Article Figures & Data
Figures
- FIG. 1.
2D structures of the compounds tested in this study. (A) Structures of the known LasR binding compounds used in the 2D similarity search against the SuperNatural and SuperDrug databases (OdDHL, TP-1, furanone C30, and patulin) and included in the SB-VS for reference (OdDHL) and comparison (all). (B) Structures of the six compounds tested for the ability to inhibit LasR in P. aeruginosa.
- FIG. 2.
Dose-response curves of the three drugs salicylic acid (A), nifuroxazide (B), and chlorzoxazone (C) when incubated together with PAO1 lasB::gfp(ASV). The three compounds reduce the RFU level (GFP fluorescence units divided by OD450) at various concentrations (right), indicating inhibition of LasR, while displaying no effect on growth (left). Growth is displayed as the increase in OD450 compared to the initial level. Salicylic acid and nifuroxazide were tested at concentrations of 400 (▪), 200 (Δ), 50 (•), 12.5 (□), 3.125 (▴), and 0 (○) μg/ml, while chlorzoxazone was tested at 200 (▪), 100 (▵), 20 (•), 6.25 (□), 1.5625 (▴), and 0 (○) μg/ml. Results are representative of three independent experiments.
- FIG. 3.
Expression of rhlA::gfp(ASV) (A) and pqsA::gfp(ASV) (B) in wild-type and lasR mutant P. aeruginosa PAO1 treated with the three identified LasR inhibitors. Results are average RFU values taken from a single time point measurement corresponding to maximal induction of the reporters in the late log phase of growth. Inhibitors were added at concentrations of 200 μg/ml for salicylic acid and nifuroxazide and 100 μg/ml for chlorzoxazone. Averages and SDs of eight replicates are shown.
- FIG. 4.
Inhibition of exogenous protease production (A), pyoverdine production (B), and rhamnolipid production (C) in P. aeruginosa treated with the three identified LasR inhibitors. 1, untreated; 2, lasI rhlI mutant (QS deficient); 3, salicylic acid treated; 4, nifuroxazide treated; 5, chlorzoxazone treated. Results were taken after 18 h of growth at 37°C. Averages and SDs of five replicates are shown. Relat., relative; activ., activity; prod., production.
- FIG. 5.
CLSM pictures of 4-day-old P. aeruginosa biofilms. A, untreated; B, lasI rhlI mutant (QS deficient); C, salicylic acid treated; D, nifuroxazide treated; E, chlorzoxazone treated. The strains were GFP tagged for visualization. The main pictures are top-down 3D projections, while the flanking pictures are vertical sections. Bars, 20 μm.
- FIG. 6.
Results of COMSTAT analysis for total biomass calculation of the individual biofilms. 1, untreated; 2, lasI rhlI mutant (QS deficient); 3, salicylic acid treated; 4, nifuroxazide treated; 5, chlorzoxazone treated. Averages and SDs from analysis of 12 images taken at random positions in three different biofilms are shown.
- FIG. 7.
Scenes displaying the conformations (magenta) of the identified LasR inhibitors and known LasR inhibitors as proposed by MVD after docking of the compounds. A, salicylic acid; B, nifuroxazide; C, chlorzoxazone; D, 4-NPO, the known LasR inhibitor used as cutoff in the docking; E, furanone C30, a potent LasR and QSI; F, OdDHL, the natural LasR ligand, as it is present in the LasR crystal structure with its hydrogen bonds (black lines) to the protein (green residues). In scenes A to E, hydrogen bonds proposed by MVD to occur upon binding are shown as black dotted lines together with the interaction residues (green). The conformation of OdDHL (white) has been included in scenes A to E for comparison, and the backbone of the LasR ligand binding domain is shown in blue-white with α-helical residues 65 to 72 omitted for clarity.
Tables
- TABLE 1.
Names and types of LasR binding compounds used as references and for comparison in the SB-VS
Compound Type Reference 3-Oxo-C12-HSL Natural ligand 45 3-Oxo-C12-(2-aminophenol) Inhibitor 62 4-Nitropyridine-N-oxide Inhibitor 50 Furanone C30 Inhibitor 29 Patulin Inhibitor 51 Penicillic acid Inhibitor 51 TP-1 Activator 42 TP-5 Inhibitor 42 - TABLE 2.
Rerank scores, similarity scores, and molecular weights of the acquired compounds, the reference ligand, and the known LasR binding compounds included for comparisona
Compound Mol wt Similarity score Rerank score Acquired compounds Indoramine 340.4 −491.29 −121.281 Nifuroxazide 274.2 −447.124 −86.3983 Tiaprofenic acid 258.3 −396.031 −71.4197 Donepezil 370.4 −499.284 −70.7016 Chlorzoxazone 169.6 −344.493 −69.6008 Salicylic acid 138.1 −336.595 −66.156 Reference, 3-oxo-C12-HSL 297.4 −129.739 Known LasR binders 3-Oxo-C12-(2-aminophenol) 305.4 −470.817 −93.8255 Patulin 154.1 −354.068 −70.1662 4-NPO 140.1 −305.698 −63.1366 Furanone C30 257.9 −265.816 −47.9107 Penicillic acid 170.2 −315.516 75.4073 TP-1 568.6 −497.462 220.864 TP-5 385.2 −398.532 431.434 ↵a The compounds were ranked on the basis of their rerank scores. The scores of the individual compounds are the best of five scores derived from independent docking rounds.
Supplemental material
Files in this Data Supplement:
- Supplemental file 1
-
Supplemental table showing settings used in Molegro Virtual Docker for import of molecules, template creation, and docking
of the compound database. Legends for supplemental figures.
Zipped Word document, 8K. - Supplemental file 2
-
Figure showing template assignment to the individual heavy atoms of the natural ligand 3-oxo-C12-HSL present in the LasR
ligand binding domain used for the docking.
Zipped TIFF image, 374K. - Supplemental file 3
-
Figure showing a representative result of the drop-collapse biosurfactant assay used to qualitatively estimate the production
of rhamnolipid.
Zipped TIFF image, 1.5MB.
- Supplemental file 1
-
Supplemental table showing settings used in Molegro Virtual Docker for import of molecules, template creation, and docking
of the compound database. Legends for supplemental figures.
