Research reported in this publication was supported by the National Institute of General Medical Sciences of the National Institutes of Health under award number R35GM124698 to JVK. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

The details of the reagents and the equipment used for the study are listed in the Table of Materials.
1. Design and synthesis of thiophenesulfonamide libraries
NOTE: Thiophenesulfonamide inhibitors such as 3-phenyl-1-(thiophen-2-ylsulfonyl)-1H-pyrazole (PTSP) are synthesized via the one-step base-promoted condensation8,9, as shown in Figure 2. To design new compound libraries for study, researchers must procure appropriate amines and sulfonyl chlorides and follow the steps summarized below. If the structure of the amine differs greatly from the aromatic pyrazole derivative, then alternative reaction conditions may need to be identified by searching the literature. This procedure is robust, and bases such as sodium hydroxide, triethyl amine, and pyridine have also worked well. If this is performed in a course, students may be given the freedom to choose their own procedure with likely favorable outcomes.
<ol>
	<li>Synthesis and isolation of PTSP 
	NOTE: This can be completed in one 3 h lab period.
	<ol>
		<li>Dissolve 822 mg of phenylpyrazole (5.7 mmol of the amine, 1.5 eq.) in 15 mL of tetrahydrofuran in a 50 mL round bottom flask with a stir bar.</li>
		<li>Slowly add 306 mg of sodium hydride (60% in oil, 7.65 mmol, 2 eq.) to create a suspension. Let this suspension stir on a stir plate for 10 min.</li>
		<li>In a vial, prepare a solution of thiophenesulfonyl chloride (3.82 mmol of the sulfonyl chloride, 1 eq.) in 5 mL of THF.</li>
		<li>Add the solution of sulfonyl chloride prepared in step 1.1.3 to the suspension prepared in step 1.1.2 and allow the resulting suspension to stir for an additional 30 min.</li>
		<li>Add 20 mL of DI water to the reaction mixture in the 50 mL round bottom flask.</li>
		<li>Transfer all contents of the reaction flask except the stir bar into a 250 mL separatory funnel10.</li>
		<li>Add 20 mL of ethyl acetate (EtOAc) to the separatory funnel and shake.</li>
		<li>Remove the bottom (aqueous) layer and set aside.</li>
		<li>Remove the top (organic) layer and set aside in a 200 mL Erlenmeyer flask.</li>
		<li>Return the aqueous layer to the separatory funnel and extract with 20 mL of ethyl acetate.</li>
		<li>Repeat steps 1.1.8 and 1.1.9.</li>
		<li>Return the combined organic layers to the separatory funnel and wash with 20 mL of saturated NaCl (brine).</li>
		<li>Remove the bottom (aqueous) layer and set aside.</li>
		<li>Drain the top (organic) layer back into the Erlenmeyer flask.</li>
		<li>Dry the combined organic layers over MgSO4 in the Erlenmeyer flask and filter into a 250 mL round bottom flask using qualitative filter paper10.</li>
		<li>Remove the organic solvent on a rotary evaporator.</li>
	</ol>
	</li>
	<li>Analyze the crude reaction mixture by 1H NMR to ensure the product has formed10. 
	NOTE: The time necessary for this step will depend on the student&#39;s comfort level with NMR spectroscopy.</li>
	<li>Purify the product by silica gel column chromatography (10:1 hexanes: ethyl acetate)10. 
	NOTE: This can be completed in one 3 h lab period.</li>
	<li>Characterize the product spectroscopically. 
	NOTE: The time necessary for this step will depend on the student&#39;s comfort level with NMR spectroscopy. 
	NOTE: Characterization data for 3-phenyl-1-(thiophen-2-ylsulfonyl)-1H-pyrazole (PTSP)10: 
	1H NMR (400 MHz, CDCl 3 ):&#160;&#948; 8.11 (d, J = 2.8 Hz, 1H), 7.88 - 7.79 (m, 3H), 7.68 (dd, J = 5.0, 1.4 Hz, 1H), 7.44 - 7.31 (m, 3H), 7.07 (dd, J = 5.0, 3.9 Hz, 1H), 6.71 (d, J = 2.8 Hz, 1H). 
	13C NMR (101 MHz, CDCl 3 ):&#160;&#948; 157.17, 136.84, 135.40, 135.36, 132.55, 131.28, 129.36, 128.72, 127.79, 126.47, 106.90. 
	HRMS (ESI): Calculated for C13H10O2N2NaS2 [M+Na+]: 313.0076. Found: 313.0078.</li>
</ol>
2. Structural modeling to predict the binding of thiophenesulfonamides to Vibrio LuxR/HapR regulator
NOTE: This protocol utilizes the web-based version of AutoDock Vina called Webina11 to dock small molecule inhibitors (PTSP in this case) into the ligand binding pocket of the LuxR/HapR homolog called SmcR from Vibrio vulnificus12. The outcomes of this protocol are (1) calculated binding affinities and (2) a structure file that can be opened in PyMol or a related program to visualize the ligand-protein interactions. This protocol can be adapted for any small molecule and any protein with a ligand binding pocket. It takes minutes to dock into SmcR because the search area for this receptor is defined below. If the search area needs to be defined for a new receptor (steps 2.15-2.20), this can be completed in one 3 h lab period. Once the search area is defined, subsequent dockings will take minutes.
<ol>
	<li>Download the AutoDock Tools. Select the appropriate file for the operating system and install it on the computer. 
	NOTE: This protocol utilizes a web-based version of AutoDock Vina. The docking can also be performed locally if this program is installed (see the manufacturer&#39;s instructions).</li>
	<li>Download the structure for apo SmcR.</li>
	<li>Navigate to the Protein Data Bank.</li>
	<li>In the search bar, enter the PDB ID 3KZ9.</li>
	<li>Click on the Download Files from the dropdown menu toward the top right corner of the page and select pdb format.</li>
	<li>After downloading the file, save it to a new folder dedicated to the docking work.</li>
	<li>Create a .mol structure file of the small molecule inhibitor (PTSP).</li>
