Evaluation of enoyl-acyl carrier protein reductase inhibitors as Pseudomonas aeruginosa quorum-quenching reagents

Abstract

Pseudomonas aeruginosa is an opportunistic pathogen which is responsible for a wide range of infections. Production of virulence factors and biofilm formation by P. aeruginosa are partly regulated by cell-to-cell communication quorum-sensing systems. Identification of quorum-quenching reagents which block the quorum-sensing process can facilitate development of novel treatment strategies for P. aeruginosa infections. We have used molecular dynamics simulation and experimental studies to elucidate the efficiencies of two potential quorum-quenching reagents, triclosan and green tea epigallocatechin gallate (EGCG), which both function as inhibitors of the enoyl-acyl carrier protein (ACP) reductase (ENR) from the bacterial type II fatty acid synthesis pathway. Our studies suggest that EGCG has a higher binding affinity towards ENR of P. aeruginosa and is an efficient quorum-quenching reagent. EGCG treatment was further shown to be able to attenuate the production of virulence factors and biofilm formation of P. aeruginosa.

Figure 1

Pairwise protein sequence alignment of ENR from P. aeruginosa (PaENR) with ENR from E. coli (EcENR). Alignment was performed by Discovery Studio Visualizer 2.0 (Accelrys) and conserved residues are shown in dark blue with a white background.

Figure 2

Hydrogen bond interaction analysis by Molegro Molecular Viewer. A: PaENR-triclosan; B: PaENR-EGCG; C: PaENR-NADH.

Figure 3

Expression of lasB:gfp(ASV) in wild-type P. aeruginosa treated with triclosan (black bar) and EGCG (gray bar). Results are average values of green fluorescence divided by OD₆₀₀ 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 0 µM, 1 µM, 25 µM and 250 µM. Averages and SDs of three replicates are shown.

Figure 4

Expression of pqsA:gfp(ASV) in wild-type P. aeruginosa treated with EGCG. Results are average values of green fluorescence divided by OD₆₀₀ 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 0 µM, 1 µM, 25 µM and 250 µM. Averages and SDs of three replicates are shown.

Figure 5

Swarming motility of wild-type P. aeruginosa strain on agar plates containing 0 µM (A), 1 µM (B), 25 µM (C) and 250 µM EGCG (D).

Figure 6

4-days-old biofilms of Gfp-tagged wild-type grown in FAB medium without (A) or with 25 µM EGCG (B). Biofilms were further treated with 50 µg/ml ciprofloxacin for 24 h (C: biofilm grown in FAB without EGCG; D: biofilm grown in FAB with 25 µM EGCG), after which they were stained with propidium iodide and images were acquired by CLSM. Live cells appear green and dead cells appear red.