Ulrik Hillaert

Use of quorum sensing inhibitors to interfere with biofilm formation and development in Burkholderia multivorans and Burkholderia cenocepacia

Burkholderia cepacia complex strains are opportunistic pathogens causing life-threatening infections in cystic fibrosis patients. B. cepacia complex strains are resistant to many antimicrobial agents and commonly produce biofilms in vitro and in vivo. This contributes to their virulence and makes Burkholderia infections difficult to treat. Recently, the quorum sensing (QS) system of Burkholderia spp. has been found to affect their biofilm-forming ability, making it an attractive target for antimicrobial therapy. However, detailed information about the anti-biofilm effect of these compounds is still lacking. In the present study, we evaluated the anti-biofilm effect of several known QS inhibitors. The effect on Burkholderia spp. biofilm formation was examined using crystal violet, resazurin and SYTO9 staining, confocal laser scanning microscopy as well as plating. When used at subinhibitory concentrations, several compounds interfered with biofilm formation by Burkholderia spp. Our results suggest that the QS inhibitors affect later stages of biofilm formation and detachment.

Structure-Activity Relationship of Cinnamaldehyde Analogs as Inhibitors of AI-2 Based Quorum Sensing and Their Effect on Virulence of Vibrio spp

Background Many bacteria, including Vibrio spp., regulate virulence gene expression in a cell-density dependent way through a communication process termed quorum sensing (QS). Hence, interfering with QS could be a valuable novel antipathogenic strategy. Cinnamaldehyde has previously been shown to inhibit QS-regulated virulence by decreasing the DNA-binding ability of the QS response regulator LuxR. However, little is known about the structure-activity relationship of cinnamaldehyde analogs. Methodology/Principal Findings By evaluating the QS inhibitory activity of a series of cinnamaldehyde analogs, structural elements critical for autoinducer-2 QS inhibition were identified. These include an α,β unsaturated acyl group capable of reacting as Michael acceptor connected to a hydrophobic moiety and a partially negative charge. The most active cinnamaldehyde analogs were found to affect the starvation response, biofilm formation, pigment production and protease production in Vibrio spp in vitro, while exhibiting low cytotoxicity. In addition, these compounds significantly increased the survival of the nematode Caenorhabditis elegans infected with Vibrio anguillarum, Vibrio harveyi and Vibrio vulnificus. Conclusions/Significance Several new and more active cinnamaldehyde analogs were discovered and they were shown to affect Vibrio spp. virulence factor production in vitro and in vivo. Although ligands for LuxR have not been identified so far, the nature of different cinnamaldehyde analogs and their effect on the DNA binding ability of LuxR suggest that these compounds act as LuxR-ligands.

6′-Derivatised α-GalCer Analogues Capable of Inducing Strong CD1d-Mediated Th1-Biased NKT Cell Responses in Mice

α-Galactosyl ceramide (α-GalCer, also known as KRN 7000) is known as the prototypical antigen for invariant natural killer T (NKT) cells. Stimulation of NKT cells by CD1d-mediated α-GalCer presentation leads to rapid release of Th1 and Th2 cytokines. Since Th1 and Th2 cytokines antagonize each other’s effects, α-GalCer analogues that induce a biased Th1/Th2 response are highly awaited. With the exception of a C-glycoside (α-C-GalCer), most of the known α-GalCer analogues able to induce polarized cytokine responses are characterized by modifications of the phytosphingosine or fatty acyl chains, expected to alter the affinity for CD1d. Herein we describe the synthesis of 6′-modified α-GalCer analogues with an intact phytoceramide moiety that are capable to skew the cytokine release profile to Th1, while maintaining strong antigenic activity. These analogues are characterized by the presence of an aromatic moiety that is connected via an amide or an urea linkage to C′-6 of the galactopyranose ring.

Fungicidal activity of truncated analogues of dihydrosphingosine.

The minimal fungicidal concentration (MFC) of dihydrosphingosine (DHS), phytosphingosine (PHS), and five short-chain DHS derivatives was determined for Candida albicans and Candida glabrata. In this respect, a C15- and a C17-homologue of DHS showed a 2- to 10-fold decreased MFC as compared to native DHS (i.e. C18-DHS). DHS derivatives that were active, that is, comprising 12, 15, 17, or 18 carbon atoms, induced accumulation of reactive oxygen species (ROS) in C. albicans.

A "Clickable" MTX Reagent as a Practical Tool for Profiling Small-Molecule-Intracellular Target Interactions via MASPIT

We present a scalable synthesis of a versatile MTX reagent with an azide ligation handle that allows rapid γ‐selective conjugation to yield MTX fusion compounds (MFCs) appropriate for MASPIT, a three‐hybrid system that enables the identification of mammalian cytosolic proteins that interact with a small molecule of interest. We selected three structurally diverse pharmacologically active compounds (tamoxifen, reversine, and FK506) as model baits. After acetylene functionalization of these baits, MFCs were synthesized via a CuAAC reaction, demonstrating the general applicability of the MTX reagent. In analytical mode, MASPIT was able to give concentration‐dependent reporter signals for the established target proteins. Furthermore, we demonstrate that the sensitivity obtained with the new MTX reagent was significantly stronger than that of a previously used non‐regiomeric conjugate mixture. Finally, the FK506 MFC was explored in a cellular array screen for targets of FK506. Out of a pilot collection of nearly 2000 full‐length human ORF preys, FKBP12, the established target of FK506, emerged as the prey protein that gave the highest increase in luciferase activity. This indicates that our newly developed synthetic strategy for the straightforward generation of MFCs is a promising asset to uncover new intracellular targets using MASPIT cellular array screening.

2,5-Bis-(2-hydroxybenzoylamino)pentanoic acid, a salicylic acid-metabolite isolated from chicken: characterization and independent synthesis.

From excreta of chickens that had been treated with sodium salicylate, a new compound was detected and identified as a double conjugated ornithine metabolite. The structural assignment of this metabolite was further confirmed by an independent efficient 3-step synthesis from ornithine.