Ellagic Acid Derivatives from <em>Terminalia chebula</em> Retz. Downregulate the Expression of Quorum Sensing Genes to Attenuate <em>Pseudomonas aeruginosa</em> PAO1 Virulence

<div><h3>Background</h3><p>Burgeoning antibiotic resistance in <em>Pseudomonas aeruginosa</em> has necessitated the development of anti pathogenic agents that can quench acylhomoserine lactone (AHL) mediated QS with least risk of resistance. This study explores the anti quorum sensing potential of <em>T. chebula</em> Retz. and identification of probable compounds(s) showing anti QS activity and the mechanism of attenuation of <em>P. aeruginosa</em> PAO1 virulence factors.</p> <h3>Methods and Results</h3><p>Methanol extract of <em>T. chebula</em> Retz. fruit showed anti QS activity using <em>Agrobacterium tumefaciens</em> A136. Bioactive fraction (F7), obtained by fractionation of methanol extract using Sephadex LH20, showed significant reduction (p<0.001) in QS regulated production of extracellular virulence factors in <em>P. aeruginosa</em> PAO1. Biofilm formation and alginate were significantly (p<0.05) reduced with enhanced (20%) susceptibility to tobramycin. Real Time PCR of F7 treated <em>P. aeruginosa</em> showed down regulation of autoinducer synthase (<em>lasI</em> and <em>rhlI</em>) and their cognate receptor (<em>lasR</em> and <em>rhlR</em>) genes by 89, 90, 90 and 93%, respectively. Electrospray Ionization Mass Spectrometry also showed 90 and 64% reduction in the production of 3-oxo-C₁₂HSL and C₄HSL after treatment. Decrease in AHLs as one of the mechanisms of quorum quenching by F7 was supported by the reversal of inhibited swarming motility in F7-treated <em>P. aeruginosa</em> PAO1 on addition of C₄HSL. F7 also showed antagonistic activity against 3-oxo-C₁₂HSL-dependent QS in <em>E. coli</em> bioreporter. <em>C. elegans</em> fed on F7-treated <em>P. aeruginosa</em> showed enhanced survival with LT50 increasing from 24 to 72 h. LC-ESI-MS of F7 revealed the presence of ellagic acid derivatives responsible for anti QS activity in <em>T. chebula</em> extract.</p> <h3>Conclusions</h3><p>This is the first report on anti QS activity of <em>T. chebula</em> fruit linked to EADs which down regulate the expression of <em>lasIR</em> and <em>rhlIR</em> genes with concomitant decrease in AHLs in <em>P. aeruginosa</em> PAO1 causing attenuation of its virulence factors and enhanced sensitivity of its biofilm towards tobramycin.</p> </div

Schematic representation of bioassay guided fractionation of <i>T. chebula</i> fruit extract.

<p>Schematic representation of bioassay guided fractionation of <i>T. chebula</i> fruit extract.</p

Comparative effect of F7 and ellagic acid on the production of virulence factors at 0.5 mg/ml.

<p>Elastase OD_{490 nm/600 nm} 0.654,Pyocyanin OD _{540 nm/600 nm} 1.08, Rhamnolipids OD _{570 nm/600 nm} 0.456 and Protease OD _{400 nm/600 nm}0.876 were taken as 100% in untreated <i>P. aeruginosa</i> PAO1.</p

CLSM images of biofilm formed by <i>P. aeruginosa</i> PAO1 (63X magnification) A)Untreated B) Treated with 1 mg/ml F7.

<p>CLSM images of biofilm formed by <i>P. aeruginosa</i> PAO1 (63X magnification) A)Untreated B) Treated with 1 mg/ml F7.</p

Effect of F7 (1 mg/ml) and tobramycin (20 µg/ml) on biofilm formation by <i>P. aeruginosa</i> PAO1.

<p>Effect of F7 (1 mg/ml) and tobramycin (20 µg/ml) on biofilm formation by <i>P. aeruginosa</i> PAO1.</p

<i>C. elegans</i>- <i>P. aeruginosa</i> killing assay A) LT ₅₀ of <i>C. elegans</i> increased from 24 to 72 h when fed on <i>P. aeruginosa</i> PAO1 treated with 0.5 mg/ml of F7.

<p>B) Microscopic images of <i>C. elegans</i> (100X) fed on 1) <i>P. aeruginosa</i> PAO1 2) <i>P. aeruginosa +</i>0.5 mg/ml F7 3) <i>E. coli</i> OP50<i>+</i>0.5 mg/ml F7.</p

Relative expression of <i>lasIR</i> and <i>rhlIR</i> genes of <i>P. aeruginosa</i> PAO1 in the presence of 0.5 mg/ml F7 as determined by qRT PCR.

<p>Relative expression of <i>lasIR</i> and <i>rhlIR</i> genes of <i>P. aeruginosa</i> PAO1 in the presence of 0.5 mg/ml F7 as determined by qRT PCR.</p

Swarming motility of <i>P. aeruginosa</i> PAO1 a) Untreated b) Treated with 0.5 mg/ml of F7 c) reversal of inhibited swarming motility by the addition of exogenous C₄HSL(2 µM).

<p>Swarming motility of <i>P. aeruginosa</i> PAO1 a) Untreated b) Treated with 0.5 mg/ml of F7 c) reversal of inhibited swarming motility by the addition of exogenous C₄HSL(2 µM).</p

Putative anti QS compounds as shown by LC-ESI-MS fragmentation data for the bioactive fraction.

<p>Putative anti QS compounds as shown by LC-ESI-MS fragmentation data for the bioactive fraction.</p

Bacterial strains and plasmids used in this study.

<p>Bacterial strains and plasmids used in this study.</p