Phytomedicine 21 (2014) 286–289
Contents lists available at ScienceDirect
Phytomedicine journal homepage: www.elsevier.de/phymed
Short Communication
Resveratrol – A potential inhibitor of biofilm formation in Vibrio cholerae Nimmy Augustine a , A.K. Goel b , K.C. Sivakumar c , R. Ajay Kumar d , Sabu Thomas a,∗ a
Cholera and Environmental Microbiology Laboratory, Rajiv Gandhi Centre for Biotechnology, Thiruvananthapuram 695 014, Kerala, India Defence Research & Development Establishment, Gwalior 474 002, Madhya Pradesh, India Distributed Information Sub-Centre, Rajiv Gandhi Centre for Biotechnology, Thiruvananthapuram 695 014, Kerala, India d Mycobacterium Research Group, Rajiv Gandhi Centre for Biotechnology, Thiruvananthapuram 695 014, Kerala, India b c
a r t i c l e
i n f o
Article history: Received 30 May 2013 Received in revised form 8 August 2013 Accepted 19 September 2013 Keywords: Resveratrol V. cholerae Biofilm inhibition AphB protein Molecular docking
a b s t r a c t Resveratrol, a phytochemical commonly found in the skin of grapes and berries, was tested for its biofilm inhibitory activity against Vibrio cholerae. Biofilm inhibition was assessed using crystal violet assay. MTT assay was performed to check the viability of the treated bacterial cells and the biofilm architecture was analysed using confocal laser scanning microscopy. The possible target of the compound was determined by docking analysis. Results showed that subinhibitory concentrations of the compound could significantly inhibit biofilm formation in V. cholerae in a concentration-dependent manner. AphB was found to be the putative target of resveratrol using docking analysis. The results generated in this study proved that resveratrol is a potent biofilm inhibitor of V. cholerae and can be used as a novel therapeutic agent against cholera. To our knowledge, this is the first report of resveratrol showing antibiofilm activity against V. cholerae. © 2013 Elsevier GmbH. All rights reserved.
Introduction Cholera, characterized by watery diarrhoea, still continues to be a life-threatening disease in resource-poor countries (WHO 2012). Role of biofilm formation in Vibrio cholerae pathogenesis is well established as it provides the bacterium with enhanced tolerance to antimicrobial agents and transforms it into a hyperinfectious form (Kierek and Watnick 2003; Tamayo et al. 2010; Yang et al. 2010; Sambanthamoorthy et al. 2012). The escalation of antimicrobial resistance among bacterial pathogens worldwide is becoming a critical concern and there is an urgent need to look for alternative strategies to combat microbial infections by reducing virulence rather than by killing the bacteria. Resveratrol (3,5,4 -trihydroxy-trans-stilbene) is a natural polyphenol and a phytoalexin (Fig. 1). It is mainly found in the skin of grapes, berries, etc. and also is a main component of red wine. There are several reports showing its medicinal properties such as antiinflammatory, antiviral, antioxidant, antimicrobial, neuroprotective and a potent chemopreventive against cancer (Baur and Sinclair 2006). Very limited work has been done to exhibit its antipathogenic activity. Wang et al. (2006) have reported the inhibition of swarming and expression of virulence factors in Proteus mirabilis and Thimothe et al. (2007) showed reduction in
∗ Corresponding author. Tel.: +91 471 2529521; fax: +91 471 2346333. E-mail address: [email protected] (S. Thomas). 0944-7113/$ – see front matter © 2013 Elsevier GmbH. All rights reserved. http://dx.doi.org/10.1016/j.phymed.2013.09.010
virulence properties of Streptococcus mutans on treatment with resveratrol. Morinaga et al. (2010) demonstrated the inhibition of cholera toxin induced cAMP accumulation in Vero cells on treatment with resveratrol and Coenye et al. 2012 reported the biofilm inhibitory activity of the same against Propionibacterium acnes. So far no literature is available demonstrating its antibiofilm activity against V. cholerae. In this study, we have investigated the biofilm inhibitory activity of resveratrol against V. cholerae and have tried to explore its possible target through computational analysis. Materials and methods Bacteria and culture conditions V. cholerae O1 (MCVO9), a clinical strain isolated during a cholera outbreak in Kerala (2009) was used as the test strain in this study. This strain showed resistance to all major antibiotics used for the treatment of cholera (Kumar et al. 2009). The strain was grown in Luria Bertani (LB) medium at 30 ◦ C. Minimum inhibitory concentration (MIC) The MIC of the resveratrol was determined using a microdilution process according to the guidelines of Clinical and Laboratory Standards Institute (CLSI 2010) and all the further analyses were done at sub-MIC levels.