	<li>Navigate to molview and click on the trash can icon to remove the structure that is there.</li>
	<li>Draw PTSP in the structure window.</li>
	<li>Click on 2D to 3D.</li>
	<li>Click on Tools &#8594; export&#160;&#8594;&#160;MOL file.</li>
	<li>A file titled &#34;Molview.mol&#34; will be downloaded to the computer. Give the file a name that makes sense and save it to the folder that contains the protein pdb file.</li>
	<li>Define the search area.</li>
	<li>Define the area in Webina as the binding pocket of the protein (SmcR) to facilitate docking in the correct region. 
	NOTE: Coordinates for SmcR are provided and generated using the following protocol. If SmcR is being used, then steps 2.15-2.20&#160;can be skipped. The protocol can be adapted for any protein with a known ligand binding pocket. Coordinates: center_x = 17, center_y = 40, center_z = 56, size_x = 12, size_y = 12, size_z = 12.</li>
	<li>In AutoDock Tools, click on File &#8594; Read Molecule and open the pdb protein file downloaded in step 2.5.</li>
	<li>Navigate to the dashboard and click on the blue arrow next to the name of the protein. This will bring up a list of chains. Click on the blue arrow next to one of the protein chains.</li>
	<li>To highlight the amino acids of the binding pocket, find the amino acid of interest in the list that appeared. Next to the amino acid, go to the triangle in the Cl (color) column at the far right. Click on the triangle and select by rainbow. 
	NOTE: Use this method to highlight the following amino acids:12 Phe75, Phe78, Leu79, Ile96, Met100, Trp114, Phe129, Asn133, Gln137, Val140, Ala163, Phe166, His167, Cys170 (If Met100 is missing, this can be skipped).</li>
	<li>Next, click on Grid &#8594; GridBox. A box should appear over the protein structure with a red, green, and blue face. Before making any changes, change Spacing (angstrom) to 1. This will ensure the box is correctly scaled for the next steps.
	<ol>
		<li>Then, change the Center Grid Box &#60;offset&#62; dials to translate the box in the x, y, and z directions to place it over the highlighted amino acids. Click and drag on the protein to rotate the structure in three dimensions. 
		NOTE: This will help ensure that the box is in the correct position. If necessary, use the top three dials for the number of points to change the size of the box.</li>
	</ol>
	</li>
	<li>Once the box position and dimensions are set, click on File&#160;&#8594; Close Saving Current.</li>
	<li>In the menu, click on Grid&#160;&#8594; Output&#160;&#8594; Save GPF. Name the file and save it in the same folder as the two structure files. This GPF file has the box coordinates.</li>
	<li>Perform &#34;Docking&#34; in Webina to generate a calculated binding energy.</li>
	<li>Open the folder that contains the .mol ligand file and the .pdb protein file. Webina requires .pdbqt files, and will do the file conversion.</li>
	<li>Navigate to Webina.</li>
	<li>Receptor: Drag over the file downloaded from the pdb called 3kz9.pdb. Click on Add hydrogen atoms, and click on convert. Click on Ok on the next pop-up to work with a monomer.</li>
	<li>Ligand: Drag over the .mol file for PTSP (or other small molecule inhibitor). Ensure no boxes are selected and click on Convert. Leave &#34;Correct Pose&#34; empty and scroll down to the Docking Box. Enter the appropriate box parameters (either those listed above for SmcR or a set that was generated by the protocol in steps 2.15-2.20&#160;for a different protein).</li>
	<li>Click on Start Webina and wait.</li>
	<li>A series of calculated binding energies will be reported [Affinity (kcal/mol)] directly below a visualization of the docked molecule. Ensure that the first value is the most negative.</li>
	<li>To visualize the docked ligand in pymol or another similar program, download the Output PDBQT File by clicking on the appropriate download button. This file is just the ligand (PTSP) in the docked coordinates.</li>
	<li>Visualize the docked ligand-SmcR complex in PyMol13.
	<ol>
		<li>Download and open PyMol. 
		NOTE: Full-time students and educators may receive a PyMol License free of charge. When asked for a license after starting PyMol for the first time, click on Buy License. Select Student/Teacher.</li>
		<li>Open the pdb file called 3kz9.pdb.</li>
		<li>Open the pdbqt&#160;output file created by Webina.</li>
		<li>Visualize the best conformation (the one with the lowest binding affinity) by clicking on the small pointing looking arrows at the bottom right of the PyMol screen. The first conformation will be the most favorable and will be showing when the file is opened.</li>
		<li>Manipulate the protein with the docked ligand in PyMol using standard commands14. 
		NOTE: The previous study by Newman et al. compared the predicted binding affinities of several compounds determined by Webina to in vitro and in vivo assays9. These binding affinities serve as references for compounds that are likely to be good inhibitors with high affinities (which correlate to lower kcal/mol numbers) for the binding pocket. An example of the Webina output data is included in Figure 3A,B for PTSP binding in the pocket of SmcR.</li>
	</ol>
	</li>
</ol>
3. Biological assessment of thiophenesulfonamides in quorum sensing inhibition
