Zingerone silences quorum sensing and attenuates virulence of Pseudomonas aeruginosa

FITOTE-03122; No of Pages 12 Fitoterapia xxx (2015) xxx–xxx

Contents lists available at ScienceDirect

Fitoterapia

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journal homepage: www.elsevier.com/locate/fitote

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Lokender Kumar a, Sanjay Chhibber a, Rajnish Kumar b, Manoj Kumar b, Kusum Harjai a,⁎

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Zingerone silences quorum sensing and attenuates virulence of Pseudomonas aeruginosa

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Department of Microbiology, Panjab University, Chandigarh 160014, India University Institute of Pharmaceutical Sciences, Panjab University, Chandigarh 160014, India

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Article history: Received 19 November 2014 Accepted in revised form 10 February 2015 Available online xxxx

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Keywords: Zingerone Virulence Quorum sensing Phytochemical Biofilm formation Antibiotic resistance

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1. Introduction

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Pseudomonas aeruginosa causes serious eye [1], ear [2] burn wounds [3], urinary tract [4], respiratory tract infections (cystic fibrosis) [5] and is also frequently associated with sepsis and septic shock [6]. Chronic lung infections with P. aeruginosa are associated with disease progression in cystic fibrosis patients [7]. Availability of antibiotic to treat such deadly infections is still limited and Pseudomonas continues to fail the current

Quorum sensing in Pseudomonas aeruginosa plays an imperative role in virulence factor, biofilm formation and antimicrobial resistance. Blocking quorum sensing pathways are viewed as viable anti-virulent therapy in association with traditional antimicrobial therapy. Anti-quorum sensing dietary phytochemicals with may prove to be a safe and viable choice as anti-virulent drug candidates. Previously, our lab proved zingerone as potent anti-biofilm agent hence; further its anti-virulent and anti-quorum activities were evaluated. Zingerone, besides decreasing swimming, swarming and twitching phenotypes of P. aeruginosa PAO1, reduced biofilm forming capacity and production of virulence factors including rhamnolipid, elastase, protease, pyocyanin, cell free and cell bound hemolysin (p b 0.001) indicating anti-virulent property attributing towards attenuation of virulence of P. aeruginosa. Further zingerone not only had marked effect on the production of quorum sensing signal molecules by clinical isolates of P. aeruginosa but also showed significant interference with the activation of QS reporter strains. To study the mechanism of blocking quorum sensing cascade, in silico analysis was carried out. Anti-QS activity was attributed to interference with the ligand receptor interaction of zingerone with QS receptors (TraR, LasR, RhlR and PqsR). Zingerone showed a good comparative docking score to respective autoinducer molecules which was even higher than that of vanillin, a proven anti-quorum sensing phytochemical. The results of the present study revealed the anti-quorum sensing activity of zingerone targeting ligand–receptor interaction, hence proposing zingerone as a suitable antivirulent drug candidate against P. aeruginosa infections. © 2015 Published by Elsevier B.V.

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⁎ Corresponding author at: Dept. of Microbiology, BMS Block, Panjab University, Chandigarh 160014, India. Tel.: +91 172 2534142; fax: +91 172 2547170. E-mail address: [email protected] (K. Harjai).

chemotherapy by developing new resistance [8]. Nosocomial pathogen P. aeruginosa [9] controls host colonization, biofilm formation, motility, antibiotic resistance and regulate the expression of enormous virulence determinants using quorum sensing dependent pathways [10]. Quorum sensing (QS) is a cell to cell communication network that permits P. aeruginosa to coordinate gene expression in response to high population density [11]. Chemical signal molecules termed autoinducers are involved in bacterial communication [12]. This sophisticated signaling regulates expression of approximately 350 genes, in which 30% encodes for virulence factor production and biofilm formation [13]. Inefficacy of antibiotic therapy against P. aeruginosa infections has been linked to persistence of biofilm during infections and emergence of multi drug resistance

http://dx.doi.org/10.1016/j.fitote.2015.02.002 0367-326X/© 2015 Published by Elsevier B.V.

Please cite this article as: Kumar L, et al, Zingerone silences quorum sensing and attenuates virulence of Pseudomonas aeruginosa, Fitoterapia (2015), http://dx.doi.org/10.1016/j.fitote.2015.02.002

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Fig. 1. Chemical structure of zingerone [4-(4-hydroxy-3-methoxyphenyl) butan-2-one].

