Small-molecule control of pyocyanin production in P. aeruginosa PA14. (A) Structures of autoinducers and inhibitors discussed in this study. (B) A simplified schematic of the major components of the P. aeruginosa quorum-sensing circuit. (C) Pyocyanin production was measured at OD = 695 nm in cell-free culture fluids prepared from WT P. aeruginosa PA14, lasR, and rhlR single and double mutants and in WT treated with 100 μM CL, CTL, mCTL, and mBTL. Error bars represent SD for two replicates. (D) Pyocyanin inhibition titrations were performed with WT P. aeruginosa PA14 in triplicate with CL (inverted triangles), CTL (squares), mCTL (diamonds), and mBTL (asterisks). Error bars represent SD of three replicates.

mBTL binds and inhibits LasR and RhlR, and the primary in vivo target of mBTL is RhlR. (A) LasR activation of expression of rsaL–gfp in E. coli. (B) RhlR activation of expression of rhlA–gfp in E. coli. In each panel, gfp expression in the presence of the cognate autoinducer (100 nM 3OC12–HSL or 20 μM C4–HSL) is set to 100%. mBTL was tested for inhibition at 1 mM. Agonism was examined at 100 nM for LasR and at 20 μM for RhlR. Error bars represent SD of three replicates. (C) SDS/PAGE analysis of whole cell (WC) and soluble (S) extracts from E. coli cultures expressing LasR in the presence of DMSO, 100 μM 3OC12–HSL, or 100 μM mBTL. An uninduced control (UN) is shown for comparison. (D) Same as C with RhlR in the presence of DMSO, 100 μM C4–HSL, or 100 μM mBTL. (E) Hierarchical clustering, heat maps, and the RMS of the fold change (log₁₀) of mBTL-treated (+) or DMSO-treated (−) WT P. aeruginosa, lasR, rhlR, and rhlI mutants. Dendrogram to the left of the map indicates average Euclidean linkage distances between the gene expression profiles. Blue and yellow indicate decreased and increased expression, respectively. Data are the average of three independent biological experiments, one in which the Cy3 and Cy5 dyes were swapped.

mBTL inhibits P. aeruginosa PA14 virulence toward C. elegans and human A549 lung cells. (A) C. elegans were applied to lawns of E. coli HB101 (circles), WT P. aeruginosa PA14 (squares), lasR mutant (triangles), rhlR mutant (inverted triangles), and lasR, rhlR double mutant (diamonds) strains. The percent live worms was calculated every hour for the first 5 h and again at 24 h. Error bars represent SEM of three replicates. (B) Same as A. E. coli HB101 (circles), WT P. aeruginosa PA14 (squares), and WT P. aeruginosa in the presence of 50 μM mBTL (asterisks). (C) The percent cell death was calculated using propidium iodide uptake into A549 lung cells after 8 h and normalized to cells lysed with detergent. Error bars represent SEM of three replicates.

mBTL inhibits quorum-sensing–regulated clogging of microfluidic chambers and biofilm formation in static cultures. (A) Time to clogging was measured for the designated P. aeruginosa PA14 strains and for the WT in the presence of 100 μM mBTL. Error bars represent SD of six replicates. (B) Biofilms were grown in static cultures at the base of a glass-bottom microtiter plate in the presence or absence of 100 μM mBTL. Biofilm thickness was measured using confocal microscopy. Error bars indicate SD of five to eight biological replicates.