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.

Pseudomonas aeruginosa uses N-acylated L-homoserine lactone signals and a triumvirate of LuxR-type receptor proteins – LasR, RhlR, and QscR – for quorum sensing (QS). Each of these receptors can contribute to QS activation or repression, and thereby, the control of myriad virulence phenotypes in this pathogen. LasR has traditionally been considered at the top of the QS receptor hierarchy in P. aeruginosa; however, recent reports suggest that RhlR plays a more prominent role in infection than originally predicted, in some circumstances superseding LasR. Herein, we report the characterization of a set of synthetic, small molecule agonists and antagonists of RhlR. Using E. coli reporter strains, we demonstrate that many of these compounds can selectively activate or inhibit RhlR instead of LasR and QscR. Moreover, several molecules maintain their activities in P. aeruginosa at concentrations analogous to native RhlR-signal levels. These compounds represent useful chemical probes to study the role of RhlR in the complex QS circuitry of P. aeruginosa, its direct (and indirect) effects on virulence, and its overall merit as a target for anti-infective therapy.

Many bacteria regulate gene expression through a cell-cell signaling process called quorum sensing (QS). In proteobacteria, QS is largely mediated by signaling molecules known as N-acylated L-homoserine lactones (AHLs) and their associated intracellular LuxR-type receptors. The design of non-native small molecules capable of inhibiting LuxR-type receptors, and thereby QS, in proteobacteria is an active area of research, and numerous lead compounds are AHL derivatives that mimic native AHL signals. Much of this past work has focused on the pathogen Pseudomonas aeruginosa, which controls an arsenal of virulence factors and biofilm formation through QS. The MexAB-OprM drug efflux pump has been shown to play a role in the secretion of the major AHL signal in P. aeruginosa, N-(3-oxododecanoyl) L-homoserine lactone. In the current study, we show that a variety of non-native AHLs and related derivatives capable of inhibiting LuxR-type receptors in P. aeruginosa display significantly higher potency in a P. aeruginosa Δ(mexAB-oprM) mutant, suggesting that MexAB-OprM also recognizes these compounds as substrates. We also demonstrate that the potency of 5,6-dimethyl-2-aminobenzimidazole, recently shown to be a QS and biofilm inhibitor in P. aeruginosa, is not affected by the presence or absence of the MexAB-OprM pump. These results have implications for the use of non-native AHLs and related derivatives as QS modulators in P. aeruginosa and other bacteria, and provide a potential design strategy for the development of new QS modulators that are resistant to active efflux.

In an effort to identify novel antibacterial chemotypes, we performed a whole‐cell screen for inhibitors of Staphylococcus aureus growth and pursued those compounds with previously uncharacterized antibacterial activity. This process resulted in the identification of a benzothiazolium salt, ABTZ‐1, that displayed potent antibacterial activity against Gram‐positive pathogens. Several clinically desirable qualities were demonstrated for ABTZ‐1 including potent activity against multidrug‐resistant clinical isolates of methicillin‐resistant S. aureus (MRSA) and vancomycin‐resistant enterococci (VRE), retention of this activity in human serum, and low hemolytic activity. The antibacterial activity of ABTZ‐1 was attributed to its inhibition of bacterial translation, as this compound prevented the incorporation of [35S]methionine into S. aureus proteins, and ABTZ‐1‐resistant strains were cross‐resistant to known inhibitors of bacterial translation. ABTZ‐1 represents a promising new class of antibacterial agents.

The inside cover picture shows the inhibition of ribosomal protein synthesis in Staphylococcus aureus by the benzothiazolium salt, ABTZ-1. This antibacterial, which inhibits the growth of multidrug-resistant Gram-positive bacteria, prevents the incorporation of radiolabeled methionine into S. aureus protein.Strains resistant to ABTZ-1 also display cross-resistance to fusidic acid and streptogramins. For more information on the discovery and characterization of ABTZ-1 by P. J. Hergenrother et al., see p. 574 ff.