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2.1. Bacterial strains

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P. aeruginosa standard strain PAO1 and five uroisolates of P. aeruginosa were used in this study. Five clinical isolates of P. aeruginosa consecutively isolated from catheterized patients having UTI, attending Government Medical College and Hospital, Chandigarh, India and maintained in our laboratory. Agrobacterium tumefaciens A136 [31] was used for the detection of long chain AHLs and maintained on LB agar with spectinomycin (50 μg/ml) and tetracycline (4.5 μg/ml). Two reporter strains, Escherichia coli MG4 (λ1.4) and PAO-JP2 (pECP61.5) [32], were also used for the detection of OdDHL and BHL, respectively. These reporter strains were grown under antibiotic pressure of ampicillin (100 mg/ml) and carbenicillin (250 mg/ml) respectively. Standard strain PAO1 and reporter strains (A. tumefaciens A136, E. coli MG4 (λ1.4), PAO-JP2) were obtained from Dr. Barbara H. Iglewski, Department of Microbiology and Immunology, University of Rochester, New York. (U.S.A.). P. aeruginosa strain PAO-R1 (pTS400) [33] was used as reporter strain for estimation of PQS which was generously provided by Everett Pesci, East Carolina University School of Medicine, Greenville, USA. Reporter PAO-R1 strain was maintained in Luria Bertani broth in the presence of carbenicillin (60 μg/ml). Only quorum sensing reporter strains were grown under antibiotic pressure. All strains were maintained as 10% glycerol stocks and stored at − 80 °C. Fresh subcultures were prepared for each new experiment from glycerol stocks.

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2.2. Drugs and chemicals

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Pure zingerone [4-(4-hydroxy-3-methoxyphenyl)-butan2-one] was obtained from Gogia Chemical Industries, India. Antibiotics were purchased from Himedia Chemicals, India. All other reagents and chemicals used were of analytical grade.

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2.3. MIC determination and sub-MIC selection

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The MIC of zingerone ([4-(4-hydroxy-3-methoxyphenyl)butan-2-one]) (Gogia Chemicals) was determined according to NCCLS (2002) guidelines against the standard P. aeruginosa strain, PAO1. Briefly, different dilutions of zingerone were prepared; 6 h culture was used as the inoculum. The

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2. Materials and methods

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the present study was to evaluate the mechanism of antivirulence by means of blocking quorum sensing cascade. Recently there is an upsurge in use of computer aided drug design methodologies such as ligand and structure based techniques to aid in discovery of novel quorum sensing inhibitors [24]. Biomolecular docking studies provide a concrete in silico molecular evidence to prove ligand receptor interaction. Molecular docking studies were performed to validate in vitro anti-quorum sensing activity of zingerone and to get insight into the mechanism of inhibition of zingerone. The results of in silico studies were compared with vanillin, which is a known quorum sensing inhibitor and with respective autoinducer of the known quorum sensing receptors (TraR, LasR, RhlR and PqsR) of P. aeruginosa [30].

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mechanisms [14,15]. Global threat of antibiotic resistance in P. aeruginosa poses a serious concern to healthcare community and pharmaceutical industry since drug resistance continues to raise exponentially [16]. In P. aeruginosa, the autoinducers belong to acyl homoserine lactones (AHL's). Quorum sensing network consist of mainly three interdependent systems, the lasI–lasR, rhlI–rhlR [17] and Pqs systems [18]. lasI–lasR system regulate the production of N-(3-oxododecanoyl) homoserine lactone (3O-C12-HSL) and rhlI–rhlR system regulate the production of N-butyryl homoserine lactone (C4-HSL) [19]. Pqs signaling system produce 2-heptyl-3-hydroxy-4-quinolone (PQS) molecule that plays an interconnecting role in quorum sensing hierarchy of P. aeruginosa between las and rhl systems [18]. Emerging research has suggested that quorum sensing mutant strains form thinner biofilms and show decreased expression of virulence factors than wild type strains [14]. Recent reports have also shown the role of AHL in regulating motility [20] pyocyanin [21] rhamnolipid [22] and exopolysaccharide production [23]. The respective signal molecule (3O-C12-HSL, C4-HSL and PQS) binds to cognate quorum sensing receptor (LasR, RhlR and PqsR) to form a ligand–receptor complex that activates the expression of genes required for virulence factor production and biofilm formation [24]. Blocking quorum sensing may disarm pathogen hence making them highly susceptible for killing by immune system or low doses of antibiotics. Natural or synthetic agents possessing quorum sensing inhibitory property may act as anti-virulent drugs which do not pose selective pressure on pathogen for survival hence may reduce the rapid emergence of drug resistance. Recently, researchers have shown that dietary phytochemicals possess anti-quorum sensing activity [25]. Ginger (Zingiber officinale) has been widely used as an herbal medicine that contains potent anti-inflammatory compounds such as gingerols, shogaols, paradols, gingerdiols, and zingerone [26]. Zingerone (4-para methoxy-4-hydroxyphenyl2butanone, vanillyl acetone) (Fig. 1) is mainly found in dry ginger root. Zingerone effectively modulate the biofilm architecture of P. aeruginosa [27] thus making it more susceptible to antibiotic treatment [28]. Recently, it was also observed in our lab that zingerone attenuates the cell surface properties making P. aeruginosa highly susceptible for killing with antibiotics and arms of innate immunity (serum sensitivity and phagocytosis) [29]. Further, to validate anti-virulence properties of zingerone, motility phenotypes, biofilm formation capacity (BFC) and production of virulence factors and quorum sensing signal molecules of P. aeruginosa were investigated. Further the aim of

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Please cite this article as: Kumar L, et al, Zingerone silences quorum sensing and attenuates virulence of Pseudomonas aeruginosa, Fitoterapia (2015), http://dx.doi.org/10.1016/j.fitote.2015.02.002

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2.5. Motility assay

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Standard strain PAO1 and five clinical isolates were evaluated for motility phenotypes and virulence factor production in vitro and without supplementation of sub MIC of zingerone.

