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

ABSTRACT: 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.

SUBMITTER: Wysoczynski-Horita CL

PROVIDER: S-EPMC5914528 | biostudies-literature | 2018 May

REPOSITORIES: biostudies-literature

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

Wysoczynski-Horita Christina L CL Boursier Michelle E ME Hill Ryan R Hansen Kirk K Blackwell Helen E HE Churchill Mair E A MEA

Molecular microbiology 20180311 3

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 appr ...[more]

PMID: 29437248

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Project description:Many Proteobacteria govern responses to changes in cell density by using acyl-homoserine lactone (AHL) quorum-sensing (QS) signaling. Similar to the LuxI-LuxR system described in Vibrio fischeri, a minimal AHL QS circuit comprises a pair of genes, a luxI-type synthase gene encoding an enzyme that synthesizes an AHL and a luxR-type AHL-responsive transcription regulator gene. In most bacteria that utilize AHL QS, cognate luxI and luxR homologs are found in proximity to each other on the chromosome. However, a number of recent reports have identified luxR homologs that are not linked to luxI homologs; in some cases luxR homologs have been identified in bacteria that have no luxI homologs. A luxR homolog without a linked luxI homologs is termed an orphan or solo. One of the first reports of an orphan was on QscR in Pseudomonas aeruginosa. The qscR gene was revealed by whole genome sequencing and has been studied in some detail. P. aeruginosa encodes two AHL synthases and three AHL responsive receptors, LasI-LasR form a cognate synthase-receptor pair as do RhlI-RhlR. QscR lacks a linked synthase and responds to the LasI-generated AHL. QS regulation of gene expression in P. aeruginosa employs multiple signals and occurs in the context of other interconnected regulatory circuits that control diverse physiological functions. QscR affects virulence of P. aeruginosa, and although it shows sensitivity to the LasI-generated AHL, 3-oxo-dodecanoylhomoserine lactone, it's specificity is relaxed compared to LasR and can respond equally well to several AHLs. QscR controls a set of genes that overlaps the set regulated by LasR. QscR is comparatively easy to purify and study in vitro, and has become a model for understanding the biochemistry of LuxR homologs. In fact there is a crystal structure of QscR bound to the LasI-generated AHL. Here, we review the current state of research concerning QscR and highlight recent advances in our understanding of its structure and biochemistry.

Project description:Pseudomonas aeruginosa uses two acyl-homoserine lactone signals and two quorum sensing (QS) transcription factors, LasR and RhlR, to activate dozens of genes. LasR responds to N-3-oxo-dodecanoyl-homoserine lactone (3OC12-HSL) and RhlR to N-butanoyl-homoserine lactone (C4-HSL). There is a third P. aeruginosa acyl-homoserine-lactone-responsive transcription factor, QscR, which acts to dampen or delay activation of genes by LasR and RhlR by an unknown mechanism. To better understand the role of QscR in P. aeruginosa QS, we performed a chromatin immunoprecipitation analysis, which showed this transcription factor bound the promoter of only a single operon of three genes linked to qscR, PA1895 to PA1897. Other genes that appear to be regulated by QscR in transcriptome studies were not direct targets of QscR. Deletion of PA1897 recapitulates the early QS activation phenotype of a QscR-null mutant, and the phenotype of a QscR-null mutant was complemented by PA1895-1897 but not by PA1897 alone. We conclude that QscR acts to modulate quorum sensing through regulation of a single operon, apparently raising the QS threshold of the population and providing a "brake" on QS autoinduction.IMPORTANCE Quorum sensing, a cell-cell communication system, is broadly distributed among bacteria and is commonly used to regulate the production of shared products. An important consequence of quorum sensing is a delay in production of certain products until the population density is high. The bacterium Pseudomonas aeruginosa has a particularly complicated quorum sensing system involving multiple signals and receptors. One of these receptors, QscR, downregulates gene expression, unlike the other receptors in P. aeruginosa QscR does so by inducing the expression of a single operon whose function provides an element of resistance to a population reaching a quorum. This finding has importance for design of quorum sensing inhibitory strategies and can also inform design of synthetic biological circuits that use quorum sensing receptors to regulate gene expression.

Project description:Bacterial populations co-ordinate gene expression collectively through quorum sensing (QS), a cell-to-cell communication mechanism employing diffusible signal molecules. The LysR-type transcriptional regulator (LTTR) protein PqsR (MvfR) is a key component of alkyl-quinolone (AQ)-dependent QS in Pseudomonas aeruginosa. PqsR is activated by 2-alkyl-4-quinolones including the Pseudomonas quinolone signal (PQS; 2-heptyl-3-hydroxy-4(1H)-quinolone), its precursor 2-heptyl-4-hydroxyquinoline (HHQ) and their C9 congeners, 2-nonyl-3-hydroxy-4(1H)-quinolone (C9-PQS) and 2-nonyl-4-hydroxyquinoline (NHQ). These drive the autoinduction of AQ biosynthesis and the up-regulation of key virulence determinants as a function of bacterial population density. Consequently, PqsR constitutes a potential target for novel antibacterial agents which attenuate infection through the blockade of virulence. Here we present the crystal structures of the PqsR co-inducer binding domain (CBD) and a complex with the native agonist NHQ. We show that the structure of the PqsR CBD has an unusually large ligand-binding pocket in which a native AQ agonist is stabilized entirely by hydrophobic interactions. Through a ligand-based design strategy we synthesized and evaluated a series of 50 AQ and novel quinazolinone (QZN) analogues and measured the impact on AQ biosynthesis, virulence gene expression and biofilm development. The simple exchange of two isosteres (OH for NH?) switches a QZN agonist to an antagonist with a concomitant impact on the induction of bacterial virulence factor production. We also determined the complex crystal structure of a QZN antagonist bound to PqsR revealing a similar orientation in the ligand binding pocket to the native agonist NHQ. This structure represents the first description of an LTTR-antagonist complex. Overall these studies present novel insights into LTTR ligand binding and ligand-based drug design and provide a chemical scaffold for further anti-P. aeruginosa virulence drug development by targeting the AQ receptor PqsR.

Dataset Information

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

Publications

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

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