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.

Chemical strategies to block quorum sensing (QS) could provide a route to attenuate virulence in bacterial pathogens. Considerable research has focused on this approach in Pseudomonas aeruginosa, which uses the LuxR-type receptor LasR to regulate much of its QS network. Non-native ligands that antagonize LasR have been developed, yet we have little understanding of the mode by which these compounds interact with LasR and alter its function, as the receptor is unstable in their presence. Herein, we report an approach to circumvent this challenge through the study of a series of synthetic LasR agonists with varying levels of potency. Structural investigations of these ligands with the LasR ligand-binding domain reveal that certain agonists can enforce a conformation that deviates from that observed for other, often more potent agonists. These results, when combined with cell-based and biophysical analyses, suggest a functional model for LasR that could guide future ligand design.

 Virulence in the Gram-negative pathogen Pseudomonas aeruginosa relies in part on the efficient functioning of two LuxI/R dependent quorum sensing (QS) cascades, namely, the LasI/R and RhlI/R systems that generate and respond to N-(3-oxo)-dodecanoyl-l-homoserine lactone and N-butyryl-l-homoserine lactone, respectively. The two acyl homoserine lactone (AHL) synthases, LasI and RhlI, use 3-oxododecanoyl-ACP and butyryl-ACP, respectively, as the acyl-substrates to generate the corresponding autoinducer signals for the bacterium. Although AHL synthases represent excellent targets for developing QS modulators in P. aeruginosa, and in other related bacteria, the identification of potent and signal synthase specific inhibitors has represented a significant technical challenge. In the current study, we sought to test the utility of AHL analogs as potential modulators of an AHL synthase and selected RhlI in P. aeruginosa as an initial target. We systematically varied the chemical functionalities of the AHL headgroup, acyl chain tail, and head-to-tail linkage to construct a small library of signal analogs and evaluated them for RhlI modulatory activity. Although the native N-butyryl-l-homoserine lactone did not inhibit RhlI, we discovered that several of our long-chain, unsubstituted acyl-d-homoserine lactones and acyl-d-homocysteine thiolactones inhibited while a few of the 3-oxoacyl-chain counterparts activated the enzyme. Additional mechanistic investigations with acyl-substrate analogs and docking experiments with AHL analogs revealed two distinct inhibitor and activator binding pockets in the enzyme. This study provides the first evidence of the yet untapped potential of AHL analogs as signal synthase modulators of QS pathways.