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2.6. Swarming motility

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Nutrient agar (8 g/l) supplemented with glucose (5 g/l) along with 1 mg/ml zingerone was prepared and plates were point inoculated with sterile tooth pick from overnight culture of P. aeruginosa strains. After incubation at 37 °C for 24 h, swarming motility was determined by measuring circular turbid zones.

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2.7. Swimming motility

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Media plates containing 1% tryptone, 0.5% NaCl and 0.3% agarose along with 1 mg/ml zingerone were point inoculated with sterile tooth pick from overnight culture of P. aeruginosa strains. After incubation at 30 °C for 24 h, swimming motility was determined by measuring the radius of circular expansion of bacterial migration from the point of inoculation.

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2.9.2. Pyocyanin estimation Pyocyanin was estimated using the method of Huerta et al. [34]. Briefly P. aeruginosa strains were grown in the presence of zingerone. Chloroform was added to cell free culture supernatant and OD at 690 nm of chloroform layer was taken.

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2.9.3. Hemolysin production Quantitative determination of cell free and cell bound hemolysin was done using 2% suspensions of washed human erythrocytes following the method of Lankisch et al. [35]. The amount of haemolysin released was determined using lyophilized hemoglobin to calibrate a standard curve. Results were expressed as hemoglobin released in mg per ml (Hb released mg/ml).

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2.9.4. Elastase production Elastolytic activity was measured by using elastin-congo red (Sigma Chemical Company, USA) as substrate following the method of Visca et al. [36]. Optical density was taken at 495 nm and results were expressed in units per liter (U/l).

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2.9.5. Protease production For proteolytic activity, culture supernatants were diluted in 10 mM Tris (pH 7.5) and incubated with 15 mg Remazol Brilliant Blue R–Hide (Sigma Chemical Company, USA) at 37 °C for 1 h. Absorbance was measured at 595 nm and results were expressed in units per liter (U/l) as described by Visca et al. [36].

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2.9.6. Rhamnolipid estimation Rhamnolipid in the culture supernatant was quantified using orcinol method as described by Zhu et al. [37]. Briefly culture supernatant was diluted in orcinol reagent and heated in water bath for 80 °C for 30 min and optical density was measure at 421 nm.

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Biofilm-forming capacity was determined by a microtitre plate assay. Biofilms were allowed to develop in 96 well standard microtiter plate having U shaped wells (Thermo Scientific) with and without sub-MIC of zingerone. The plate was incubated at 37 °C for 24 h under static conditions. After 24 h, the wells were drained and washed three times with sterile PBS to remove free cells. The wells were stained with crystal violet (0.1%, w/v) for 15 min at room temperature. Excess dye was removed by washing the wells with sterile PBS. Dye taken up by the biofilm cells was extracted with 95% (v/v) ethanol. Absorbance was measured at 570 nm and biofilmforming capacity was calculated as A570 of the strain in the presence of zingerone/A570 of the strain in the absence of zingerone. Sterile media and sterile media with zingerone served as control.

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2.9.1. Preparation of cell free supernatant (CFS) P. aeruginosa strains were grown with and without the supplementation of zingerone. Next day culture supernatants were collected from cells after adjusting the absorbance (A540) to 1 for estimation of virulence factors. Sterile media and sterile media with zingerone served as control.

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concentration of zingerone resulting in no visible growth was selected as the MIC. Sterile media and sterile media with zingerone served as control. For all further experiments, a subMIC of zingerone was used for all strains.

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2.10. Effect of zingerone on activation of quorum sensing reporter 262 strain 263 264

2.8. Twitching motility

2.10.1. Extraction of AHLs P. aeruginosa PAO1 was grown overnight in Luria broth for 14–16 h at 37 °C. Overnight grown culture supernatants (10 ml) were extracted twice with equal volume of acidified ethyl acetate and used for further experiments.

Media plates containing Luria agar along with 1 mg/ml zingerone were prepared and stabbed with toothpick up to bottom of the Petri dish from overnight culture of P. aeruginosa strains. After incubation at 37 °C for 48 h, a hazy zone of growth at the interface between the agar and polystyrene surface was observed.

2.10.2. Quantitative assay for estimation of quorum sensing signal molecules The reporter culture was diluted 1:1 in Z buffer [Na2HPO4·7H2O (0.06 M), NaH2PO4·H2O (0.04 M), KCl (0.01 M), MgSO4·7H2O (0.001 M), pH 7] and assayed for β-galactosidase activity by using ONPG as substrate. After the development of

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Please cite this article as: Kumar L, et al, Zingerone silences quorum sensing and attenuates virulence of Pseudomonas aeruginosa, Fitoterapia (2015), http://dx.doi.org/10.1016/j.fitote.2015.02.002