N. Augustine et al. / Phytomedicine 21 (2014) 286–289
287
DNA binding residues was done using BindN software (Wang and Brown 2006). Results and discussion
Fig. 1. Chemical structure of resveratrol.
Biofilm assay The biofilm assay was performed by standardizing the overnight culture to an OD600 of 0.05 and 200 l of the diluted culture was added into the 96 well polystyrene microtiter plate. The biofilm was grown under static conditions at 30 ◦ C for 24 h. To evaluate the biofilm inhibitory activity of resveratrol, the culture was grown in the presence of the compound at different concentrations (10, 15, 20, 25, 30 g/ml). Resveratrol was obtained from Calbiochem and a stock solution of 50 mg/ml was prepared in dimethyl sulfoxide (DMSO). The biofilm formation was quantified using crystal violet assay (O’Toole 2011). Visualization of biofilms
In the present investigation MIC of the resveratrol was found to be 60 g/ml for V. cholerae and the sub-inhibitory concentrations could inhibit its biofilm formation significantly. Biofilm inhibition activity was found to be in a concentration-dependent manner – ∼85% inhibition was observed at 30 g/ml, ∼79% at 25 g/ml, ∼75% at 20 g/ml and ∼64% at 15 g/ml while at 10 g/ml the inhibition was not significant (Fig. 2A). The inhibitory activity was again confirmed with cover slip assay which showed the reduction in adhesion of the cells onto the cover slip surface. Measurement of biofilm thickness and z-stack imaging revealed a significant difference in biofilm thickness of resveratrol treated and untreated cultures. The untreated culture could produce ∼70 m thick mature biofilms, whereas the culture treated with 15 g/ml and 20 g/ml of resveratrol could produce only 18 m and 15 m thick biofilms respectively after 24 h incubation (Fig. 2B). MTT assay was performed to prove that activity was solely due to biofilm inhibition and not due to growth inhibition. The results showed that sub-MIC of the compound did not change the viability of the cells till 30 g/ml and as a result bacterial growth was not inhibited (Fig. 3). Results gathered in this study proved that resveratrol is a potent biofilm inhibitor of V. cholerae. We investigated the binding affinity of resveratrol for its potential inhibitory action on various receptors using AutoDock 4.2.