NOTE: The procedure specifically for assaying the Vibrio campbellii protein LuxR is described here. However, this procedure can be adapted for use with any of the Vibrio LuxR/HapR proteins, all of which are available on ectopically replicating plasmids that are compatible with the reporter plasmid pJV0649. The &#34;red-green screen&#34; assay is performed in a heterologous bacterial background using E. coli cells containing the two plasmids. The LuxR/HapR expression plasmid (conferring kanamycin resistance) is available expressing different LuxR genes (Figure 4A). The pJV064 plasmid (conferring chloramphenicol resistance) encodes a gfp gene under the control of a LuxR-activated promoter and a mCherry gene under the control of a LuxR-repressed promoter (Figure 4A). Both promoters were chosen due to their identification as LuxR-regulated genes with large changes in expression in vivo15. The LuxR/HapR E. coli strains and plasmid pJV064 have been published previously9,15.
<ol>
	<li>Liquid media preparation: Prepare 1 L of LB medium.
	<ol>
		<li>Weigh out 10 g of NaCl, 10 g of bactotryptone, and 5 g of yeast extract.</li>
		<li>Mix the components in a beaker or graduated cylinder using a stir plate.</li>
		<li>Bring the total volume to 1 L using graduated cylinders.</li>
		<li>If desired, aliquot into 100 mL per bottle before autoclaving.</li>
		<li>Autoclave for 15 min for each 1 L of media. Alternatively, if an autoclave is not available, an Instant Pot may be used16.</li>
	</ol>
	</li>
	<li>Strain inoculation (Day 1)
	<ol>
		<li>Add antibiotics to the LB medium at final concentrations of kanamycin (40 &#956;g/mL) and chloramphenicol (10 &#956;g/mL).</li>
		<li>Aliquot 5 mL of LB/Kan40/CM10 medium into sterile test tubes with caps.</li>
		<li>Inoculate E. coli strains from freezer stocks: E. coli culture expressing LuxR/HapR and pJV064 plasmid (LuxR+)9,15; E. coli empty vector control&#160;culture and pJV064 plasmid (LuxR-)9,15.</li>
		<li>Shake the culture overnight at 275 rpm at 30 &#176;C for 16-18 h.</li>
	</ol>
	</li>
	<li>Assay of thiophenesulfonamides (Day 2)
	<ol>
		<li>Weigh out ~30 mg of the compound. Resuspend to 100 mM in DMSO and vortex to mix. Next, prepare a 10 mM stock.</li>
		<li>Mix 20 &#956;L of the 100 mM stock with 180 &#956;L of DMSO to dilute it (1:10) to yield a 10 mM working stock. Vortex to mix.</li>
		<li>Back-dilute LuxR+ culture and LuxR- cultures (1:100) in 10 mL of LB/Kan/CM media into 15 mL conical tubes and vortex briefly to mix.</li>
		<li>Use a black-well, clear-bottomed, sterile 96-well plate for the assay.</li>
		<li>Prepare a dilution series for all columns. A diagram of the 4-fold dilution series is included as a reference (Figure 4B). 
		NOTE: A digital multi-channel pipet is recommended for this process. This method is for a 4-fold dilution series (1:4). However, different dilution series may be used (e.g., 1:10, 1:5).
		<ol>
			<li>Pipet 200 &#181;L of the LuxR+ culture mix into a 96-well plate in row A, 1-6 (Figure 4B; green).</li>
			<li>Pipet 200 &#181;L of the LuxR- culture mix into a 96-well plate in row A, 7-12 (Figure 4B; orange).</li>
			<li>Pipet 150 &#181;L of the LuxR+ culture mix into a 96-well plate in rows B-E, 1-6 (Figure 4B; green).</li>
			<li>Pipet 150 &#181;L of the LuxR- culture mix into a 96-well plate in rows B-E, 7-12 (Figure 4B; orange).</li>
			<li>Add 2 &#181;L of the compound or DMSO to each well of row A according to Figure 4B.</li>
			<li>For every column, pipet up and down 3 times, then transfer 50 &#181;L from row A into row B, in the same column.</li>
			<li>Repeat the mixing and pipetting for rows B, C, D, E. After mixing row E, remove 50 &#181;L and put in waste. In the end, one should have 150 &#956;L in each well.</li>
		</ol>
		</li>
		<li>Cover the plate with microporous tape.</li>
		<li>Incubate the plate by shaking at 275 rpm at 30 &#176;C overnight for 16-18 h.</li>
		<li>Measure the fluorescence and optical density of the assay plates (Day 3).
		<ol>
			<li>Remove the tape carefully, and do not spill liquid out of the wells.</li>
			<li>On a plate reader, read the optical density at 600 nm (OD600), GFP, and mCherry. 
			NOTE: A set gain for both fluorescence channels is recommended to enable comparisons between assays.</li>
		</ol>
		</li>
		<li>Record data as normalized fluorescence per cell: GFP/OD600 and mCherry/OD600.</li>
		<li>Plot the data, as shown in Figure 5, to illustrate the results for the DMSO control in comparison to the test samples.</li>
	</ol>
	</li>
</ol>
4. Washing the black assay plates for re-use
<ol>
	<li>Soak the plates in bleach.
	<ol>
		<li>Label a 1 L plastic beaker as &#34;biological waste&#34;. Dump liquid from plates into this waste container (carefully clean up any drips with 70% EtOH). Rinse the plates with DI water from the squirt bottle over the waste and dump it into it.</li>
		<li>Label another 1 L plastic beaker as &#34;30% bleach&#34;. Put plates/lids in this container and cover with 30% bleach. Wrap the top with a plastic sheet and weigh the plates with a tip box or something similar. Let sit submerged in the bleach solution overnight.</li>
	</ol>
	</li>
	<li>Rinse and soak in ethanol.
	<ol>
		<li>Thoroughly rinse the plates with DI water into the sink so that no bleach remains. Dump out any remaining water from wells into the sink.</li>
		<li>Add 70% EtOH to all wells and lids using a spray bottle. Let the plates sit upright with the lid on top but askew so that the EtOH can evaporate, but nothing falls into the wells. Let the plate sit overnight.</li>
	</ol>
	</li>
	<li>Allow the plates to dry.
	<ol>
		<li>Dump out any remaining EtOH.</li>
		<li>Flip over the plate onto a paper towel and let the rest of the EtOH evaporate. Leave the plates this way in a fume hood. The plates will be ready for the next use.</li>
	</ol>
	</li>
</ol>