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2.10.5. Molecular docking studies Docking is a computational process that predicts the preferred confirmation of one molecule to a second when bound to each other to form a stable complex. Molecular docking studies were performed using Glide, version 5.8, Schrodinger, 2012. The crystal structure of TraR (PDB ID: 1H0M), LasR protein (PDB ID: 2UV0), and PqsR (PDB ID: 4JVI) were downloaded from http://www.pdb.org and the modeled RhlR protein (ID: P54292.1) was obtained from http://www. proteinmodelportal.org/query/uniprot/P54292 because of unavailability of its crystal structure. The protein structures obtained were not suitable for immediate use in molecular docking studies. Thus the protein structures were prepared using protein preparation wizard of Schrodinger molecular modeling suite. The structures were processed to assign bond orders, add hydrogen, create disulfide bond, and fill in missing side chains using Prime and Delete water beyond 5 Å from hetero groups to fix any irregularity in the structure. Finally the structures were refined through restrained minimization using the OPLS2005 forcefield. The chemical structures of the ligands were constructed using 2-D sketcher of Maestro implemented in Schrodinger. The 3-D structures were generated and optimized using Ligprep. Conformers were generated with maximum number of conformers as 1000 using OPLS 2005 forcefield and root mean square deviation (RMSD) as 1 Å. For TraR, LasR and PqsR docking grid was generated using the receptor active site where autoinducers were bound while in case of RhlR site map was used to predict the binding site of BHL to RhlR receptor. These grids were used for docking studies. Docking was performed using Glide with enabling the “write XP descriptor information” option and keeping parameters for number of poses and energy window, RMSD, atomic displacement and strain correction as default. Docking posses of each ligand were analyzed by examining their total energy score (Glide XP score). Glide Score XP is a harder function that exacts severe penalties for poses that violate established

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Growth profile of P. aeruginosa in the presence of different concentrations of zingerone was studied (Data not shown). Sub MIC (10 mg/ml) of zingerone not showing any significant growth inhibition was selected and used for further experiments.

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3.2. Zingerone affects biofilm forming capacity

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3. Results

The results demonstrated that standard strain PAO1 and clinical isolates showed high biofilm forming capacity (BFC) (Fig. 2). Zingerone significantly reduced biofilm forming capacity of PAO1 and all strains (P b 0.001). PAO1 and L-4 strain showed maximum BFC of 1.1 and 1.0 respectively and zingerone significantly (p b 0.001) reduced the BFC to 0.5 to and 0.45 (Fig. 2).

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2.10.4. Effect of zingerone on quorum sensing reporter strains Quorum sensing reporter strains, A. tumefaciens 136, E. coli MG4 (λ1.4), PAO-JP2 and PAO-R1 that detect specific signal molecules were incubated with equal volume of overnight extracted culture supernatant along with different concentration of zingerone (100 μg/ml, 500 μg/ml and 1000 μg/ml). Deactivation of reporter strains by zingerone was checked by quantitative assay.

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2.10.3. Effect of zingerone on quorum sensing signal molecule production Standard strain PAO1 and five clinical isolates were grown in the presence of sub-MIC (10 mg/ml) of zingerone and overnight culture supernatant was evaluated for presence of long chain AHL molecules, specific quorum sensing signal molecules including BHL (rhl system), OdDHL (las system) and PQS (PQS system). The effect of zingerone on production of quorum sensing signal molecule production was checked using quantitative assay.

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physical chemistry principles such as that charged and strongly polar groups be adequately exposed to solvent. It is more adept at minimizing false positives and can be especially useful in lead optimization or other studies in which only a limited number of compounds will be considered experimentally and each computationally identified compound needs to be as high in quality as possible. The best orientation was selected on the basis of docking score.

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3.3. Zingerone affects motility phenotypes of P. aeruginosa

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PAO1 and five clinical isolates showed characteristic motility phenotypes and showed unique dendritic appearance in swarming and smooth spreading of colony in swimming motility (Fig. 3). These organisms also showed twitching motility by showing a circular zone of cells on the surface of Petri dish (Fig. 3). Presence of zingerone in the media significantly reduced zone of diameter of swarming swimming and twitching motility of P. aeruginosa (Table 1).

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Zingerone at sub-MIC concentration significantly affected 365 the production of virulence factors of P. aeruginosa. PAO1 366

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yellow color, 1 ml of 1 M Na2CO3 was added to stop the reaction. OD of the reaction samples was taken at 420 and 550 nm. Units of β-galactosidase were calculated as 1000 × A420 nm − (1.75 × A550 nm) / time × volume × A600 nm.

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Fig. 2. Effect of zingerone on biofilm forming capacity of PAO1 and five clinical isolates (*p b 0.05, **p b 0.01 and ***p b 0.001).

Please cite this article as: Kumar L, et al, Zingerone silences quorum sensing and attenuates virulence of Pseudomonas aeruginosa, Fitoterapia (2015), http://dx.doi.org/10.1016/j.fitote.2015.02.002

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Fig. 3. Effect of zingerone on motility phenotypes (swarming, swimming and twitching) of PAO1 and five clinical isolates.