Confocal laser scanning microscopic analysis Biofilms were grown on borosilicate cover slips in 50 ml Falcon tubes as described earlier. The biofilms were stained with LIVE/DEAD® BacLightTM Bacterial Viability Kit (Invitrogen) according to manufacturer’s instructions. The slides were observed under 40× objective using a Nikon Eclipse Ti Confocal laser scanning inverted microscope (Nikon, Melville, NY). The excitation/emission wavelengths for Syto9 and Propidium Iodide (PI) were 488/525 and 595/561 nm. The thickness of biofilm was measured using NISElements AR software, version 4.00.04. MTT assay The MTT assay was performed as previously described to assess the viability of the cells (Mshana et al. 1998). MTT (3(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) was obtained from Sigma and a solution of 5 mg/ml was prepared in phosphate buffered saline (pH 7.2). Molecular docking analysis Docking studies were performed to identify the possible target of resveratrol at the molecular level. The involvement of many proteins in biofilm formation in V. cholerae has been reported previously (Yang et al. 2010). Of them, AphA (PDB ID: 1YG2), AphB (PDB ID: 3SZP), LuxO (Uniprot ID: Q9KT84), TcpA (PDB ID: 3HRV), VpsT (PDB ID: 3KLN) and VpsR (Uniprot ID: Q9AQ41) were selected for our docking studies. The 3D structure of all proteins except LuxO and VpsR were obtained from RCSB protein data bank. The structure of LuxO and VpsR was modelled using SWISS-MODEL. The 3D structure of resveratrol was obtained from Pubchem database (http://pubchem.ncbi.nlm.nih.gov). All the proteins analysed are transcriptional regulators except for TcpA which is a pilus protein. Individual PDB files were prepared for docking using the prepare ligand4.py script from MGLTools 1.5.4. Charges and nonpolar hydrogen atoms were added using the prepare receptor4.py script. AutoDock 4.2 was used for all docking studies. Prediction of
Fig. 2. (A) Graph showing percentage inhibition of biofilm at different concentrations of resveratrol. (B) CLSM images of V. cholerae biofilm formed on coverslips after 24 h incubation. (a) Biofilm formed by untreated culture. (b and c) Biofilm formation by culture treated with 15 and 20 g/ml of resveratrol. (d) Three dimensional view of biofilm formed by untreated culture. (e and f) Three dimensional view of biofilm formed by cultures treated with 15 and 20 g/ml. Image construction and measurement of biofilm thickness were performed using NIS-elements AR software.
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Fig. 3. Graph showing quantitative measurement of bacterial viability of untreated and treated cultures.
Table 1 Summary of the binding affinity of resveratrol with different proteins. Protein
Binding affinity (kcal/mol)
AphA TcpA VpsT AphB LuxO VpsR
−6.1 −5.9 −6.5 −8.0 −6.7 −6.7
AutoDock provides the overall docking energy of a given ligand molecule expressed as the sum of intermolecular interaction energies including van der Waals attraction and repulsive energies, coulombic electrostatic energy and the internal steric energy of the ligand (Wang et al. 2003). The molecular docking study revealed that resveratrol exhibited fairly good binding score with different receptor targets. Table 1 shows the binding free energy (binding affinity) obtained for the various receptors considered in this study. Among them AphB is observed to have remarkable lowest binding free energy of −8.0 kcal/mol and forms two hydrogen bonds with Thr31 and Val144 (Fig. 4). Taylor et al. (2012) have reported that three polar residues on ␣3 helix of AphB (Thr31 , Thr33 , Ser42 ) are involved in the formation of hydrogen bonds with DNA. In V. cholerae, expression of genes responsible for virulence factors and toxin-coregulated pilus is initiated by tcpPH promoter on Vibrio pathogenicity island (VPI), which in turn is regulated by transcriptional factors AphA, a winged helix DNA binding protein and AphB, a LysR-type regulator. AphA facilitates the binding of AphB to the promoter. AphB has a conserved structure with a N-terminal DNA binding domain and a C-terminal regulatory domain (Kovacikova
et al. 2010; Liu et al. 2010). From the results generated in this analysis, it was predicted that resveratrol could possibly interact with T31 (Thr31 ) and showed AphB as the putative target of resveratrol in V. cholerae. In this study, we have demonstrated for the first time the antibiofilm activity of resveratrol against V. cholerae, the causative agent of cholera. As this compound acts against biofilm formation and virulence factors rather than changing the viability of the bacteria, the selective pressure for developing resistance would be less. It is a well-characterized natural compound. In this context, we propose that resveratrol is a potential molecule for the development of a novel drug which may be used alone or in combination with other antibiotics to tackle the disease, cholera. Further studies including gene expression and in vivo models are needed to explore the molecular mechanism of action in detail. Conflict of interest There is no conflict of interest among authors. Acknowledgements This study was supported by a research grant from Defence Research Development Organisation (DRDO), Ministry of Defence, Govt. of India, New Delhi. The authors are thankful to Prof. M. Radhakrishna Pillai, Director, Rajiv Gandhi Centre for Biotechnology (RGCB), for the facilities provided. Authors are thankful to Mr. Anurup K.G., RGCB, for providing with technical help in carrying out CLSM analysis. References
Fig. 4. Docking conformation of resveratrol with AphB. Residues interacting with ligand are represented with lines.