Biological Assessment of Thiophenesulfonamides in Quorum Sensing Inhibition

<ol>
	<li>Ng, W. L., Bassler, B. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacterial+quorum-sensing+network+architectures.">Bacterial quorum-sensing network architectures.</a> Annu Rev Genet. 43, 197-222 (2009).</li><li>Barrasso, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dual-function+quorum-sensing+systems+in+bacterial+pathogens+and+symbionts.">Dual-function quorum-sensing systems in bacterial pathogens and symbionts.</a> PLoS Pathog. 16, e1008934 (2020).</li><li>Ball, A. S., Chaparian, R. R., van Kessel, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quorum+sensing+gene+regulation+by+LuxR/HapR+master+regulators+in+Vibrios.">Quorum sensing gene regulation by LuxR/HapR master regulators in Vibrios.</a> J Bacteriol. 199 (19), 00105-00117 (2017).</li><li>Ramos, J. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+TetR+family+of+transcriptional+repressors.">The TetR family of transcriptional repressors.</a> Microbiol Mol Biol Rev. 69, 326-356 (2005).</li><li>Kim, B. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=QStatin,+a+selective+inhibitor+of+quorum+sensing+in+Vibrio+species.">QStatin, a selective inhibitor of quorum sensing in Vibrio species.</a> mBio. 9 (1), 02262 (2018).</li><li>Kim, S. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=LuxR+homologue+SmcR+is+essential+for+Vibrio+vulnificus+pathogenesis+and+biofilm+detachment,+and+its+expression+is+induced+by+host+cells.">LuxR homologue SmcR is essential for Vibrio vulnificus pathogenesis and biofilm detachment, and its expression is induced by host cells.</a> Infect Immun. 81, 3721-3730 (2013).</li><li>Hasegawa, H., Hase, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TetR-type+transcriptional+regulator+VtpR+functions+as+a+global+regulator+in+Vibrio+tubiashii.">TetR-type transcriptional regulator VtpR functions as a global regulator in Vibrio tubiashii.</a> Appl Environ Microbiol. 75, 7602-7609 (2009).</li><li>Dorn, S. K., Newman, J. D., Van Kessel, J. C., Brown, L. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthesis+and+biological+assay+of+small-molecule+quorum+sensing+inhibitors:+A+three-week+course-based+undergraduate+research+experience.">Synthesis and biological assay of small-molecule quorum sensing inhibitors: A three-week course-based undergraduate research experience.</a> J Chem Educ. 98, 3533-3541 (2021).</li><li>Newman, J. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amino+acid+divergence+in+the+ligand-binding+pocket+of+Vibrio+LuxR/HapR+proteins+determines+the+efficacy+of+thiophenesulfonamide+inhibitors.">Amino acid divergence in the ligand-binding pocket of Vibrio LuxR/HapR proteins determines the efficacy of thiophenesulfonamide inhibitors.</a> Mol Microbiol. 116 (4), 1173-1188 (2021).</li><li>Mohrig, J. R. H., Schatz, P. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Techniques in Organic Chemistry. 3. End. , (2001).</li><li>Kochnev, Y., Hellemann, E., Cassidy, K. C., Durrant, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Webina:+An+open-source+library+and+web+app+that+runs+AutoDock+Vina+entirely+in+the+web+browser.">Webina: An open-source library and web app that runs AutoDock Vina entirely in the web browser.</a> Bioinformatics. 36, 4513-4515 (2020).</li><li>Kim, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crystal+structure+of+SmcR,+a+quorum-sensing+master+regulator+of+Vibrio+vulnificus,+provides+insight+into+its+regulation+of+transcription.">Crystal structure of SmcR, a quorum-sensing master regulator of Vibrio vulnificus, provides insight into its regulation of transcription.</a> J Biol Chem. 285, 14020-14030 (2010).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The PyMOL molecular graphics system v. 2.0. , (2023).</li><li>. PyMol wiki commands Available from: <a target="_blank" href="https://pymolwiki.org/index.php/Category:Commands">https://pymolwiki.org/index.php/Category:Commands</a> (2023)</li><li>van Kessel, J. C., Ulrich, L. E., Zhulin, I. B., Bassler, B. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+activator+and+repressor+functions+reveals+the+requirements+for+transcriptional+control+by+LuxR,+the+master+regulator+of+quorum+sensing+in+Vibrio+harveyi.">Analysis of activator and repressor functions reveals the requirements for transcriptional control by LuxR, the master regulator of quorum sensing in Vibrio harveyi.</a> mBio. 4 (4), 00378 (2013).</li><li>Swenson, V. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessment+and+verification+of+commercially+available+pressure+cookers+for+laboratory+sterilization.">Assessment and verification of commercially available pressure cookers for laboratory sterilization.</a> PLoS One. 13, e0208769 (2018).</li></ol>

JVK and LCB disclose financial interests in Quornix, LLC, that may benefit from the outcomes of research on thiophenesulfonamide compounds.