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(6.4 μg/ml) and three strains i.e. L-1 (5.8 μg/ml), L-2 (4.8 μg/ml) and L-4 (5.8 μg/ml) showed very high levels of pyocyanin. Isolates L-3 (3.9 μg/ml) and L-5 (2.9 μg/ml) showed comparatively less amount of pyocyanin in culture supernatant (Fig. 4a). Zingerone was found to be highly effective in suppressing pyocyanin production. Zingerone treated PAO1, L-1, L-2 and L-4 strains showed significantly very low pyocyanin levels (p b 0.01) (Fig. 4a). Comparative amount of cell bound hemolysin was observed in all the strains. PAO1 (0.33 mg/ml), L-1 (0.23 mg/ml) and L-4 (0.30 mg/ml) showed slightly high amount of cell bound hemolysin production (Fig. 4b). Zingerone significantly (p b 0.05) suppressed cell bound hemolysin production and all the strains showed comparatively less amount of hemolysin production. Similarly effect of zingerone on cell free hemolysin was significantly very high (p b 0.01). PAO1 (0.41 mg/ml), L-1 (0.33 mg/ml), and L-4 (0.39 mg/ml), were found to produce maximum hemolysin production and zingerone was able to reduce the levels to (0.16 mg/ml), (0.11 mg/ml) and (0.11 mg/ml) respectively (Fig. 4c). Extracellular enzymes; protease and elastase were also found to be significantly reduced with zingerone treatment. Elastase production was highly decreased as compared to protease in strains treated with zingerone. Protease production in PAO1, L-1 and L-4 was significantly reduced (p b 0.01) below 100 U/l in all the strains (Fig. 4d). PAO1 and all clinical isolates were found to be high producer of elastase. PAO1, L-1 and L-4 showed maximum production. Zingerone treatment showed marked reduction in the elastase activity (Fig. 4f). Rhamnolipid production was also evaluated in the

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Table 1 Effect of zingerone supplementation on swarming, swimming and twitching motility phenotypes (zone of expansion) (*p b 0.05, **p b 0.01 and ***p b 0.001).

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PAO1 L-1 L-2 L-3 L-4 L-5

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± ± ± ± ± ±

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± ± ± ± ± ±

0.2** 0.14** 0.14*** 0.14* 0.07* 0.42**

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PAO1 showed increase in long chain AHL molecule production (732.72 MU) in culture supernatant as compared to the clinical isolates as detected by A. tumefaciens (Fig. 5a). Isolates L-4 (555.54 MU) produced high levels of long chain AHL molecules as compared to other isolates. Zingerone treatment significantly (p b 0.001) reduced the amount of AHL molecules in PAO1 and other clinical isolates (Fig. 5a). PAO1 showed high BHL molecule production (203.6 MU) in culture supernatant as compared to the clinical isolates (Fig. 5b). Two isolates, L-2 and L-4 produced high levels of QS signal molecules as compared to L-1, L-3 and L-5. Zingerone treatment could significantly (p b 0.001) reduce the amount of BHL molecules in PAO1 (105.5 MU) as well as in all the clinical isolates (Fig. 5b). Production of OdDHL showed similar trend and PAO1 was found to produce maximum amount of OdDHL molecules (375.4 MU) (Fig. 7c). All clinical isolates also showed high amount of OdDHL production but L-2 (217.4) and L-4 (311.1) produced relatively very high amount of signal molecules as compared to other strains. Zingerone significantly reduced production of OdDHL molecules in PAO1 (148.1), L-2 (102.5) and L-4 (140.2) respectively (Fig. 5c). Similarly, PQS

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presence of zingerone and significant reduction was observed in all the strains. Zingerone suppressed rhamnolipid production in PAO1, L-1, L-4 from 1.7, 1.5, 1.4 to 0.7, 0.5 and 0.9 respectively (Fig. 4e).

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± ± ± ± ± ±

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± ± ± ± ± ±

0.28*** 0.10** 0.1*** 0.1*** 0.07** 0.1***

± ± ± ± ± ±

0.21 0.14 0.14 0.15 0.14 0.21

± ± ± ± ± ±

0.3*** 0.14** 0.2* 0.1** 0.1*** 0.2***

Please cite this article as: Kumar L, et al, Zingerone silences quorum sensing and attenuates virulence of Pseudomonas aeruginosa, Fitoterapia (2015), http://dx.doi.org/10.1016/j.fitote.2015.02.002

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Three concentrations of zingerone below sub-MIC 431 (100 μg/ml, 500 μg/ml and 1000 μg/ml) showed concentration 432 dependent decrease in the ability of reporter strain to detect 433

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production was significantly high in P. aeruginosa PAO1 and zingerone was able to suppress PQS production significantly (Fig. 5d). PAO1 produced the highest amount of PQS (685.2 MU) but among isolates, L-3 and L-4 produced high amount of PQS. Zingerone treatment could significantly reduce PQS production and levels were found to be below 300 MU (Fig. 5d).

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Fig. 4. Effect of zingerone on virulence factor production by PAO1 and five clinical isolates in vitro (*p b 0.05, **p b 0.01 and ***p b 0.001).

Fig. 5. Effect of zingerone on quorum sensing signal molecules production by PAO1 and five clinical isolates ($p b 0.05, $$p b 0.01 and $$$p b 0.001).

