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Ellagic acid derivatives from Terminalia chebula Retz. downregulate the expression of quorum sensing genes to attenuate Pseudomonas aeruginosa PAO1 virulence.

Sarabhai S, Sharma P, Capalash N - PLoS ONE (2013)

Bottom Line: Biofilm formation and alginate were significantly (p<0.05) reduced with enhanced (20%) susceptibility to tobramycin.Electrospray Ionization Mass Spectrometry also showed 90 and 64% reduction in the production of 3-oxo-C(12)HSL and C(4)HSL after treatment.This is the first report on anti QS activity of T. chebula fruit linked to EADs which down regulate the expression of lasIR and rhlIR genes with concomitant decrease in AHLs in P. aeruginosa PAO1 causing attenuation of its virulence factors and enhanced sensitivity of its biofilm towards tobramycin.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, Panjab University, Chandigarh, India.

ABSTRACT

Background: Burgeoning antibiotic resistance in Pseudomonas aeruginosa 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 T. chebula Retz. and identification of probable compounds(s) showing anti QS activity and the mechanism of attenuation of P. aeruginosa PAO1 virulence factors.

Methods and results: Methanol extract of T. chebula Retz. fruit showed anti QS activity using Agrobacterium tumefaciens 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 P. aeruginosa PAO1. Biofilm formation and alginate were significantly (p<0.05) reduced with enhanced (20%) susceptibility to tobramycin. Real Time PCR of F7 treated P. aeruginosa showed down regulation of autoinducer synthase (lasI and rhlI) and their cognate receptor (lasR and rhlR) genes by 89, 90, 90 and 93%, respectively. Electrospray Ionization Mass Spectrometry also showed 90 and 64% reduction in the production of 3-oxo-C(12)HSL and C(4)HSL 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 P. aeruginosa PAO1 on addition of C(4)HSL. F7 also showed antagonistic activity against 3-oxo-C(12)HSL-dependent QS in E. coli bioreporter. C. elegans fed on F7-treated P. aeruginosa 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 T. chebula extract.

Conclusions: This is the first report on anti QS activity of T. chebula fruit linked to EADs which down regulate the expression of lasIR and rhlIR genes with concomitant decrease in AHLs in P. aeruginosa PAO1 causing attenuation of its virulence factors and enhanced sensitivity of its biofilm towards tobramycin.

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Effect of F7 (1 mg/ml) and tobramycin (20 µg/ml) on biofilm formation by P. aeruginosa PAO1.
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pone-0053441-g003: Effect of F7 (1 mg/ml) and tobramycin (20 µg/ml) on biofilm formation by P. aeruginosa PAO1.

Mentions: Prophylactic efficiency of F7 was shown by significant reduction (65%, p<0.05) in biofilm formation in 24 h at 1 mg/ml (Fig. 3) that was increased to 85% at 5 mg/ml (p<0.001) (data not shown) with concomitant increase in planktonic bacterial cells by 1.2 log folds (from 2.75±0.78×106 to 6.7±0.34×107 CFU/ml). CLSM images of biofilm formed by GFP tagged P. aeruginosa in the presence of 1 mg/ml of F7 and stained with PI showed bacterial cells scattered singly on the adherent surface. Z stack analysis of the images showed 1–1.5 µm thickness that corresponds to size of single bacterial cell. However, in untreated sample, bacterial aggregation and formation of bacterial microcolonies was observed that measured to 10 µm. Non significant signal of PI indicated the absence of antibacterial effect of F7 and inhibition of biofilm formation was thus due to its anti QS activity (Fig. 4).


Ellagic acid derivatives from Terminalia chebula Retz. downregulate the expression of quorum sensing genes to attenuate Pseudomonas aeruginosa PAO1 virulence.

Sarabhai S, Sharma P, Capalash N - PLoS ONE (2013)

Effect of F7 (1 mg/ml) and tobramycin (20 µg/ml) on biofilm formation by P. aeruginosa PAO1.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3539995&req=5

pone-0053441-g003: Effect of F7 (1 mg/ml) and tobramycin (20 µg/ml) on biofilm formation by P. aeruginosa PAO1.
Mentions: Prophylactic efficiency of F7 was shown by significant reduction (65%, p<0.05) in biofilm formation in 24 h at 1 mg/ml (Fig. 3) that was increased to 85% at 5 mg/ml (p<0.001) (data not shown) with concomitant increase in planktonic bacterial cells by 1.2 log folds (from 2.75±0.78×106 to 6.7±0.34×107 CFU/ml). CLSM images of biofilm formed by GFP tagged P. aeruginosa in the presence of 1 mg/ml of F7 and stained with PI showed bacterial cells scattered singly on the adherent surface. Z stack analysis of the images showed 1–1.5 µm thickness that corresponds to size of single bacterial cell. However, in untreated sample, bacterial aggregation and formation of bacterial microcolonies was observed that measured to 10 µm. Non significant signal of PI indicated the absence of antibacterial effect of F7 and inhibition of biofilm formation was thus due to its anti QS activity (Fig. 4).

Bottom Line: Biofilm formation and alginate were significantly (p<0.05) reduced with enhanced (20%) susceptibility to tobramycin.Electrospray Ionization Mass Spectrometry also showed 90 and 64% reduction in the production of 3-oxo-C(12)HSL and C(4)HSL after treatment.This is the first report on anti QS activity of T. chebula fruit linked to EADs which down regulate the expression of lasIR and rhlIR genes with concomitant decrease in AHLs in P. aeruginosa PAO1 causing attenuation of its virulence factors and enhanced sensitivity of its biofilm towards tobramycin.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, Panjab University, Chandigarh, India.

ABSTRACT

Background: Burgeoning antibiotic resistance in Pseudomonas aeruginosa 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 T. chebula Retz. and identification of probable compounds(s) showing anti QS activity and the mechanism of attenuation of P. aeruginosa PAO1 virulence factors.

Methods and results: Methanol extract of T. chebula Retz. fruit showed anti QS activity using Agrobacterium tumefaciens 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 P. aeruginosa PAO1. Biofilm formation and alginate were significantly (p<0.05) reduced with enhanced (20%) susceptibility to tobramycin. Real Time PCR of F7 treated P. aeruginosa showed down regulation of autoinducer synthase (lasI and rhlI) and their cognate receptor (lasR and rhlR) genes by 89, 90, 90 and 93%, respectively. Electrospray Ionization Mass Spectrometry also showed 90 and 64% reduction in the production of 3-oxo-C(12)HSL and C(4)HSL 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 P. aeruginosa PAO1 on addition of C(4)HSL. F7 also showed antagonistic activity against 3-oxo-C(12)HSL-dependent QS in E. coli bioreporter. C. elegans fed on F7-treated P. aeruginosa 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 T. chebula extract.

Conclusions: This is the first report on anti QS activity of T. chebula fruit linked to EADs which down regulate the expression of lasIR and rhlIR genes with concomitant decrease in AHLs in P. aeruginosa PAO1 causing attenuation of its virulence factors and enhanced sensitivity of its biofilm towards tobramycin.

Show MeSH
Related in: MedlinePlus