This CURE was originally developed as an abbreviated two-stage, three-week protocol (design/synthesis and assay) and was implemented in five semesters as part of an upper-level organic laboratory course8. Since the original report, the computer modeling module was added, and the E. coli assay was optimized for novice researchers. The resulting three-stage, two-semester protocol has been implemented three times as part of Indiana University&#39;s Arts and Sciences Undergraduate Research Ex...

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.

<table><tbody><tr><td>2-thiophensulfonyl chloride</td><td>Ambeed</td><td>A258464</td><td></td></tr><tr><td>3-Phenyl-1H-pyrazole</td><td>Ambeed</td><td>A104401</td><td>98%</td></tr><tr><td>96-well clear bottom black plates</td><td>USA Scientific</td><td>5665-5090Q</td><td>96-well polystyrene uClear black TC plate with lid, clear flat bottom, sterile, 8/sleeve, 32/case&amp;nbsp;</td></tr><tr><td>Autodock Tools</td><td></td><td></td><td>http://mgltools.scripps.edu/downloads</td></tr><tr><td>Autodock Vina</td><td></td><td></td><td>https://vina.scripps.edu&amp;nbsp;</td></tr><tr><td>Chloramphenicol</td><td></td><td></td><td></td></tr><tr><td>DMSO</td><td></td><td></td><td></td></tr><tr><td>ethyl acetate</td><td>Fisher Scientific</td><td>AA31344M4</td><td>Reagent grade</td></tr><tr><td>hexanes</td><td>Fisher Scientific</td><td>H291</td><td></td></tr><tr><td>Kanamycin</td><td></td><td></td><td></td></tr><tr><td>magnesium sulfate</td><td>Fisher Scientific</td><td>M65-500</td><td>Anhydrous</td></tr><tr><td>Microporous Film</td><td>USA Scientific</td><td>2920-1010</td><td>Microporous Film, -20degC to +80degC, 50/box, Sterilized</td></tr><tr><td>molview</td><td></td><td></td><td>molview.org</td></tr><tr><td>NaCl</td><td></td><td></td><td></td></tr><tr><td>Protein Databank</td><td></td><td></td><td>https://www.rcsb.org/</td></tr><tr><td>Pymol</td><td></td><td></td><td>https://pymol.org/2/</td></tr><tr><td>Qualitative filter paper</td><td>Fisher Scientific</td><td>09-805-342</td><td>Cytiva Whatman&amp;trade; Qualitative Filter Paper: Grade 1 Circles, 47 mm</td></tr><tr><td>Silica gel</td><td>Sorbtech</td><td>30930M-25</td><td>Silica Gel, Standard Grade, 60A, 40-63um (230 x 400 mesh)</td></tr><tr><td>Sodium hydride</td><td>Millipore Sigma</td><td>452912</td><td>60 % dispersion in mineral oil</td></tr><tr><td>Tetrahydrofuran</td><td>Fisher Scientific</td><td>MTX02847</td><td>Tetrahydrofuran, anhydrous, 99.9%, ACS Grade, DriSolv</td></tr><tr><td>TLC Plates</td><td>Sorbtech</td><td>1634067</td><td>Silica gel TLC plates, aluminum backed</td></tr><tr><td>Tryptone</td><td></td><td></td><td></td></tr><tr><td>webina</td><td></td><td></td><td>https://durrantlab.pitt.edu/webina/</td></tr><tr><td>Yeast Extract</td><td></td><td></td><td></td></tr></tbody></table>

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.

As representative results, data is included from three thiophenesulfonamide compounds synthesized by undergraduate students for compounds 1A, 2B, and 3B (Figure 5A-C; described in detail in Newman et al.9). Each compound was tested in the E. coli strain expressing V. campbellii LuxR and using the pJV064 reporter plasmid. The normalized fluorescence per cell is shown for each assay. The assay was performed wi...

Author Spotlight: Advancing Therapeutics to Treat Vibriosis in Humans and Aquatic Organisms

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.

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 ...

synthesis-and-assay-of-vibrio-quorum-sensing-inhibitors

Research

JoVE Journal

Biology

384 Views.  Indiana University. Ons onderzoek richt zich op het ontwerpen, synthetiseren en analyseren van potentiële remmers van Vibrio-bacteriën die ziekten veroorzaken bij zeedieren en mensen. Onze eerdere studies identificeerden thiofeensulfonamideverbindingen als krachtige remmers van pathogeniteit in Vibrio-soorten. We streven ernaar om verbindingen te ontwikkelen voor de behandeling van de ziekte van Vibrio.We hebben een panel van thiofeensulfonamidemoleculen gesynthetiseerd...

Video: Auteur in de schijnwerpers: geavanceerde therapieën voor de behandeling van vibriose bij mensen en waterorganismen

