Regulation of Burkholderia cenocepacia biofilm formation by RpoN and the c-di-GMP effector BerB.

Knowledge about the molecular mechanisms that are involved in the regulation of biofilm formation is essential for the development of biofilm-control measures. It is well established that the nucleotide second messenger cyclic diguanosine monophosphate (c-di-GMP) is a positive regulator of biofilm formation in many bacteria, but more knowledge about c-di-GMP effectors is needed. We provide evidence that c-di-GMP, the alternative sigma factor RpoN (σ54), and the enhancer-binding protein BerB play a role in biofilm formation of Burkholderia cenocepacia by regulating the production of a biofilm-stabilizing exopolysaccharide. Our findings suggest that BerB binds c-di-GMP, and activates RpoN-dependent transcription of the berA gene coding for a c-di-GMP-responsive transcriptional regulator. An increased level of the BerA protein in turn induces the production of biofilm-stabilizing exopolysaccharide in response to high c-di-GMP levels. Our findings imply that the production of biofilm exopolysaccharide in B. cenocepacia is regulated through a cascade involving two consecutive transcription events that are both activated by c-di-GMP. This type of regulation may allow tight control of the expenditure of cellular resources.

Dissecting the Multiple Roles of PqsE in Pseudomonas aeruginosa Virulence by Discovery of Small Tool Compounds.

Pseudomonas aeruginosa uses quorum sensing (QS) as a cell-to-cell communication system to orchestrate the expression of virulence determinants. The biosynthesis of the important Pseudomonas quinolone signal (PQS) requires the pqsABCDE operon. Here, PqsE acts as a pathway-specific thioesterase, but it also contributes to the regulation of bacterial virulence via an unknown mechanism. In this manuscript, we report the discovery of PqsE inhibitors as tool compounds to gain further insights into its different functions. Differential scanning fluorimetry (DSF) was used to screen a fragment library, and isothermal titration calorimetry (ITC) was employed as a secondary filter. As proven by X-ray crystallography, hit molecules bound to the active center inhibiting PqsE's thioesterase activity in cell-based and in vitro assays. Notably, the ligands did not affect the levels of the PqsE-regulated virulence factor pyocyanin. These findings indicate that the regulatory function of PqsE is not linked to its thioesterase activity and must be encoded outside of the active center. This study highlights the potential of fragment-based screening for the discovery of tool compounds. This approach provided novel insight into complex biological systems, which could not be obtained by knockout studies.

Exploring the chemical space of ureidothiophene-2-carboxylic acids as inhibitors of the quorum sensing enzyme PqsD from Pseudomonas aeruginosa.

Pseudomonas aeruginosa employs a quorum sensing (QS) communication system that makes use of small diffusible molecules. Among other effects, the QS system coordinates the formation of biofilm which decisively contributes to difficulties in the therapy of Pseudomonas infections. The present work deals with the structure-activity exploration of ureidothiophene-2-carboxylic acids as inhibitors of PqsD, a key enzyme in the biosynthetic pathway of signal molecules in the Pseudomonas QS system. We describe an improvement of the inhibitory activity by successfully combining features from two different PqsD inhibitor classes. Furthermore the functional groups, which are responsible for the inhibitory potency, were identified. Moreover, the inability of the new inhibitors, to prevent signal molecule formation in whole cell assays, is discussed.

Catechol-based substrates of chalcone synthase as a scaffold for novel inhibitors of PqsD.

A new strategy for treating Pseudomonas aeruginosa infections could be disrupting the Pseudomonas Quinolone Signal (PQS) quorum sensing (QS) system. The goal is to impair communication among the cells and, hence, reduce the expression of virulence factors and the formation of biofilms. PqsD is an essential enzyme for the synthesis of PQS and shares some features with chalcone synthase (CHS2), an enzyme expressed in Medicago sativa. Both proteins are quite similar concerning the size of the active site, the catalytic residues and the electrostatic surface potential at the entrance of the substrate tunnel. Hence, we evaluated selected substrates of the vegetable enzyme as potential inhibitors of the bacterial protein. This similarity-guided approach led to the identification of a new class of PqsD inhibitors having a catechol structure as an essential feature for activity, a saturated linker with two or more carbons and an ester moiety bearing bulky substituents. The developed compounds showed PqsD inhibition with IC50 values in the single-digit micromolar range. The binding mode of these compounds was investigated by Surface Plasmon Resonance (SPR) experiments revealing that their interaction with the protein is not influenced by the presence of the anthranilic acid bound to active site cysteine. Importantly, some compounds reduced the signal molecule production in cellulo.

Composing compound libraries for hit discovery--rationality-driven preselection or random choice by structural diversity?

AIMS: In order to identify new scaffolds for drug discovery, surface plasmon resonance is frequently used to screen structurally diverse libraries. Usually, hit rates are low and identification processes are time consuming. Hence, approaches which improve hit rates and, thus, reduce the library size are required. METHODS: In this work, we studied three often used strategies for their applicability to identify inhibitors of PqsD. In two of them, target-specific aspects like inhibition of a homologous protein or predicted binding determined by virtual screening were used for compound preselection. Finally, a fragment library, covering a large chemical space, was screened and served as comparison. RESULTS & CONCLUSION: Indeed, higher hit rates were observed for methods employing preselected libraries indicating that target-oriented compound selection provides a time-effective alternative.