Baur, J.A., Sinclair, D.A., 2006. Therapeutic potential of resveratrol: the in vivo evidence. Nature Reviews Drug Discovery 5, 493–506. CLSI, 2010. Performance standards for antimicrobial susceptibility testing; Twentieth Informational Supplement: CLSI document M100-S20. Clinical and Laboratory Standard Institute, 940 West Valley Road, Suite 1400, Wayne Pennsylvania 19087-1898, USA. Coenye, T., Brackman, G., Rigole, P., De Witte, E., Honraet, K., Rossel, B., Nelis, H.J., 2012. Eradication of Propionibacterium acnes biofilms by plant extracts and putative identification of icariin, resveratrol and salidroside as active compounds. Phytomedicine 19, 409–412. Kierek, K., Watnick, P.I., 2003. Environmental determinants of Vibrio cholerae biofilm development. Applied and Environmental Microbiology 69, 5079–5088. Kovacikova, G., Lin, W., Skorupski, K., 2010. The LysR-type virulence activator AphB regulates the expression of genes in Vibrio cholerae in response to low pH and anaerobiosis. Journal of Bacteriology 192, 4181–4191. Kumar, P., Wilson, P.A., Bhai, R., Thomas, S., 2009. Characterization of an SXT variant Vibrio cholerae O1 Ogawa isolated from a patient in Trivandrum, India. FEMS Microbiology Letters 303, 132–136. Liu, Z., Yang, M., Peterfreund, G.L., Tsou, A.M., Selamoglu, N., Daldal, F., Zhong, Z., Kan, B., Zhu, J., 2010. Vibrio cholerae anaerobic induction of virulence gene expression
N. Augustine et al. / Phytomedicine 21 (2014) 286–289 is controlled by thiol-based switches of virulence regulator AphB. Proceedings of the National Academy of Sciences 108, 810–815. Morinaga, N., Yahiro, K., Noda, M., 2010. Resveratrol, a natural polyphenolic compound, inhibits cholera toxin-induced cyclic AMP accumulation in vero cells. Toxicon 56, 29–35. Mshana, R.N., Tadesse, G., Abate, G., MiÖrner, H.K., 1998. Use of 3-(4,5dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide for rapid detection of rifampin-resistant Mycobacterium tuberculosis. Journal of Clinical Microbiology 36, 1214–1219. O’Toole, G.A., 2011. Microtiter dish biofilm formation assay. Journal of Visualized Experiments: JoVE 47, 2437. Sambanthamoorthy, K., Sloup, R.E., Parashar, V., Smith, J.M., Kim, E.E., Semmelhack, M.F., Neiditch, M.B., Waters, C.M., 2012. Identification of small molecules that antagonize diguanylatecyclase enzymes to inhibit biofilm formation. Antimicrobial Agents and Chemotherapy 56, 5202–5211. Tamayo, R., Patimalla, B., Camilli, A., 2010. Growth in a biofilm induces a hyperinfectious phenotype in Vibrio cholerae. Infection and Immunity 78, 3560–3569. Taylor, J.L., De Silva, R.S., Kovacikova, G., Lin, W., Taylor, R.K., Skorupski, K., Kull, F.J., 2012. The crystal structure of AphB, a virulence gene activator from Vibrio