Please cite this article as: Kumar L, et al, Zingerone silences quorum sensing and attenuates virulence of Pseudomonas aeruginosa, Fitoterapia (2015), http://dx.doi.org/10.1016/j.fitote.2015.02.002

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3.8. TraR ligand–receptor interactions

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Subsequently, zingerone was found to interact with active site of TraR and showed dock score of −6.7 as compared to the autoinducer molecule and vanillin which showed dock score of −6.8 and −5.8 respectively. It was observed that phenolic group of zingerone formed H-bond with ASP70 at a distance of 1.97 Å and the methoxyl oxygen acted as H-bond acceptor and

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Fig. 6. Interference of zingerone with activation of quorum sensing reporter strains (A136, MG 4 (λ 1.4), JP-2 and PAO-R1) by respective quorum sensing signal molecules (Long chain AHLs, OdDHL, BHL and PQS) (*p b 0.05, **p b 0.01 and ***p b 0.001).

3.9. LasR ligand–receptor interactions

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Zingerone, vanillin and autoinducers (3-oxo-C8-HSL, 3oxo-C12-HSL, C4-HSL and, PQS) for cognate receptors (TraR, LasR, RhlR and PqsR) were docked using the Glide 5.5. The docking scores of these compounds were compared with the docking scores of respective autoinducer molecules.

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The docking studies showed that zingerone effectively binds in to the active site of LasR with SER 129, TYR93, and TYR56 residues and showed significantly high dock score of − 7.6. Respective autoinducer 3O-C12-HSL and vanillin showed dock scores of − 9.0 and − 6.9 respectively. The phenolic group act as hydrogen bond acceptor with SER 129 and terminal carbonyl group formed H-bond with TYR 93 and acting as hydrogen bond donor (Fig. 8-A, B). TYR56 showed π–π interactions with aromatic group thus strengthening the interactions. The major ligand receptor interactions are represented in Fig. 8. The lactone group of autoinducer formed H-bond with TRP60 at a distance of 1.899 Å that acts as H bond donor. The amide group acted as H bond acceptor and formed H-bond with ASP 73 at a distance of 2.189 Å. The amide carbonyl group formed a bridge of 2H-bonds respectively with TYR56 and SER129 at a distance of 1.80 Å and 1.959 Å (Fig. 8-C, D).

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3.10. RhlR ligand–receptor interactions

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Zingerone showed interaction with the ligand binding site of RhlR and showed dock score of −4.2. Autoinducer C4-HSL and vanillin showed dock scores of −4.8 and −4.2 respectively. The phenolic group of zingerone form H-bonds with GLY73 at a distance of 2.321 Å of ligand binding pocket of RhlR. The aromatic group of zingerone occupied HIS 61, VAL60, ASP81, TRP68 and LEU69 (Fig. 9-A, B). Autoinducer, C-4 HSL interacts with RhlR and occupied by TRP108, LEU107, ALA 111, TRP 96, ASP 81, ALA 44, THR212, VAL 133, ALA83, ILE84, SER135, TYR64, TYR 68, and TYR 72. TYR 68 and ASP 81 formed H-bond with autoinducer molecule at a distance of 1.92 Å and 2.06 Å respectively (Fig. 9-C, D). The major ligand receptor interactions are represented in Fig. 9.

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Zingerone was found to interact with the ligand binding pocket of PqsR receptor and showed dock score of −7.1. Autoinducer (PQS) and vanillin showed dock scores of −8.0 and −6.9, respectively. Phenolic group of zingerone formed Hbonds with LEU208 and GLN194 at a distance of 2.1 Å to 2 Å and 2.061 Å respectively. The aromatic group occupied MET 224 ILE236 and LEU207, VAL211, PRO210 ARG209 and SER196 (Fig. 10-A, B). The quinolone ring of PQS molecule was occupied by PRO238, PHE221, ALA237, MET224, LEU197, LEU208 and ILE236 residues. ARG 209 was found to have some minor ionic interactions with amine (Fig. 10-C, D). The major ligand receptor interactions are represented in Fig. 10.

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3.7. In silico analysis of ligand–receptor interaction of zingerone using molecular docking studies

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formed H-bond with TRP57 at a distance of 1.952 Å. The aromatic group of zingerone showed π–π interactions with TRY53 and TRY61 and sandwiched between the two amino acids thus showed highly significant interactions with the TraR receptor (Fig. 7-A, B). The lactone group of autoinducer acted as H-bond acceptor and formed H-bond with TRP57 at a distance of 1.918 Å and the amide group was found to form Hbond with ASP70 at a distance of 1.889 Å that acts as H-bond donor (Fig. 7-C, D). The major ligand receptor interactions are represented in Fig. 7.

