Inhibition of Quorum-Sensing Regulator from Pseudomonas aeruginosa Using a Flavone Derivative

Abstract

Quorum sensing (QS) is a cell-to-cell communication process that controls bacterial collective behaviors. The QS network regulates and coordinates bacterial virulence factor expression, antibiotic resistance and biofilm formation. Therefore, inhibition of the QS system is an effective strategy to suppress the bacterial virulence. Herein, we identify a phosphate ester derivative of chrysin as a potent QS inhibitor of the human pathogen Pseudomonas aeruginosa (P. aeruginosa) using a designed luciferase reporter assay. In vitro biochemical analysis shows that the chrysin derivative binds to the bacterial QS regulator LasR and abrogates its DNA-binding capability. In particular, the derivative exhibits higher anti-virulence activity compared to the parent molecule. All the results reveal the potential application of flavone derivative as an anti-virulence compound to combat the infectious diseases caused by P. aeruginosa.

Keywords: Pseudomonas aeruginosa; flavone derivative; inhibitor; las system; quorum sensing.

Figure 1

Schematic illustration of the E. coli luciferase-based screening assay. (a) The construction of the Escherichia coli luciferase reporter strain. (b) The procedure of screening of potential inhibitors toward the P. aeruginosa las system. (c) Chemical structures of the identified six compounds that attenuate the bioluminescence of E. coli reporter strain, including three xanthone derivatives (D5, D6, D7) and three flavone derivatives (E5, E6, E7).

Figure 2

Electrophoretic mobility shift assay of full-length LasR protein binding to lasB promoter DNA. Furanone C-30 is a synthetic furanone bacterial quorum-sensing inhibitor [28]. * Indicates the free DNA band, ** indicates the DNA–LasR complex band.

Figure 3

(a) Inhibition of P. aeruginosa biofilm formation by different compounds. The biofilm content of the control group was set as 100%. The biofilm content in other groups was normalized to the control. (b) Inhibition of P. aeruginosa virulence factor by different compounds. All experiments were performed in triplicate. Results are shown as mean ± sd. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Figure 4

(a) Inhibition of P. aeruginosa virulence factor expression by increasing gradient concentrations of compound E6 (5 to 100 μM). (b) Quantitative transcript analysis of QS-regulated genes treated with or without 50 μM compound E6. (c) The swimming and swarming motility assays of P. aeruginosa with and without 50 μM of compound E6. (d) Docking model of compound E6 binding to LasR–LBD; hydrogen bonds are indicated by the gray dashed line. The molecular docking model indicates the plausible allosteric inhibitory mechanism. Results are shown as mean ± sd. * p < 0.05, ** p < 0.01, **** p < 0.0001.

Figure 5

Inhibitory mechanism of flavone derivative toward P. aeruginosa las quorum-sensing system. The scheme shows the inhibition of different regulatory pathways for P. aeruginosa virulence expression.