Vibrio cholerae: Model Organism to Study Bacterial Pathogenesis - Interview

I am Es.In MindUP, we study biology of human pathogens in non hosts environments. Many human pathogens after exiting their human host have to survive in the environment before infecting another host. So our goal is to better understand processes that control survival of human pathogens in the environment.And our goal is to then better understand transmission dynamics of infectious agents. So we are using Vibrio cholera, the cost of agent of diarrhea, disease, cholera as a model organism. Vibrio cholera is a facultative human pathogen and it has an aquatic lifecycle.Therefore, it provides us great opportunity to study the survival of human pathogen in a natural environment. There are many tools to study this organism. Its genome is completely sequenced and there are micro array representing every gene in the genome.And furthermore, there are many tools that we can manipulate the genome of this organism. My research program takes advantage of these tools and integrates them to study aquatic lifecycle of cholera. So in our experiments, we try to simulate the aquatic environments that organism lives in.And then we would like to know how does this organism so senses fluctuations in its environment with respect to nutrient load, temperature, salinity, or other biological stressors such as pages and tzo? So in aquatic environment, RIA cholera is found either in a free living form or in a surface attached form. And the surface attached forms are known as biofilms, which are simply composed of microorganisms and their extra polymeric substances, which can be exo polysaccharide and proteins and sometimes DNA.And this matrix acts as a glue and hold the organism together. And when grown in biofilm mode, these organisms are actually resistant to fluctuating parameters of their environments and they can be better protected from, for example, phage and proteasome probations. And these are factors that are controlling abundance of microorganisms in the environment.Some of the recent techno technological advance that helped us in our studies are the completion of the genome sequence of the organism and development of microray technology. Using these technologies, we can determine at the whole genome level how this organism responds to major fluctuations in its environment such as nutrient deprivation or changes in temperature. Or one of our major goal is to integrate our whole genome expression studies to a network analysis.So we would like to identify genetic regulatory network that controls response of to fluctuating environmental parameters. We would like to be able to model behavior of organism in the environment. And also we still do not know when in the environment with whom Vibra Cide interacts with.We do not know much about ecology of this organism, and we also do not know relevance of the genes and processes we identified to be important for survival of this organism in the laboratory to the real world. No are the genes that we identified express when Ria cholera is present in natural environment. We also would like to be able to identify and culture organisms that Vibrio Cora interacts with because in the environment, Vibrio Cora is a part of the complex microbial community.So I think understanding biology of Vibrio Cora in natural environment would be a, it's a challenging and it's critical for us to understand biology of the organism. Within the next five years, I hope that we would be able to better characterize op polysaccharide components of biofilms. For example, in Vibrio cholera, we still do not know structure of the op polysaccharide that is holding the cells together.We also do not know all the components of biofilm metrics. Eventually I think it be great to be able to quantify components of the biofilm metrics in natural biofilms of these organisms. I think another major challenge is to study, be OC in natural environment.And I hope that we would be able to take advantage of comparative genomic studies and combine whole genomic expression profiling comparative genomics to better understand evolution of IBR cholera as a pathogen.

Anti-virulent Disruption of Pathogenic Biofilms using Engineered Quorum-quenching Lactonases

The rapid emergence of multi-drug resistant bacteria has accelerated the need for novel therapeutic approaches to counter life-threatening infections. The persistence of bacterial infection is often associated with quorum-sensing-mediated biofilm formation. Thus, the disruption of this signaling circuit presents an attractive anti-virulence strategy. Quorum-quenching lactonases have been reported to be effective disrupters of quorum-sensing circuits. However, there have been very few reports of the effective use of these enzymes in disrupting bacterial biofilm formation. This protocol describes a method to disrupt biofilm formation in a clinically relevant A. baumannii S1 strain through the use of an engineered quorum-quenching lactonase. Acinetobacter baumannii is a major human pathogen implicated in serious hospital-acquired infections globally and its virulence is attributed predominantly to its biofilm's tenacity. The engineered lactonase treatment achieved significant A. baumannii S1 biofilm reduction. This study also showed the possibility of using engineered quorum-quenching enzymes in future treatment of biofilm-mediated bacterial diseases. Lastly, the method may be used to evaluate the competency of promising quorum-quenching enzymes.

Quorum-quenching enzymes are anti-virulent and anti-bacterial options that can mitigate pathogenesis without risk of incurring resistance, by preventing the expression of virulence factors and genes associated with antibiotic resistance and biofilm formation. In this study, we report a method that demonstrates the efficacy of quorum-quenching enzymes in bacterial biofilm disruption.

The rapid emergence of multi-drug resistant bacteria has accelerated the need for novel therapeutic approaches to counter life-threatening infections. The ...

Immunology and Infection

Development of a More Sensitive and Specific Chromogenic Agar Medium for the Detection of Vibrio parahaemolyticus and Other Vibrio Species

Foodborne infections in the US caused by Vibrio species have shown an upward trend. In the genus Vibrio, V. parahaemolyticus is responsible for the majority of Vibrio-associated infections. Thus, accurate differentiation among Vibrio spp. and detection of V. parahaemolyticus is critically important to ensure the safety of our food supply. Although molecular techniques are increasingly common, culture-depending methods are still routinely done and they are considered standard methods in certain circumstances. Hence, a novel chromogenic agar medium was tested with the goal of providing a better method for isolation and differentiation of clinically relevant Vibrio spp. The protocol compared the sensitivity, specificity and detection limit for the detection of V. parahaemolyticus between the new chromogenic medium and a conventional medium. Various V. parahaemolyticus strains (n=22) representing diverse serotypes and source of origins were used. They were previously identified by Food and Drug Administration (FDA) and Centers for Disease Control and Prevention (CDC), and further verified in our laboratory by tlh-PCR. In at least four separate trials, these strains were inoculated on the chromogenic agar and thiosulfate-citrate-bile salts-sucrose (TCBS) agar, which is the recommended medium for culturing this species, followed by incubation at 35-37 &#176;C for 24-96 hr. Three V. parahaemolyticus strains (13.6%) did not grow optimally on TCBS, nonetheless exhibited green colonies if there was growth. Two strains (9.1%) did not yield the expected cyan colonies on the chromogenic agar. Non-V. parahaemolyticus strains (n=32) were also tested to determine the specificity of the chromogenic agar. Among these strains, 31 did not grow or exhibited other colony morphologies. The mean recovery of V. parahaemolyticus on the chromogenic agar was ~96.4% relative to tryptic soy agar supplemented with 2% NaCl. In conclusion, the new chromogenic agar is an effective medium to detect V. parahaemolyticus and to differentiate it from other vibrios.

