A Quorum-Sensing Antagonist Targets Both Membrane-Bound and Cytoplasmic Receptors and Controls Bacterial Pathogenicity

Abstract

Summary Quorum sensing is a process of bacterial communication involving production and detection of secreted molecules called autoinducers. Gram-negative bacteria use acyl-homoserine lactone (AHL) autoinducers, which are detected by one of two receptor types. First, cytoplasmic LuxR-type receptors bind accumulated intracellular AHLs. AHL-LuxR complexes bind DNA and alter gene expression. Second, membrane-bound LuxN-type receptors bind accumulated extracellular AHLs. AHL-LuxN complexes relay information internally by phosphorylation cascades that direct gene-expression changes. Here we show that a small molecule, previously identified as an antagonist of LuxN-type receptors, is also a potent antagonist of the LuxR family, despite differences in receptor structure, localization, AHL specificity, and signaling mechanism. Derivatives were synthesized and optimized for potency, and in each case, we characterized the mode of action of antagonism. The most potent antagonist protects Caenorhabditis elegans from quorum-sensing-mediated killing by Chromobacterium violaceum, validating the notion that targeting quorum sensing has potential for antimicrobial drug development.

Exploiting Quorum Sensing To Confuse Bacterial Pathogens

“…In another example of cross-inhibition, a high-throughput chemical screen of approximately 35,000 compounds led to the identification of 15 small molecules that inhibited the LuxN/AI-1 QS circuit in V. harveyi (129). Despite the fact that these molecules were initially identified in a screen for inhibition of the membrane-bound sensor kinase LuxN, one of these molecules, 4606-4237 (Table 3), which carries a phenoxyl group at the terminus of the acyl chain and has a homocysteine thiolactone group in place of the homoserine lactone moiety, was later demonstrated to act as an antagonist of the cytoplasmic receptor CviR of C. violaceum (128). Thus, despite the differences in sequence and/or signal transduction mechanisms by AHL receptor proteins, the similarities of their ligand-binding pockets can be sufficient to allow for cross-inhibition by the same inhibitor compound.…”

“…Interestingly, the mechanism by which analogues antagonize LuxR-type receptor activity also appears to be variable depending on the ligand and the receptor. Swem et al generated and tested various analogues of the previously identified CviR antagonist 4606-4237, leading to the identification of C10HSL, CTL, and CL as additional CviR antagonists (Table 3) (128). Studies using purified CviR protein complexed with these antagonists indicated that three of the antagonists function by preventing receptor association with target DNA, while C10HSL functions instead by preventing a productive interaction with RNA polymerase while having limited impact on DNA binding by CviR (128).…”

“…Swem et al generated and tested various analogues of the previously identified CviR antagonist 4606-4237, leading to the identification of C10HSL, CTL, and CL as additional CviR antagonists (Table 3) (128). Studies using purified CviR protein complexed with these antagonists indicated that three of the antagonists function by preventing receptor association with target DNA, while C10HSL functions instead by preventing a productive interaction with RNA polymerase while having limited impact on DNA binding by CviR (128). More recently, structure-function studies and cocrystallization were used to further elucidate the mechanism by which these analogues inhibit CviR activity.…”

“…(186). Furthermore, studies with the CviR antagonist CL have shown promise in in vivo infection models, as administration of CL at a concentration of 20 M was able to increase the life span of C. elegans following infection with WT C. violaceum from 2 days to 7 days (128). Given that a cviI mutant deficient for QS is able to kill C. elegans in an average of 12 days, the 5-day increase in survival afforded by CL treatment is substantial (128).…”

“…Furthermore, studies with the CviR antagonist CL have shown promise in in vivo infection models, as administration of CL at a concentration of 20 M was able to increase the life span of C. elegans following infection with WT C. violaceum from 2 days to 7 days (128). Given that a cviI mutant deficient for QS is able to kill C. elegans in an average of 12 days, the 5-day increase in survival afforded by CL treatment is substantial (128). Obviously, the testing of other AHL receptor antagonists in vivo will be a crucial step in evaluating their therapeutic potential, especially for AHL-like molecules containing a lactone ring moiety that may be subject to the same degradation or inactivation as native AHLs in the host.…”