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Flavones as Quorum Sensing Inhibitors Identified by a Newly Optimized Screening Platform Using Chromobacterium violaceum as Reporter Bacteria.

ABSTRACT: Quorum sensing (QS) is the process by which bacteria produce and detect signal molecules to coordinate their collective behavior. This intercellular communication is a relevant target for anti-biofilm therapies. Here we have optimized a screening-applicable assay to search for new quorum sensing inhibitors from natural compound libraries. In this system, QS is correlated with the production of violacein, which is directly controlled by the LuxI/LuxR system in Chromobacterium violaceum ATCC 31532. The parallel use of C. violaceum Tn5-mutant CV026, which depends on auto-inducer addition, allows simultaneous discrimination of compounds that act as quenchers of the AHL signal (quorum quenchers). The incorporation of a redox stain into the platform allowed further distinction between QS inhibitors, quorum quenchers and antibacterial compounds. A pilot screening was performed with 465 natural and synthetic flavonoids. All the most active compounds were flavones and they displayed potencies (IC50) in the range of 3.69 to 23.35 ?M. These leads were particularly promising as they inhibited the transition from microcolonies into mature biofilms from Escherichia coli and Pseudomonas aeruginosa strains. This approach can be very effective in identifying new antimicrobials posing lesser risks of resistance.

SUBMITTER: Skogman ME

PROVIDER: S-EPMC6273190 | biostudies-literature | 2016 Sep

REPOSITORIES: biostudies-literature

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Flavones as Quorum Sensing Inhibitors Identified by a Newly Optimized Screening Platform Using Chromobacterium violaceum as Reporter Bacteria.

Skogman Malena E ME Kanerva Sonja S Manner Suvi S Vuorela Pia M PM Fallarero Adyary A

Molecules (Basel, Switzerland) 20160910 9

Quorum sensing (QS) is the process by which bacteria produce and detect signal molecules to coordinate their collective behavior. This intercellular communication is a relevant target for anti-biofilm therapies. Here we have optimized a screening-applicable assay to search for new quorum sensing inhibitors from natural compound libraries. In this system, QS is correlated with the production of violacein, which is directly controlled by the LuxI/LuxR system in Chromobacterium violaceum ATCC 31532 ...[more]

PMID: 27626397

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Project description:Iron is a transition metal used as a cofactor in many biochemical reactions. In bacteria, iron homeostasis involves Fur-mediated de-repression of iron uptake systems, such as the iron-chelating compounds siderophores. In this work, we identified and characterized novel regulatory systems that control siderophores in the environmental opportunistic pathogen Chromobacterium violaceum. Screening of a 10,000-transposon mutant library for siderophore halos identified seven possible regulatory systems involved in siderophore-mediated iron homeostasis in C. violaceum. Further characterization revealed a regulatory cascade that controls siderophores involving the transcription factor VitR acting upstream of the quorum-sensing (QS) system CviIR. Mutation of the regulator VitR led to an increase in siderophore halos, and a decrease in biofilm, violacein, and protease production. We determined that these effects occurred due to VitR-dependent de-repression of vioS. Increased VioS leads to direct inhibition of the CviR regulator by protein-protein interaction. Indeed, insertion mutations in cviR and null mutations of cviI and cviR led to an increase of siderophore halos. RNA-seq of the cviI and cviR mutants revealed that CviR regulates CviI-dependent and CviI-independent regulons. Classical QS-dependent processes (violacein, proteases, and antibiotics) were activated at high cell density by both CviI and CviR. However, genes related to iron homeostasis and many other processes were regulated by CviR but not CviI, suggesting that CviR acts without its canonical CviI autoinducer. Our data revealed a complex regulatory cascade involving QS that controls siderophore-mediated iron homeostasis in C. violaceum.IMPORTANCEThe iron-chelating compounds siderophores play a major role in bacterial iron acquisition. Here, we employed a genetic screen to identify novel siderophore regulatory systems in Chromobacterium violaceum, an opportunistic human pathogen. Many mutants with increased siderophore halos had transposon insertions in genes encoding transcription factors, including a novel regulator called VitR, and CviR, the regulator of the quorum-sensing (QS) system CviIR. We found that VitR is upstream in the pathway and acts as a dedicated repressor of vioS, which encodes a direct CviR-inhibitory protein. Indeed, all QS-related phenotypes of a vitR mutant were rescued in a vitRvioS mutant. At high cell density, CviIR activated classical QS-dependent processes (violacein, proteases, and antibiotics production). However, genes related to iron homeostasis and type-III and type-VI secretion systems were regulated by CviR in a CviI- or cell density-independent manner. Our data unveil a complex regulatory cascade integrating QS and siderophores in C. violaceum.

Project description:This study presents an inexpensive, eco-friendly, and simple green synthesis of ZnO nanoparticles using Origanum vulgare extract. These nanoparticles are non-hazardous, environmentally friendly, and cheaper than other methods of biosynthesis. Ongoing research determines the role of phytochemicals in the fabrication and biosynthesis of ZnO NPs and their role in antibacterial activity and biomedical applications. Characterizations by fourier transform infrared spectroscopy (FTIR), diffuse reflectance UV-visible spectroscopy, X-ray diffraction (XRD), scanning electron microscopy (SEM), and transmission electron microscopy (TEM) determine the successful biosynthesis of ZnO NPs. Meanwhile, TEM and X-ray diffraction studies approximated the spherical morphology and crystalline nature of biosynthesized ZnO NPs of nano size in the range of 20-30 nm. The global increase in drug resistance necessitates the search for new drugs with different mechanisms of action. Quorum sensing (QS), a cell-to-cell communication, has gained attention as an emerging drug target. It controls numerous biochemical processes in bacteria, which are essential for their survival and pathogenicity. The potential of nanomedicines has also been tested to synthesize new antibiotics to tackle drug resistance. ZnO NPs were explored for their antibacterial, antiquorum sensing, and antibiofilm activities with a bioreporter strain of Chromobacterium violaceum. Susceptibility testing results indicated the potential antibacterial activity of ZnO NPs with a minimum inhibitory concentration (MIC) of 4 µg/mL and minimum bactericidal concentration (MBC) of 16 µg/mL. Antiquorum-sensing assays revealed that these nanoparticles inhibit quorum sensing with minimum antiquorum sensing activity (MQSIC) of 1 µg/mL, without causing any bacterial growth inhibition. In addition, ZnO NPs inhibit biofilm formation at inhibitory and higher concentrations. RT-qPCR results supported the downregulation of the quorum sensing genes when C. violaceum was treated with ZnO NPs. The outcomes of this study are promising with regard to the biofilm and quorum sensing, emphasizing the potential applications of ZnO NPs against bacterial communication and biofilm formation.

Dataset Information

Flavones as Quorum Sensing Inhibitors Identified by a Newly Optimized Screening Platform Using Chromobacterium violaceum as Reporter Bacteria.

Publications

Flavones as Quorum Sensing Inhibitors Identified by a Newly Optimized Screening Platform Using Chromobacterium violaceum as Reporter Bacteria.

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