treatment of e. coli cultures with thiophenesulfonamides to assess the suppression of quorum sensing

<meta charset="utf8"></head><body>Evaluación biológica de las tiofenesulfonamidas en la inhibición de la detección de quórum

Bacteria sense population density and the type of cells nearby using a cell-cell communication process called quorum sensing (QS)1. Diverse clades of bacteria use QS to control various behaviors, such as motility, biofilm formation, virulence factor secretion, and more. The proteins and signals involved in QS differ widely among bacteria. In Vibrio species, the QS signaling system predominantly uses membrane-bound hybrid histidine-kinase receptors that recognize specific cognate small molecule signals called autoinducers2 (Figure 1). These receptors control the flow of phosphate through the system to a response regulator that transcribes small RNAs. The production of sRNAs alters the production of the master quorum sensing regulator, which is defined as a conserved group of proteins collectively called LuxR/HapR3. Thus, at low cell densities, the mRNA encoding LuxR/HapR is degraded via sRNA targeting, and at high cell density, LuxR/HapR protein is produced at maximal levels (reviewed in Ball et al.3).
The LuxR/HapR group of proteins belongs to the large group of TetR proteins, which is defined by the presence of a helix-turn-helix in the DNA binding domain, formation of a functional homodimer, and typically the inclusion of a ligand binding domain4. The Vibrio LuxR/HapR proteins fit all these criteria, though a ligand has not yet been identified. The LuxR/HapR protein in all studied Vibrio species controls numerous downstream behaviors, many of which are known to be important in pathogenesis: production of biofilms, proteases, cytotoxins, hemolysins, type III secretion, and type VI secretion complexes, and more3. Deletion of the LuxR/HapR protein leads to a decrease or loss of virulence in host systems5,6,7, leading to the hypothesis that inhibition of these proteins is a viable strategy for counteracting disease progression. Vibrio species cause vibriosis disease in marine organisms, including fish, shellfish, and corals, as well as in humans who contact or ingest certain species.
Previous research has identified a panel of thiophenesulfonamide compounds that specifically bind to the ligand binding domain of LuxR/HapR proteins in multiple Vibrio species to block their function5,8,9 (Figure 1). Using a reporter screen in E. coli, the compounds were identified and subsequently tested in the native Vibrio, which showed a high correlation between the effect in the heterologous E. coli and the efficacy in the Vibrio9. These compounds are simple to synthesize in a single step, making them ideal for small library synthesis in the context of a chemical biology lab course. A three-week course-based undergraduate research experience (CURE) that was designed around this molecular scaffold has been previously reported8. This three-week module has been further optimized, streamlined, and broadened in this year-long CURE designed to target the inhibition of LuxR/HapR proteins in diverse Vibrio species.

<meta charset="utf8"></head><body>Autor destacado: Avance terapéutico para tratar la vibriosis en humanos y organismos acuáticos

Síntesis y ensayo de inhibidores de detección de quórum Vibrio

Our research focuses on designing, synthesizing, and analyzing potential inhibitors of Vibrio bacteria that cause disease in marine animals and humans. Our previous studies identified thiophenesulfonamide compounds as potent inhibitors of pathogenicity in Vibrio species. We aim to develop compounds for the treatment of Vibrio disease.We have synthesized a panel of thiophenesulfonamide molecules active against several Vibrio species. We've solved the X-ray crystal structure of a thiophenesulfonamide molecule in the binding pocket of the target Vibrio. And we determined the thiophenesulfonamide mechanism of action in blocking the protein target and preventing pathogenicity in vivo.This protocol is designed with the novice researcher in mind. Every step was optimized for simplicity and error reduction, while still allowing for teaching concepts and the scientific method. Our protocol bridges microbiology, organic chemistry, structure modeling, and drug design in a novel, teaching-based experimental system.Novel therapeutics that target Vibrio bacteria are needed to treat the increasing cases of Vibriosis in humans and aquaculture. Because our compounds are not antibiotics and don't kill cells, they have the potential to treat disease without generating resistance. We will focus on optimizing the inhibitors of Vibrio species that do not have a therapeutically relevant inhibitor yet.We will also explore the chemical space afforded by substituted aromatic sulfonyl chloride starting materials.

