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.

Quorum sensing (QS), a bacterial cell-to-cell communication system mediated by small molecules and peptides, has received significant interest as a potential target to block infection. The common pathogen Pseudomonas aeruginosa uses QS to regulate many of its virulence phenotypes at high cell densities, and the LasR QS receptor plays a critical role in this process. Small molecule tools that inhibit LasR activity would serve to illuminate its role in P. aeruginosa virulence, but we currently lack highly potent and selective LasR antagonists, despite considerable research in this area. V-06-018, an abiotic small molecule discovered in a high-throughput screen, represents one of the most potent known LasR antagonists, but has seen little study since its initial report. Herein, we report a systematic study of the structure-activity relationships (SARs) that govern LasR antagonism by V-06-018. We synthesized a focused library of V-06-018 derivatives and evaluated the library for bioactivity using a variety of cell-based LasR reporter systems. The SAR trends revealed by these experiments allowed us to design probes with 10-fold greater potency than V-06-018 and 100-fold greater potency than other commonly used N-acyl L-homoserine lactone (AHL)-based LasR antagonists, along with high selectivities for LasR. Biochemical experiments to probe the mechanism of antagonism by V-06-018 and its analogs support these compounds interacting with the native ligand-binding site in LasR and, at least in part, stabilizing an inactive form of the protein. The compounds described herein are the most potent and efficacious antagonists of LasR known, and represent robust probes both for characterizing the mechanisms of LuxR-type QS and for chemical biology research in general in the growing QS field.

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 selfassembly 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.

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 broadspectrum 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.

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