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cholerae, reveals residues that influence its response to oxygen and pH. Molecular Microbiology 83, 457–470. Thimothe, J., Bonsi, I.A., Padilla-Zakour, O.I., Koo, H., 2007. Chemical characterization of red wine grape (Vitis vinifera and Vitis interspecific hybrids) and pomace phenolic extracts and their biological activity against Streptococcus mutans. Journal of Agricultural and Food Chemistry 55, 10200–10207. Wang, W.B., Lai, H.C., Hsueh, P.R., Chiou, R.Y.Y., Lin, S.B., Liaw, S.J., 2006. Inhibition of swarming and virulence factor expression in Proteus mirabilis by resveratrol. Journal of Medical Microbiology 55, 1313–1321. Wang, L., Brown, S.J., 2006. BindN: a web-based tool for efficient prediction of DNA and RNA binding sites in amino acid sequences. Nucleic Acids Research 34, W243–W248. Wang, R., Lu, Y., Wang, S., 2003. Comparative evaluation of 11 scoring functions for molecular docking. Journal of Medicinal Chemistry 46, 2287. World Health Organisation, 2012. Cholera 2011. Weekly Epidemiological Record 87, 289–304 http://www.who.int/wer/2012/wer8731 32.pdf Yang, M., Frey, E.M., Liu, Z., Bishar, R., Zhu, J., 2010. The virulence transcriptional activator AphA enhances biofilm formation by Vibrio cholerae by activating expression of the biofilm regulator VpsT. Infection and Immunity 78, 697–703.
Contents lists available at ScienceDirect
Phytomedicine journal homepage: www.elsevier.de/phymed
Short Communication
Resveratrol – A potential inhibitor of biofilm formation in Vibrio cholerae Nimmy Augustine a , A.K. Goel b , K.C. Sivakumar c , R. Ajay Kumar d , Sabu Thomas a,∗ a
Cholera and Environmental Microbiology Laboratory, Rajiv Gandhi Centre for Biotechnology, Thiruvananthapuram 695 014, Kerala, India Defence Research & Development Establishment, Gwalior 474 002, Madhya Pradesh, India Distributed Information Sub-Centre, Rajiv Gandhi Centre for Biotechnology, Thiruvananthapuram 695 014, Kerala, India d Mycobacterium Research Group, Rajiv Gandhi Centre for Biotechnology, Thiruvananthapuram 695 014, Kerala, India b c
a r t i c l e
i n f o
Article history: Received 30 May 2013 Received in revised form 8 August 2013 Accepted 19 September 2013 Keywords: Resveratrol V. cholerae Biofilm inhibition AphB protein Molecular docking
a b s t r a c t Resveratrol, a phytochemical commonly found in the skin of grapes and berries, was tested for its biofilm inhibitory activity against Vibrio cholerae. Biofilm inhibition was assessed using crystal violet assay. MTT assay was performed to check the viability of the treated bacterial cells and the biofilm architecture was analysed using confocal laser scanning microscopy. The possible target of the compound was determined by docking analysis. Results showed that subinhibitory concentrations of the compound could significantly inhibit biofilm formation in V. cholerae in a concentration-dependent manner. AphB was found to be the putative target of resveratrol using docking analysis. The results generated in this study proved that resveratrol is a potent biofilm inhibitor of V. cholerae and can be used as a novel therapeutic agent against cholera. To our knowledge, this is the first report of resveratrol showing antibiofilm activity against V. cholerae. © 2013 Elsevier GmbH. All rights reserved.