Development of a More Sensitive and Specific Chromogenic Agar Medium for the Detection of Vibrio parahaemolyticus and Other Vibrio Species

Detection and isolation of clinically relevant Vibrio species require selective and differential culture media. This study evaluated the ability of a new chromogenic medium to detect and identify V. parahaemolyticus and other related species. The new medium was found to have better sensitivity and specificity than the conventional medium.

Foodborne infections in the US caused by Vibrio species have shown an upward trend. In the genus Vibrio, V. parahaemolyticus is responsible for the ...

Synthesis of Quinoline-Based Ionic Liquids for Antimicrobial Applications

Green Synthesis of Quinoline-Based Ionic Liquid

The ever-growing menace of Antimicrobial Resistance (AMR) jeopardizes the potency of the prevailing antibiotics against the relentlessly sprouting infections spawned by bacteria, viruses, parasites as well as fungi, posing a great threat to human health and well-being. In this regard, several novel molecules have proved their mettle, with Ionic Liquids (ILs) being one of the most eco-friendly, non-volatile, and thermally stable alternatives to the existing antimicrobials, possessing high solvating potential as well as low vapor pressure. Moreover, the utilization of these entities in both stabilizing as well as destabilizing protein structures and enhancing enzymatic activity has further raised their potential in the biomedical industry. With this in view, we present the green synthesis and characterization of quinoline-based IL, owing to its immense antimicrobial potency, with low cytotoxicity and great artificial chaperone activity. Here, maneuvering the one-pot synthesis approach in solvent-free, greener reaction conditions not only ameliorated the reaction efficiency but also augmented the chemical yield. The purity of the synthesized IL was corroborated using 1H Nuclear Magnetic Resonance (NMR), 13C NMR, and Infrared (IR) spectroscopy. The biological potential of the synthesized compound is further validated by analyzing its Absorption, Distribution, Metabolism, Excretion, and Toxicity (ADMET) properties and authenticated using disc diffusion assay.

Author Spotlight: Advancing Antimicrobial Resistance Research with Innovative Approaches and Synthetic Compounds

In the present work, we elucidate the green synthesis of quinoline-based Ionic Liquid (IL), namely, 1-Hexadecylquinolin-1-ium bromide {[C16quin]Br} by mixing quinoline with an excess of 1-Bromohexadecane, along with its detailed characterization using Nuclear Magnetic Resonance and Infrared spectroscopic measurements.

The ever-growing menace of Antimicrobial Resistance (AMR) jeopardizes the potency of the prevailing antibiotics against the relentlessly sprouting ...

Chemistry

Coincubation Assay for Quantifying Competitive Interactions between Vibrio fischeri Isolates

This manuscript describes a culture-based, coincubation assay for detecting and characterizing competitive interactions between two bacterial populations. This method employs stable plasmids that allow each population to be differentially tagged with distinct antibiotic resistance capabilities and fluorescent proteins for selection and visual discrimination of each population, respectively. Here, we describe the preparation and coincubation of competing Vibrio fischeri strains, fluorescence microscopy imaging, and quantitative data analysis. This approach is simple, yields quick results, and can be used to determine whether one population kills or inhibits the growth of another population, and whether competition is mediated through a diffusible molecule or requires direct cell-cell contact. Because each bacterial population expresses a different fluorescent protein, the assay permits the spatial discrimination of competing populations within a mixed colony. Although the described methods are performed with the symbiotic bacterium V. fischeri using conditions optimized for this species, the protocol can be adapted for most culturable bacterial isolates.

Coincubation Assay for Quantifying Competitive Interactions between Vibrio fischeri Isolates

Bacteria encode diverse mechanisms for engaging in interbacterial competition. Here, we present a culture-based protocol for characterizing competitive interactions between bacterial isolates and how they impact the spatial structure of a mixed population.

This manuscript describes a culture-based, coincubation assay for detecting and characterizing competitive interactions between two bacterial populations. ...

High-throughput Antiviral Assays to Screen for Inhibitors of Zika Virus Replication

Antiviral drug discovery requires the development of reliable biochemical and cellular assays that can be performed in high-throughput screening (HTS) formats. The flavivirus non-structural (NS) proteins are thought to co-translationally assemble on the endoplasmic reticulum (ER) membranes, forming the replication complex (RC). The NS3 and NS5 are the most studied enzymes of the RC and constitute the main targets for drug development due to their crucial roles in viral genome replication. NS3 protease domain, which requires NS2B as its cofactor, is responsible for the cleavage of the immature viral polyprotein into the mature NS proteins, whereas NS5 RdRp domain is responsible for the RNA replication. Herein, we describe in detail the protocols used in replicon-based screenings and enzymatic assays to test large compound libraries for inhibitors of the Zika virus (ZIKV) replication. Replicons are self-replicating subgenomic systems expressed in mammalian cells, in which the viral structural genes are replaced by a reporter gene. The inhibitory effects of compounds on viral RNA replication can be easily evaluated by measuring the reduction in the reporter protein activity. The replicon-based screenings were performed using a BHK-21 ZIKV replicon cell line expressing Renilla luciferase as a reporter gene. To characterize the specific targets of identified compounds, we established in-vitro fluorescence-based assays for recombinantly expressed NS3 protease and NS5 RdRp. The proteolytic activity of the viral protease was measured by using the fluorogenic peptide substrate Bz-nKRR-AMC, while the NS5 RdRp elongation activity was directly detected by the increase of the fluorescent signal of SYBR Green I during RNA elongation, using the synthetic biotinylated self-priming template 3&#8242;UTR-U30 (5&#39;-biotin-U30-ACUGGAGAUCGAUCUCCAGU-3&#39;).

