staphylococcus aureus), and that the polymer and oil phases comprising these materials can be exploited to load and sustain the release of triclosan, a model hydrophobic and broad-spectrum antimicrobial agent, into surrounding media. This approach both improves the inherent anti-fouling properties of these materials and endows them with the ability to efficiently kill planktonic pathogens. Finally, we show that this approach can be used to fabricate dual-action SLIPS on complex surfaces, including the luminal surfaces of flexible catheter tubes. This strategy has the potential to be general; we anticipate that the materials, strategies, and concepts reported here will enable new approaches to the design of slippery surfaces with improved anti-fouling properties and open the door to new applications of slippery liquid-infused materials that host or promote the release of a variety of other active agents.

Interkingdom Responses to Bacterial Quorum Sensing Signals Regulate Frequency and Rate of Nodulation in Legume-Rhizobia Symbiosis.

Density-dependent phenotypic switching in bacteria, the phenomenon of quorum sensing (QS), is instrumental in many pathogenic and mutualistic behaviors. In many Gram-negative bacteria, QS is regulated by N-acylated-l-homoserine lactones (AHLs). Synthetic analogues of these AHLs hold significant promise for regulating QS at the host-symbiont interface. Regulation depends on refined temporal and spatial models of quorums under native conditions. Critical to this is an understanding of how the presence of these signals may affect a prospective host. We screened a library of AHL analogues for their ability to regulate the legume-rhizobia mutualistic symbiosis (nodulation) between Medicago truncatula and Sinorhizobium meliloti. Using an established QS-reporter line of S. meliloti and nodulation assays with wild-type bacteria, we identified compounds capable of increasing either the rate of nodule formation or total nodule number. Most importantly, we identified compounds with activity exclusive to either host or pathogen, underscoring the potential to generate QS modulators selective to bacteria with limited effects on a prospective host.

Characterization of structural elements in native autoinducing peptides and non-native analogues that permit the differential modulation of AgrC-type quorum sensing receptors in Staphylococcus aureus.

Staphylococcus aureus uses short macrocyclic peptides (i.e., autoinducing peptides, or AIPs) to assess its local population density in a cell-cell signaling mechanism called quorum sensing (QS). At high cell numbers, this pathogen can initiate many virulent behaviors that allow for the establishment of infection. Binding of the AIP signal to its cognate transmembrane AgrC-type receptor is a critical event in the QS signaling cascade; consequently, interference of AIP:receptor interactions may have the potential to prevent and eradicate certain S. aureus infections. To date, four pairs of AIP:AgrC receptors have been identified in S. aureus, each pair being utilized by a specific S. aureus group (I-IV). Other staphylococcal species also use closely related, but distinct, AIP:AgrC pairs to control QS. We seek to develop non-native ligands capable of intercepting AIP:AgrC binding in each S. aureus group and in related species. As these bacteria may use their respective AIP signal to attenuate the QS systems of other groups/species, such ligands would provide valuable chemical tools to probe possible interference mechanisms in a range of contexts. In the current study, we used solution-phase NMR techniques to characterize the 3-D structures of a set of known native and non-native peptides that have differential modulatory activity in certain AgrC receptors. Analysis of these structures revealed several distinct structural motifs that belay differential activity in selected S. aureus AgrC receptors (i.e., AgrC-I, AgrC-II, and AgrC-III). The results of this study can be leveraged for the design of new synthetic ligands with enhanced selectivities and potencies for these AgrC receptors.

Highly Stable, Amide-Bridged Autoinducing Peptide Analogues that Strongly Inhibit the AgrC Quorum Sensing Receptor in Staphylococcus aureus.

Blocking quorum sensing (QS) pathways has attracted considerable interest as an approach to suppress virulence in bacterial pathogens. Toward this goal, we recently developed analogues of a native autoinducing peptide (AIP-III) signal that can inhibit AgrC-type QS receptors and attenuate virulence phenotypes in Staphylococcus aureus. Application of these compounds is limited, however, as they contain hydrolytically unstable thioester linkages and have only low aqueous solubilities. Herein, we report amide-linked AIP analogues with greatly enhanced hydrolytic stabilities and solubilities relative to our prior analogues, whilst maintaining strong potencies as AgrC receptor inhibitors in S. aureus. These compounds represent powerful tools for the study of QS.

Small molecule disruption of quorum sensing cross-regulation in pseudomonas aeruginosa causes major and unexpected alterations to virulence phenotypes.

The opportunistic pathogen Pseudomonas aeruginosa uses three interwoven quorum-sensing (QS) circuits-Las, Rhl, and Pqs-to regulate the global expression of myriad virulence-associated genes. Interception of these signaling networks with small molecules represents an emerging strategy for the development of anti-infective agents against this bacterium. In the current study, we applied a chemical approach to investigate how the Las-Rhl-Pqs QS hierarchy coordinates key virulence phenotypes in wild-type P. aeruginosa. We screened a focused library of synthetic, non-native N-acyl l-homoserine lactones and identified compounds that can drastically alter production of two important virulence factors: pyocyanin and rhamnolipid. We demonstrate that these molecules act by targeting RhlR in P. aeruginosa, a QS receptor that has seen far less scrutiny to date relative to other circuitry. Unexpectedly, modulation of RhlR activity by a single compound induces inverse regulation of pyocyanin and rhamnolipid, a result that was not predicted using genetic approaches to interrogate QS in P. aeruginosa. Further, we show that certain RhlR agonists strongly repress Pqs signaling, revealing disruption of Rhl-Pqs cross-regulation as a novel mechanism for QS inhibition. These compounds significantly expand the known repertoire of chemical probes available to study RhlR in P. aeruginosa. Moreover, our results suggest that designing chemical agents to disrupt Rhl-Pqs crosstalk could be an effective antivirulence strategy to fight this common pathogen.

A Comparative Analysis of Synthetic Quorum Sensing Modulators in Pseudomonas aeruginosa: New Insights into Mechanism, Active Efflux Susceptibility, Phenotypic Response, and Next-Generation Ligand Design.

Quorum sensing (QS) is a chemical signaling mechanism that allows bacterial populations to coordinate gene expression in response to social and environmental cues. Many bacterial pathogens use QS to initiate infection at high cell densities. Over the past two decades, chemical antagonists of QS in pathogenic bacteria have attracted substantial interest for use both as tools to further elucidate QS mechanisms and, with further development, potential anti-infective agents. Considerable recent research has been devoted to the design of small molecules capable of modulating the LasR QS receptor in the opportunistic pathogen Pseudomonas aeruginosa. These molecules hold significant promise in a range of contexts; however, as most compounds have been developed independently, comparative activity data for these compounds are scarce. Moreover, the mechanisms by which the bulk of these compounds act are largely unknown. This paucity of data has stalled the choice of an optimal chemical scaffold for further advancement. Herein, we submit the best-characterized LasR modulators to standardized cell-based reporter and QS phenotypic assays in P. aeruginosa, and we report the first comprehensive set of comparative LasR activity data for these compounds. Our experiments uncovered multiple interesting mechanistic phenomena (including a potential alternative QS-modulatory ligand binding site/partner) that provide new, and unexpected, insights into the modes by which many of these LasR ligands act. The lead compounds, data trends, and mechanistic insights reported here will significantly aid the design of new small molecule QS inhibitors and activators in P. aeruginosa, and in other bacteria, with enhanced potencies and defined modes of action.