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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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Antagonistic activity of F7 against 3-oxo-C12HSL mediated QS in E. coli bioreporter strain harboring pSC11(PlasI)::lacZ) and lasR expression vector pJN105L.
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pone-0053441-g008: Antagonistic activity of F7 against 3-oxo-C12HSL mediated QS in E. coli bioreporter strain harboring pSC11(PlasI)::lacZ) and lasR expression vector pJN105L.

Mentions: Real time PCR showed 89, 90, 90 and 93% reduction in the expression of lasI, lasR, rhlI and rhlR, respectively with 0.5 mg/ml of F7(Fig. 6). This was supported by the reduction of peak intensity of 3-oxo-C12HSL by 64% (peaks corresponding to 3-oxo-C12HSL at m/z 316 ammonium and 595 dimer adduct) and 90% reduction in C4-HSL (peaks corresponding to C4HSL at m/z 159) on ESI MS analysis of AHLs after F7 exposure (Fig. S4). The reduction in both AHLs was consistence with the reduction in virulence factors controlled by 3-oxo-C12HSL (elastase and protease) and C4HSL (rhamnolipids and pyocyanin) in P. aeruginosa PAO1(Fig. 2). Growth of P. aeruginosa PAO1 was monitored in the presence of different concentrations of F7 (0–1.25 mg/ml) and it showed insignificant change in growth (p = 0.132) at 0.5 mg/ml (Fig. S5) indicating quorum sensing inhibition as the mechanism for the reduction in AHLs and not the killing of cells. Restoration of inhibited swarming motility on addition of 2 µM C4HSL further supported the observation (Fig. 7). F7 at 1 mg/ml also reduced β-galactosidase activity by 93% in E. coli bioreporter strain showing its antagonistic activity towards transcriptional regulator lasR (Fig. 8).


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)

Antagonistic activity of F7 against 3-oxo-C12HSL mediated QS in E. coli bioreporter strain harboring pSC11(PlasI)::lacZ) and lasR expression vector pJN105L.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0053441-g008: Antagonistic activity of F7 against 3-oxo-C12HSL mediated QS in E. coli bioreporter strain harboring pSC11(PlasI)::lacZ) and lasR expression vector pJN105L.
Mentions: Real time PCR showed 89, 90, 90 and 93% reduction in the expression of lasI, lasR, rhlI and rhlR, respectively with 0.5 mg/ml of F7(Fig. 6). This was supported by the reduction of peak intensity of 3-oxo-C12HSL by 64% (peaks corresponding to 3-oxo-C12HSL at m/z 316 ammonium and 595 dimer adduct) and 90% reduction in C4-HSL (peaks corresponding to C4HSL at m/z 159) on ESI MS analysis of AHLs after F7 exposure (Fig. S4). The reduction in both AHLs was consistence with the reduction in virulence factors controlled by 3-oxo-C12HSL (elastase and protease) and C4HSL (rhamnolipids and pyocyanin) in P. aeruginosa PAO1(Fig. 2). Growth of P. aeruginosa PAO1 was monitored in the presence of different concentrations of F7 (0–1.25 mg/ml) and it showed insignificant change in growth (p = 0.132) at 0.5 mg/ml (Fig. S5) indicating quorum sensing inhibition as the mechanism for the reduction in AHLs and not the killing of cells. Restoration of inhibited swarming motility on addition of 2 µM C4HSL further supported the observation (Fig. 7). F7 at 1 mg/ml also reduced β-galactosidase activity by 93% in E. coli bioreporter strain showing its antagonistic activity towards transcriptional regulator lasR (Fig. 8).

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