In this work, we describe the protocols used in replicon-based and viral enzyme-based assays to screen for inhibitors of Zika virus replication in a high-throughput screening format.

Antiviral drug discovery requires the development of reliable biochemical and cellular assays that can be performed in high-throughput screening (HTS) ...

Deferred Growth Inhibition Assay to Quantify the Effect of Bacteria-derived Antimicrobials on Competition

Competitive exclusion can occur in microbial communities when, for example, an inhibitor-producing strain outcompetes its competitor for an essential nutrient or produces antimicrobial compounds that its competitor is not resistant to. Here we describe a deferred growth inhibition assay, a method for assessing the ability of one bacterium to inhibit the growth of another through the production of antimicrobial compounds or through competition for nutrients. This technique has been used to investigate the correlation of nasal isolates with the exclusion of particular species from a community. This technique can also be used to screen for lantibiotic producers or potentially novel antimicrobials. The assay is performed by first culturing the test inhibitor-producing strain overnight on an agar plate, then spraying over the test competitor strain and incubating again. After incubation, the extent of inhibition can be measured quantitatively, through the size of the zone of clearing around the inhibitor-producing strain, and qualitatively, by assessing the clarity of the inhibition zone. Here we present the protocol for the deferred inhibition assay, describe ways to minimize variation between experiments, and define a clarity scale that can be used to qualitatively assess the degree of inhibition.

The deferred growth inhibition assay can be used to assess the competition effect by one bacterial isolate on another. Inhibition is quantified by measuring the zone of clearing around the inhibitor-producing isolate, or qualitatively assessed by determining the visible extent of inhibition.

Competitive exclusion can occur in microbial communities when, for example, an inhibitor-producing strain outcompetes its competitor for an essential ...

Quantification of Interbacterial Competition using Single-Cell Fluorescence Imaging

Interbacterial competition can directly impact the structure and function of microbiomes. This work describes a fluorescence microscopy approach that can be used to visualize and quantify competitive interactions between different bacterial strains at the single-cell level. The protocol described here provides methods for advanced approaches in slide preparation on both upright and inverted epifluorescence microscopes, live-cell and time-lapse imaging techniques, and quantitative image analysis using the open-source software FIJI. The approach in this manuscript outlines the quantification of competitive interactions between symbiotic Vibrio fischeri populations by measuring the change in area over time for two coincubated strains that are expressing different fluorescent proteins from stable plasmids. Alternative methods are described for optimizing this protocol in bacterial model systems that require different growth conditions. Although the assay described here uses conditions optimized for V. fischeri, this approach is highly reproducible and can easily be adapted to study competition among culturable isolates from diverse microbiomes.

This manuscript describes a method for using single-cell fluorescence microscopy to visualize and quantify bacterial competition in coculture.

Interbacterial competition can directly impact the structure and function of microbiomes. This work describes a fluorescence microscopy approach that can ...

A Deferred Growth Inhibition Assay to Evaluate the Effect of Bacteria-Derived Antimicrobial Compounds

Source: Moran, J. C. et al., <a href="https://app.jove.com/v/54437">Deferred Growth Inhibition Assay to Quantify the Effect of Bacteria-derived Antimicrobials on Competition.</a> J. Vis. Exp.&#160;(2016)
This video showcases the deferred growth inhibition assay, used to investigate how antimicrobial compounds produced by one type of bacteria affect another competing bacterial strain.

The following method is adapted&#160;to test competitor strains with inhibitor-producing strains.
NOTE: This protocol involves aerosol generation with ...

JoVE Encyclopedia of Experiments

Immunology

Immunodiagnostics

Specialized Assays

An Assay to Screen Bioactive Nanoparticles for Toll-Like Receptor Signaling Inhibition

Centrifuge 20 volumes of the peptide-GNP hybrid solution in 1.5-milliliter eppendorf tubes at 18,000 times g for 30 minutes. Carefully discard the supernatants. Transfer the hybrids to a single fresh tube, and wash them twice with 1 milliliter of PBS. After this, resuspend the washed hybrids in one volume of R10 medium. Mix equal volumes of the concentrated hybrids and the LPS-containing R10 medium such that the final concentration of the hybrids and LPS are 100 hundred nanomolar and 10 nanograms per milliliter respectively.
Next, remove the culture medium from the macrophage culture plate. Add 100 microliters of the hybrid-LPS mixture into each well with three replicates for each condition. Include a negative control and an LPS control. Incubate at 37 degrees Celsius for 24 hours, then, transfer the medium of each well to a separate centrifuge tube. Centrifuge the tubes at 18,000 times g in 4 degrees Celsius for 30 minutes. Transfer the supernatants into a fresh 96-well round bottom plate.
To begin the reporter assay on these samples, add 180 microliters of pre-warmed QUANTI-Blue solution to each well of a new 96-well flat bottom plate, then transfer 20 microliters of the supernatants from each sample into this plate. Incubate at 37 degrees Celsius for one to two hours until the dark blue color develops to an optical density over 1. After this, use a plate reader to record the absorption at 655 nanometers. Next, transfer 10 microliters of fresh supernatant from each sample into a 96-well clear flat-bottom white plate.
Using an automatic injector, add 50 microliters of the luciferase solution to each well and immediately record the luminescence well by well. Then, validate the inhibitory effect of the potential candidates and evaluate the TLR specificity as outlined in the text protocol.

Synthesis and Assay of Vibrio Quorum Sensing Inhibitors

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