Tratamiento de cultivos de E. coli con tiofenesulfonamidas para evaluar la supresión de la detección de quórum

treatment-e-coli-cultures-with-thiophenesulfonamides-to-assess

Research

JoVE Journal

Biology

Treatment of E. coli Cultures with Thiophenesulfonamides to Assess the Suppression of Quorum Sensing

26 vistas. Para comenzar, obtenga el caldo de lisogenia o medio LB y agregue los antibióticos al medio. Alícuota cinco mililitros del medio en tubos de ensayo estériles con tapones. Inocular el medio con cepas de Escherichia coli que expresen plásmidos tomados de existencias congeladas e incubar el cultivo en un agitador durante la noche.A continuación, prepare una solución de tiofeno sulfonamida de 10 milimolares en DMSO y vórtice para mezclar bien. Dilui...

Video: Tratamiento de cultivos de E. coli con tiofenesulfonamidas para evaluar la supresión de la detección de quórum

Synthesis and Assay of Vibrio Quorum Sensing Inhibitors

Bacteria detect local population numbers using quorum sensing, a method of cell-cell communication broadly utilized to control bacterial behaviors. In Vibrio species, the master quorum sensing regulators LuxR/HapR control hundreds of quorum sensing genes, many of which influence virulence, metabolism, motility, and more. Thiophenesulfonamides are potent inhibitors of LuxR/HapR that bind the ligand pocket in these transcription factors and block downstream quorum sensing gene expression. This class of compounds served as the basis for the development of a set of simple, robust, and educational procedures for college students to assimilate their chemistry and biology skills using a CURE model: course-based undergraduate research experience. Optimized protocols are described that comprise three learning stages in an iterative and multi-disciplinary platform to engage students in a year-long CURE: (1) design and synthesize new small molecule inhibitors based on the thiophenesulfonamide core, (2) use structural modeling to predict binding affinity to the target, and (3) assay the compounds for efficacy in microbiological assays against specific Vibrio LuxR/HapR proteins. The described reporter assay performed in E. coli successfully predicts the efficacy of the compounds against target proteins in the native Vibrio species.

Thiophenesulfonamide compounds are potent and specific inhibitors of Vibrio quorum sensing regulators LuxR/HapR that block their activity in vivo, thus preventing transcription of genes for virulence, motility, and biofilms. This protocol details how these compounds are synthesized, modeled in silico, and assayed in vivo for activity against LuxR/HapR.

Bacteria detect local population numbers using quorum sensing, a method of cell-cell communication broadly utilized to control bacterial behaviors. In ...

Investigación

Biología

To begin, obtain the Lysogeny broth or LB medium and add the antibiotics to the medium. Aliquot five milliliters of the medium into sterile test tubes with caps. Inoculate the media with Escherichia coli strains expressing plasmids taken from freezer stocks and incubate the culture in a shaker overnight.Next, prepare a 10 millimolar thiophene sulfonamide solution in DMSO and vortex to mix thoroughly. Back-dilute the cultures 1 to 100 times in 10 milliliters of media taken in 15 milliliter conical tubes. Vortex briefly to mix.Add cultures to the appropriate wells of a 96-well plate. After mixing, prepare a dilution series from rows A to E, transferring 50 microliters of the solution each time. Cover the plate with microporous tape and incubate it.After removing the tape, using a plate reader, read the optical density at 600 nanometers GFP and mCherry. Finally, record and plot the data as normalized fluorescence per cell. Data for 3-thiophene sulfonamide compounds suggested that compounds 1A and 3B were each inhibitory to LuxR/HapR to different extents, whereas 2B did not have any activity.