A rapid method for quantifying chlorogenic acid levels in potato samples.

Chlorogenic acids (CGA) are found in many plant-derived foodstuffs and are claimed to have various beneficial effects on human health. Potatoes are a major component of the human diet and contain CGA, but little is known about their abundance in these important tubers. We therefore used a rapid, sensitive, and selective method for quantifying CGAs in food using ultra-high performance LC diode array detection (UHPLC-DAD). We also established an optimized sample preparation protocol based on ultrafiltration and used these techniques to quantify the CGA contents of selected potato varieties (fresh and after storage) and potato products. The measured CGA concentrations in potato skins were 37-636 mg/100 g dry weight (DW) and were three to four times greater than those in the flesh. Storage reduced the CGA levels in potatoes by up to 81%. The studied potato purees contained 4-11 mg CGA/100 g DW. In addition, the quinic acid contents of potato flesh (11-95 mg/100 g DW) and puree (11-22 mg/100 g DW) were measured using stable isotope dilution analysis. None of the tested samples contained caffeic acid. Overall, our results demonstrated that the UHPLC-DAD method can be used to rapidly quantify CGA levels in potatoes and related food products.

Biochemical and biophysical analysis of a chiral PqsD inhibitor revealing tight-binding behavior and enantiomers with contrary thermodynamic signatures.

Antivirulence strategies addressing bacterial pathogenicity without exhibiting growth inhibition effects represent a novel approach to overcome today's crisis in antibiotic development. In recent studies, we examined various inhibitors of PqsD, an enzyme involved in formation of Pseudomonas aeruginosa cell-to-cell signaling molecules, and observed desired cellular effects for 2-nitrophenyl derivatives. Herein, we investigated the binding characteristics of this interesting compound class using several biochemical and biophysical methods. The inhibitors showed time-dependent activity, tight-binding behavior, and interactions with the catalytic center. Furthermore, isothermal titration calorimetry (ITC) experiments with separated enantiomers revealed contrary thermodynamic signatures showing either enthalpy- or entropy-driven affinity. A combination of site-directed mutagenesis and thermodynamic profiling was used to identify key residues involved in inhibitor binding. This information allowed the proposal of experimentally confirmed docking poses. Although originally designed as transition state analogs, our results suggest an altered position for both enantiomers. Interestingly, the main difference between stereoisomers was found in the orientation of the hydroxyl group at the stereogenic center. The predicted binding modes are in accordance with experimental data and, thus, allow future structure-guided optimization.

Molecular basis of HHQ biosynthesis: molecular dynamics simulations, enzyme kinetic and surface plasmon resonance studies.

BACKGROUND: PQS (PseudomonasQuinolone Signal) and its precursor HHQ are signal molecules of the P. aeruginosa quorum sensing system. They explicate their role in mammalian pathogenicity by binding to the receptor PqsR that induces virulence factor production and biofilm formation. The enzyme PqsD catalyses the biosynthesis of HHQ. RESULTS: Enzyme kinetic analysis and surface plasmon resonance (SPR) biosensor experiments were used to determine mechanism and substrate order of the biosynthesis. Comparative analysis led to the identification of domains involved in functionality of PqsD. A kinetic cycle was set up and molecular dynamics (MD) simulations were used to study the molecular bases of the kinetics of PqsD. Trajectory analysis, pocket volume measurements, binding energy estimations and decompositions ensured insights into the binding mode of the substrates anthraniloyl-CoA and ß-ketodecanoic acid. CONCLUSIONS: Enzyme kinetics and SPR experiments hint at a ping-pong mechanism for PqsD with ACoA as first substrate. Trajectory analysis of different PqsD complexes evidenced ligand-dependent induced-fit motions affecting the modified ACoA funnel access to the exposure of a secondary channel. A tunnel-network is formed in which Ser317 plays an important role by binding to both substrates. Mutagenesis experiments resulting in the inactive S317F mutant confirmed the importance of this residue. Two binding modes for ß-ketodecanoic acid were identified with distinct catalytic mechanism preferences.

Structure optimization of 2-benzamidobenzoic acids as PqsD inhibitors for Pseudomonas aeruginosa infections and elucidation of binding mode by SPR, STD NMR, and molecular docking.

Pseudomonas aeruginosa employs a characteristic pqs quorum sensing (QS) system that functions via the signal molecules PQS and its precursor HHQ. They control the production of a number of virulence factors and biofilm formation. Recently, we have shown that sulfonamide substituted 2-benzamidobenzoic acids, which are known FabH inhibitors, are also able to inhibit PqsD, the enzyme catalyzing the last and key step in the biosynthesis of HHQ. Here, we describe the further optimization and characterization of this class of compounds as PqsD inhibitors. Structural modifications showed that both the carboxylic acid ortho to the amide and 3'-sulfonamide are essential for binding. Introduction of substituents in the anthranilic part of the molecule resulted in compounds with IC50 values in the low micromolar range. Binding mode investigations by SPR with wild-type and mutated PqsD revealed that this compound class does not bind into the active center of PqsD but in the ACoA channel, preventing the substrate from accessing the active site. This binding mode was further confirmed by docking studies and STD NMR.