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the quorum sensing signal molecule. A. tumefaciens A136 (long chain AHL detector) detected 739.97 MU in P. aeruginosa (without zingerone). Zingerone could slightly decrease the activation of reporter strain at 100 μg/ml since 579.97 MU was detected. Highly significant decrease was found at 500 μg/ml (179.98 MU) and 1000 μg/ml (135.39 MU) (Fig. 6). E. coli MG4 (λ 1.4) (OdDHL detector) could detect 375.4 MU in the culture supernatant of P. aeruginosa without zingerone (Fig. 6). Zingerone at the concentration of 100 μg/ml interfered with detection activity (352.8 MU) (p N 0.05) but 500 μg (230.8) and 1000 μg (88.39) significantly (p b 0.001) reduced the detection activity of MG4 reporter strain. Similarly with JP-2 strain, BHL molecules in culture supernatant were found to be 203.6 MU which slightly reduced in the presence of 100 μg of zingerone (192.5 MU) (Fig. 6). Addition of 500 μg and 1000 μg of zingerone caused highly significant reduction in JP-2 activity resulting in decreased AHL detection of 116.6 MU and 60.1 MU respectively. PAO-R1 also showed similar trend in detection of PQS and levels were found to be reduced in the presence of zingerone. Addition of 100 μg of zingerone showed non significant (p N 0.05) reduction (601.2 MU) as compared to control (658.2) but highly significant reduced levels were observed with 500 μg and 1000 μg (420.9 and 104.8 respectively) (Fig. 6).

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Please cite this article as: Kumar L, et al, Zingerone silences quorum sensing and attenuates virulence of Pseudomonas aeruginosa, Fitoterapia (2015), http://dx.doi.org/10.1016/j.fitote.2015.02.002

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Fig. 7. Representation of ligand receptor interactions of zingerone (A, B) and autoinducer (C, D) with TraR receptor active site.

4. Discussion

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Antibiotics target a physiologically essential biochemical pathway that leads to the death of the organism; hence it poses a selective pressure on microorganism to develop drug resistance mechanism for its survival. Despite recent advancement in antimicrobial chemotherapy, antibiotic resistance in P. aeruginosa remains a challenging problem worldwide [38]. Biofilm formation and development of multidrug resistance are mainly responsible for ineffectiveness of antibiotic therapy against Pseudomonas infections [39]. Hence, there is an urgent need to find out new strategies and new drugs targeting virulence of pathogen without causing bactericidal effect that may enhance antibiotic efficacy. Virulence of P. aeruginosa is linked to quorum sensing. Quorum sensing is known to regulate motility phenotypes, virulence factors and biofilm formation [40]. Virulence attributes like swimming, swarming and twitching motilities are key regulators of biofilm formation [41]. Recently it was also

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demonstrated that Pqs quorum sensing system regulates swarming motility [42]. Strains isolated from cystic fibrosis patients showed high swimming and swarming motility suggesting their role in disease progression [43]. In our study, it was found that zingerone supplementation significantly reduced all three motility phenotypes and biofilm forming capacity of all the strains. Reduction in motility may be linked to ineffective migration of bacterial cells to newer areas and may also lead to impaired binding to the surfaces hence inhibiting or delaying biofilm formation. Quorum sensing controls expression of arsenals of virulence determinants in P. aeruginosa [44] (pyocyanin, hemolysin, protease, elastase and rhamnolipid). Zingerone was found to suppress pyocyanin and hemolysin production that contribute towards decreased virulence of P. aeruginosa. Rhamnolipid is a bio surfactant that protects pathogen against attack of immune cells and help in nutrient availability within biofilms by decreasing liquid surface tension [45]. Inhibition in rhamnolipid production may decrease the biofilm formation. Extracellular proteases

Please cite this article as: Kumar L, et al, Zingerone silences quorum sensing and attenuates virulence of Pseudomonas aeruginosa, Fitoterapia (2015), http://dx.doi.org/10.1016/j.fitote.2015.02.002

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LasA elastase, LasB elastase and alkaline protease play an important role during P. aeruginosa virulence. Marked suppression of all the virulence factors by zingerone indicated that it is an effective anti-virulent candidate for P. aeruginosa. Since most of the virulence factors are regulated by quorum sensing signal molecules, hence, drugs that inhibit signal molecule production or bind to AHL receptor may block the signal pathways that may further suppress quorum sensing dependent expression of genes. Therefore, the effect of zingerone was evaluated on AHL production as well as on the interference in the signal detection. Zingerone significantly decreased production of AHL molecules which indicated that zingerone modulate quorum sensing signaling pathway leading to inhibition of quorum sensing molecules. Anti-QS activity of phytochemicals could be attributed to various mechanisms that may include inhibition of biosynthesis of quorum sensing signal or deactivation of signaling by binding of phytochemical to the cognate receptor of AHL molecule [25]. Further, to

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Fig. 8. Representation of ligand receptor interactions of zingerone (A, B) and autoinducer (C, D) with LasR receptor active site.

evaluate interference of zingerone against AHL detection, four reporter strains A. tumefaciens A136, E. coli MG4 (λ1.4), P. aeruginosa PAO-JP2 and P. aeruginosa PAO-R1 were used. Zingerone significantly inhibited the activation of all the reporter strains when supplemented with PAO1 extract containing AHLs. It indicated that zingerone may bind with the quorum sensing receptor and inhibit the binding of signal molecule with the receptor present on reporter strains thereby, blocking downstream signaling pathway. A. tumefaciens A136 detects C-6 to C-12 acyl chain AHL molecules and is a highly sensitive strain that responds to even very low concentration of AHL molecules. In vitro results showed deactivation of A136 reporter strain with zingerone supplementation. Since, A136 contains TraR–Lac Z fusion; results indicated that zingerone might have binding potential to TraR receptor. Similarly, MG4 (λ 1.4), JP-2 and PAO-R1 specifically detects OdDHL, BHL and PQS molecules respectively. Zingerone interfered with the activation of specific response of reporter strains indicating that