Introduction Cholera, characterized by watery diarrhoea, still continues to be a life-threatening disease in resource-poor countries (WHO 2012). Role of biofilm formation in Vibrio cholerae pathogenesis is well established as it provides the bacterium with enhanced tolerance to antimicrobial agents and transforms it into a hyperinfectious form (Kierek and Watnick 2003; Tamayo et al. 2010; Yang et al. 2010; Sambanthamoorthy et al. 2012). The escalation of antimicrobial resistance among bacterial pathogens worldwide is becoming a critical concern and there is an urgent need to look for alternative strategies to combat microbial infections by reducing virulence rather than by killing the bacteria. Resveratrol (3,5,4 -trihydroxy-trans-stilbene) is a natural polyphenol and a phytoalexin (Fig. 1). It is mainly found in the skin of grapes, berries, etc. and also is a main component of red wine. There are several reports showing its medicinal properties such as antiinflammatory, antiviral, antioxidant, antimicrobial, neuroprotective and a potent chemopreventive against cancer (Baur and Sinclair 2006). Very limited work has been done to exhibit its antipathogenic activity. Wang et al. (2006) have reported the inhibition of swarming and expression of virulence factors in Proteus mirabilis and Thimothe et al. (2007) showed reduction in
∗ Corresponding author. Tel.: +91 471 2529521; fax: +91 471 2346333. E-mail address: [email protected] (S. Thomas). 0944-7113/$ – see front matter © 2013 Elsevier GmbH. All rights reserved. http://dx.doi.org/10.1016/j.phymed.2013.09.010
virulence properties of Streptococcus mutans on treatment with resveratrol. Morinaga et al. (2010) demonstrated the inhibition of cholera toxin induced cAMP accumulation in Vero cells on treatment with resveratrol and Coenye et al. 2012 reported the biofilm inhibitory activity of the same against Propionibacterium acnes. So far no literature is available demonstrating its antibiofilm activity against V. cholerae. In this study, we have investigated the biofilm inhibitory activity of resveratrol against V. cholerae and have tried to explore its possible target through computational analysis. Materials and methods Bacteria and culture conditions V. cholerae O1 (MCVO9), a clinical strain isolated during a cholera outbreak in Kerala (2009) was used as the test strain in this study. This strain showed resistance to all major antibiotics used for the treatment of cholera (Kumar et al. 2009). The strain was grown in Luria Bertani (LB) medium at 30 ◦ C. Minimum inhibitory concentration (MIC) The MIC of the resveratrol was determined using a microdilution process according to the guidelines of Clinical and Laboratory Standards Institute (CLSI 2010) and all the further analyses were done at sub-MIC levels.
N. Augustine et al. / Phytomedicine 21 (2014) 286–289
287
DNA binding residues was done using BindN software (Wang and Brown 2006). Results and discussion
Fig. 1. Chemical structure of resveratrol.
Biofilm assay The biofilm assay was performed by standardizing the overnight culture to an OD600 of 0.05 and 200 l of the diluted culture was added into the 96 well polystyrene microtiter plate. The biofilm was grown under static conditions at 30 ◦ C for 24 h. To evaluate the biofilm inhibitory activity of resveratrol, the culture was grown in the presence of the compound at different concentrations (10, 15, 20, 25, 30 g/ml). Resveratrol was obtained from Calbiochem and a stock solution of 50 mg/ml was prepared in dimethyl sulfoxide (DMSO). The biofilm formation was quantified using crystal violet assay (O’Toole 2011). Visualization of biofilms
In the present investigation MIC of the resveratrol was found to be 60 g/ml for V. cholerae and the sub-inhibitory concentrations could inhibit its biofilm formation significantly. Biofilm inhibition activity was found to be in a concentration-dependent manner – ∼85% inhibition was observed at 30 g/ml, ∼79% at 25 g/ml, ∼75% at 20 g/ml and ∼64% at 15 g/ml while at 10 g/ml the inhibition was not significant (Fig. 2A). The inhibitory activity was again confirmed with cover slip assay which showed the reduction in adhesion of the cells onto the cover slip surface. Measurement of biofilm thickness and z-stack imaging revealed a significant difference in biofilm thickness of resveratrol treated and untreated cultures. The untreated culture could produce ∼70 m thick mature biofilms, whereas the culture treated with 15 g/ml and 20 g/ml of resveratrol could produce only 18 m and 15 m thick biofilms respectively after 24 h incubation (Fig. 2B). MTT assay was performed to prove that activity was solely due to biofilm inhibition and not due to growth inhibition. The results showed that sub-MIC of the compound did not change the viability of the cells till 30 g/ml and as a result bacterial growth was not inhibited (Fig. 3). Results gathered in this study proved that resveratrol is a potent biofilm inhibitor of V. cholerae. We investigated the binding affinity of resveratrol for its potential inhibitory action on various receptors using AutoDock 4.2.
