Pseudomonas aeruginosa Biofilm Formation and Persistence, along with the Production of Quorum Sensing-Dependent Virulence Factors, Are Disrupted by a Triterpenoid Coumarate Ester Isolated from Dalbergia trichocarpa, a Tropical Legume.

Tsiry Rasamiravaka,Pierre Duez,Mondher El Jaziri,Olivier M Vandeputte,Martin Kiendrebeogo,Caroline Stévigny,Abel Andriantsimahavandy,Joelle Huet,Laurent Pottier,Andry Rasamindrakotroka,Christian Rabemanantsoa,Suzanne Fleiszig

doi:10.1371/journal.pone.0132791

Abstract

Recently, extracts of Dalbergia trichocarpa bark have been shown to disrupt P. aeruginosa PAO1 quorum sensing (QS) mechanisms, which are key regulators of virulence factor expression and implicated in biofilm formation. One of the active compounds has been isolated and identified as oleanolic aldehyde coumarate (OALC), a novel bioactive compound that inhibits the formation of P. aeruginosa PAO1 biofilm and its maintenance as well as the expression of the las and rhl QS systems. Consequently, the production of QS-controlled virulence factors including, rhamnolipids, pyocyanin, elastase and extracellular polysaccharides as well as twitching and swarming motilities is reduced. Native acylhomoserine lactones (AHLs) production is inhibited by OALC but exogenous supply of AHLs does not restore the production of virulence factors by OALC-treated cultures, indicating that OALC exerts its effect beyond AHLs synthesis in the QS pathways. Further experiments provided a significant inhibition of the global virulence factor activator gacA by OALC. OALC disorganizes established biofilm structure and improves the bactericidal activity of tobramycin against biofilm-encapsulated PAO1 cells. Finally, a significant reduction of Caenorhabditis elegans paralysis was recorded when the worms were infected with OALC-pre-treated P. aeruginosa. Taken together, these results show that triterpenoid coumarate esters are suitable chemical backbones to target P. aeruginosa virulence mechanisms.

Highlights

Over the last decades, pathogenic bacteria have presented an increasing multi-drug resistance prevalence all over the world [1, 2], a situation that has stimulated the search for new potential antibacterial drug targets [3, 4]
To identify the Quorum sensing (QS) inhibitory (QSI) compound(s) occurring in the D. trichocarpa bark (DTB) extract, the chromatographic fractionation was bio-guided by quantification of lasB and rhlA
The procedures and results that led to the isolation of the active compound are detailed in S1 Text and S1 Fig. The chemical structure of the isolated active compound was elucidated through spectroscopic analyzes, including UV, mass and NMR spectral data which are detailed in S2 Text, S2 and S3 Figs, leading to its identification as a 3β-hydroxyolean-12-en-28-al 3-p-coumarate or oleanolic aldehyde coumarate (OALC, MW: 586.52, C39H54O4; Fig 1 and S2 Table)

Summary

Introduction

Pathogenic bacteria have presented an increasing multi-drug resistance prevalence all over the world [1, 2], a situation that has stimulated the search for new potential antibacterial drug targets [3, 4]. Quorum sensing (QS), a bacterial cell-to-cell communication, is used by many bacteria to detect their critical cell density by producing and perceiving diffusible signal molecules in order to coordinate a common behavior such as the expression and regulation of virulence factors, motility and biofilm formation [8, 9]. The PQS system is incorporated into the QS hierarchy in times of cell stress, and acts as a link between the las and rhl quorum-sensing systems [18]. In this QS regulatory cascade, the las and rhl systems are positively influenced by the global activator GacA and the global regulator Vfr at both the transcriptional and post-transcriptional levels [19, 20]

Methods

Results

Conclusion