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Crystallization and preliminary crystal structure analysis of the ligand-binding domain of PqsR (MvfR), the Pseudomonas quinolone signal (PQS) responsive quorum-sensing transcription factor of Pseudomonas aeruginosa.

ABSTRACT: The opportunistic bacterial pathogen Pseudomonas aeruginosa employs three transcriptional regulators, LasR, RhlR and PqsR, to control the transcription of a large subset of its genes in a cell-density-dependent process known as quorum sensing. Here, the recombinant production, crystallization and structure solution of the ligand-binding domain of PqsR (MvfR), the LysR-type transcription factor that responds to the Pseudomonas quinolone signal (PQS), a quinolone-based quorum-sensing signal that is unique to P. aeruginosa and possibly a small number of other bacteria, is reported. PqsR regulates the expression of many virulence genes and may therefore be an interesting drug target. The ligand-binding domain (residues 91-319) was produced as a fusion with SUMO, and hexagonal-shaped crystals of purified PqsR_91-319 were obtained using the vapour-diffusion method. Crystallization in the presence of a PQS precursor allowed data collection to 3.25 Å resolution on a synchrotron beamline, and initial phases have been obtained using single-wavelength anomalous diffraction data from seleno-L-methionine-labelled crystals, revealing the space group to be P6(5)22, with unit-cell parameters a = b = 116-120, c = 115-117 Å.

SUBMITTER: Xu N

PROVIDER: S-EPMC3433192 | biostudies-literature | 2012 Sep

REPOSITORIES: biostudies-literature

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Crystallization and preliminary crystal structure analysis of the ligand-binding domain of PqsR (MvfR), the Pseudomonas quinolone signal (PQS) responsive quorum-sensing transcription factor of Pseudomonas aeruginosa.

Xu Ningna N Yu Shen S Moniot Sébastien S Weyand Michael M Blankenfeldt Wulf W

Acta crystallographica. Section F, Structural biology and crystallization communications 20120830 Pt 9

The opportunistic bacterial pathogen Pseudomonas aeruginosa employs three transcriptional regulators, LasR, RhlR and PqsR, to control the transcription of a large subset of its genes in a cell-density-dependent process known as quorum sensing. Here, the recombinant production, crystallization and structure solution of the ligand-binding domain of PqsR (MvfR), the LysR-type transcription factor that responds to the Pseudomonas quinolone signal (PQS), a quinolone-based quorum-sensing signal that i ...[more]

PMID: 22949189

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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.

Project description:UnlabelledPseudomonas aeruginosa is a Gram-negative bacterium that is ubiquitous in the environment, and it is an opportunistic pathogen that can infect a variety of hosts, including humans. During the process of infection, P. aeruginosa coordinates the expression of numerous virulence factors through the production of multiple cell-to-cell signaling molecules. The production of these signaling molecules is linked through a regulatory network, with the signal N-(3-oxododecanoyl) homoserine lactone and its receptor LasR controlling the induction of a second acyl-homoserine lactone signal and the Pseudomonas quinolone signal (PQS). LasR-mediated control of PQS occurs partly by activating the transcription of pqsR, a gene that encodes the PQS receptor and is necessary for PQS production. We show that LasR interacts with a single binding site in the pqsR promoter region and that it does not influence the transcription of the divergently transcribed gene, nadA. Using DNA affinity chromatography, we identified additional proteins that interact with the pqsR-nadA intergenic region. These include the H-NS family members MvaT and MvaU, and CysB, a transcriptional regulator that controls sulfur uptake and cysteine biosynthesis. We show that CysB interacts with the pqsR promoter and that CysB represses pqsR transcription and PQS production. Additionally, we provide evidence that CysB can interfere with the activation of pqsR transcription by LasR. However, as seen with other CysB-regulated genes, pqsR expression was not differentially regulated in response to cysteine levels. These findings demonstrate a novel role for CysB in influencing cell-to-cell signal production by P. aeruginosa.ImportanceThe production of PQS and other 4-hydroxy-2-alkylquinolone (HAQs) compounds is a key component of the P. aeruginosa cell-to-cell signaling network, impacts multiple physiological functions, and is required for virulence. PqsR directly regulates the genes necessary for HAQ production, but little is known about the regulation of pqsR. We identified CysB as a novel regulator of pqsR and PQS production, but, unlike other CysB-controlled genes, it does not appear to regulate pqsR in response to cysteine. This implies that CysB functions as both a cysteine-responsive and cysteine-unresponsive regulator in P. aeruginosa.

Project description:Pseudomonas aeruginosa is an infectious pathogen which has the ability to cause primary and secondary contagions in the blood, lungs, and other body parts of immunosuppressed individuals, as well as community-acquired diseases, such as folliculitis, osteomyelitis, pneumonia, and others. This opportunistic bacterium displays drug resistance and regulates its pathogenicity via the quorum sensing (QS) mechanism, which includes the LasI/R, RhlI/R, and PQS/MvfR systems. Targeting the QS systems might be an excellent way to treat P. aeruginosa infections. Although a wide array of antibiotics, namely, newer penicillins, cephalosporins, and combination drugs are being used, the use of selenium nanoparticles (SeNPs) to cure P. aeruginosa infections is extremely rare as their mechanistic interactions are weakly understood, which results in carrying out this study. The present study demonstrates a computational approach of binding the interaction pattern between SeNPs and the QS signaling proteins in P. aeruginosa, utilizing multiple bioinformatics approaches. The computational investigation revealed that SeNPs were acutely 'locked' into the active region of the relevant proteins by the abundant residues in their surroundings. The PatchDock-based molecular docking analysis evidently indicated the strong and significant interaction between SeNPs and the catalytic cleft of LasI synthase (Phe105-Se = 2.7 Å and Thr121-Se = 3.8 Å), RhlI synthase (Leu102-Se = 3.7 Å and Val138-Se = 3.2 Å), transcriptional receptor protein LasR (Lys42-Se = 3.9 Å, Arg122-Se = 3.2 Å, and Glu124-Se = 3.9 Å), RhlR (Tyr43-Se = 2.9 Å, Tyr45-Se = 3.4 Å, and His61-Se = 3.5 Å), and MvfR (Leu208-Se = 3.2 Å and Arg209-Se = 4.0 Å). The production of acyl homoserine lactones (AHLs) was inhibited by the use of SeNPs, thereby preventing QS as well. Obstructing the binding affinity of transcriptional regulatory proteins may cause the suppression of LasR, RhlR, and MvfR systems to become inactive, thereby blocking the activation of QS-regulated virulence factors along with their associated gene expression. Our findings clearly showed that SeNPs have anti-QS properties against the established QS systems of P. aeruginosa, which strongly advocated that SeNPs might be a potent solution to tackle drug resistance and a viable alternative to conventional antibiotics along with being helpful in therapeutic development to cure P. aeruginosa infections.

Dataset Information

Crystallization and preliminary crystal structure analysis of the ligand-binding domain of PqsR (MvfR), the Pseudomonas quinolone signal (PQS) responsive quorum-sensing transcription factor of Pseudomonas aeruginosa.

Publications

Crystallization and preliminary crystal structure analysis of the ligand-binding domain of PqsR (MvfR), the Pseudomonas quinolone signal (PQS) responsive quorum-sensing transcription factor of Pseudomonas aeruginosa.

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