Please cite this article as: Kumar L, et al, Zingerone silences quorum sensing and attenuates virulence of Pseudomonas aeruginosa, Fitoterapia (2015), http://dx.doi.org/10.1016/j.fitote.2015.02.002

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zingerone might also inhibit las, rhl and pqs systems by binding to their respective cognate receptors (LasR, RhlR and PqsR). To prove this hypothesis molecular docking analysis of zingerone against quorum sensing receptors (TraR, LasR RhlR and PqsR) was performed. Interestingly, from docking studies we observed binding of zingerone to the active site of TraR receptor. Zingerone formed H-bond with aspartate and tryptophan residues and molecule was sandwiched with two tyrosine amino acids of TraR receptor binding pocket. This complex also showed significantly high dock score. In case of LasR–zingerone complex, zingerone interacted with tyrosine and serine amino acid residues of LasR receptor binding pocket. Phenolic group of zingerone formed H-bond and π–π interactions that stabilized the zingerone–LasR complex hence giving high dock score of zingerone which was comparable to that of autoinducer molecule. Dock score of zingerone was much higher than vanillin which is a known anti-QS molecule. Zingerone formed

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Fig. 9. Representation of ligand receptor interactions of zingerone (A, B) and autoinducer (C, D) with RhlR receptor active site.

H-bond with glycine residue and showed high ligand receptor dock score with RhlR receptor. Similarly in PqsR docking studies zingerone showed high binding affinity with the receptor. Effect of zingerone on Pqs signaling was also significantly high. The aromatic ring contributed specifically for H-bonding with leucine and glycine residues of receptor and showed high ligand receptor interactions. Molecular docking analysis confirmed that zingerone has potent ability to bind with all the quorum sensing receptors. Binding of zingerone at the place of autoinducer may specifically block receptor–ligand interaction leading to no further activation of QS dependent gene expression for regulation of virulence factor and biofilm formation. In vitro and in silico analysis provide valid proof that zingerone bind to quorum sensing signal receptor proteins that block the signal to bind with the receptor moiety. This lead to inhibition of the signaling mechanism and therefore pathogen is not able to sense the bacterial population. Marked suppression in virulence factor

Please cite this article as: Kumar L, et al, Zingerone silences quorum sensing and attenuates virulence of Pseudomonas aeruginosa, Fitoterapia (2015), http://dx.doi.org/10.1016/j.fitote.2015.02.002

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production may be attributed to inhibition of las, rhl and pqs signaling simultaneously. This study provides valid evidence that zingerone is anti-virulent drug candidate that attenuate virulence by interfering quorum sensing signaling pathways. Zingerone is non toxic, safe and potent anti-inflammatory flavor used in food industry to provide aroma of ginger in food product. Known anti-inflammatory activity of zingerone is due to free radical scavenging effect of zingerone [46]. Methoxy group with phenolic hydroxyl group may facilitate proton release and long chain ethyl methyl ketone group help in bulk stabilization of zingerone [47] that may also enhance cell penetration and free radical neutralization effect. Since free radical induced tissue damage plays a major role in infection hence anti-inflammatory effect along with anti-virulent effect of zingerone may play a critical role in management of P. aeruginosa infection. Zingerone was found to be a potent inhibitor of ligand–receptor interaction of quorum sensing pathways, which silences cell communication network and ultimately suppresses virulence of P. aeruginosa. Besides having anti-inflammatory and antioxidant properties, the anti-virulent property of zingerone may give advantage in microbial infections where inflammation play key role in the pathogenesis of disease. Zingerone treatment suppressed production of virulence factors that help the pathogen to establish and cause

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Fig. 10. Representation of ligand receptor interactions of zingerone (A, B) and autoinducer (C, D) with PqsR receptor active site.

tissue damage. This study besides providing an insight that zingerone has the potential to be used as drug candidate against P. aeruginosa infections, also provides new approach to validate anti-virulence activity and opens a vast area to employ zingerone as anti-virulent drug candidate to suppress P. aeruginosa mediated infections. The insights obtained from molecular modeling docking studies can be used for further design of more potent anti-virulent derivatives of zingerone. Zingerone can play a critical role in the management of P. aeruginosa infections. In clinical setting catheter coating with zingerone might help reducing incidence of biofilm associated chronic infections. Novel derivatives of zingerone can also be synthesized which could be employed as antivirulence agents.

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The authors declare that they have no conflict of interest. Acknowledgment

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We acknowledge INSPIRE program of Department of 677 Science and Technology (DST) Govt of India. 678

Please cite this article as: Kumar L, et al, Zingerone silences quorum sensing and attenuates virulence of Pseudomonas aeruginosa, Fitoterapia (2015), http://dx.doi.org/10.1016/j.fitote.2015.02.002

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