Confocal laser scanning microscopic analysis Biofilms were grown on borosilicate cover slips in 50 ml Falcon tubes as described earlier. The biofilms were stained with LIVE/DEAD® BacLightTM Bacterial Viability Kit (Invitrogen) according to manufacturer’s instructions. The slides were observed under 40× objective using a Nikon Eclipse Ti Confocal laser scanning inverted microscope (Nikon, Melville, NY). The excitation/emission wavelengths for Syto9 and Propidium Iodide (PI) were 488/525 and 595/561 nm. The thickness of biofilm was measured using NISElements AR software, version 4.00.04. MTT assay The MTT assay was performed as previously described to assess the viability of the cells (Mshana et al. 1998). MTT (3(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) was obtained from Sigma and a solution of 5 mg/ml was prepared in phosphate buffered saline (pH 7.2). Molecular docking analysis Docking studies were performed to identify the possible target of resveratrol at the molecular level. The involvement of many proteins in biofilm formation in V. cholerae has been reported previously (Yang et al. 2010). Of them, AphA (PDB ID: 1YG2), AphB (PDB ID: 3SZP), LuxO (Uniprot ID: Q9KT84), TcpA (PDB ID: 3HRV), VpsT (PDB ID: 3KLN) and VpsR (Uniprot ID: Q9AQ41) were selected for our docking studies. The 3D structure of all proteins except LuxO and VpsR were obtained from RCSB protein data bank. The structure of LuxO and VpsR was modelled using SWISS-MODEL. The 3D structure of resveratrol was obtained from Pubchem database (http://pubchem.ncbi.nlm.nih.gov). All the proteins analysed are transcriptional regulators except for TcpA which is a pilus protein. Individual PDB files were prepared for docking using the prepare ligand4.py script from MGLTools 1.5.4. Charges and nonpolar hydrogen atoms were added using the prepare receptor4.py script. AutoDock 4.2 was used for all docking studies. Prediction of
Fig. 2. (A) Graph showing percentage inhibition of biofilm at different concentrations of resveratrol. (B) CLSM images of V. cholerae biofilm formed on coverslips after 24 h incubation. (a) Biofilm formed by untreated culture. (b and c) Biofilm formation by culture treated with 15 and 20 g/ml of resveratrol. (d) Three dimensional view of biofilm formed by untreated culture. (e and f) Three dimensional view of biofilm formed by cultures treated with 15 and 20 g/ml. Image construction and measurement of biofilm thickness were performed using NIS-elements AR software.
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Fig. 3. Graph showing quantitative measurement of bacterial viability of untreated and treated cultures.
Table 1 Summary of the binding affinity of resveratrol with different proteins. Protein
Binding affinity (kcal/mol)
AphA TcpA VpsT AphB LuxO VpsR
−6.1 −5.9 −6.5 −8.0 −6.7 −6.7
AutoDock provides the overall docking energy of a given ligand molecule expressed as the sum of intermolecular interaction energies including van der Waals attraction and repulsive energies, coulombic electrostatic energy and the internal steric energy of the ligand (Wang et al. 2003). The molecular docking study revealed that resveratrol exhibited fairly good binding score with different receptor targets. Table 1 shows the binding free energy (binding affinity) obtained for the various receptors considered in this study. Among them AphB is observed to have remarkable lowest binding free energy of −8.0 kcal/mol and forms two hydrogen bonds with Thr31 and Val144 (Fig. 4). Taylor et al. (2012) have reported that three polar residues on ␣3 helix of AphB (Thr31 , Thr33 , Ser42 ) are involved in the formation of hydrogen bonds with DNA. In V. cholerae, expression of genes responsible for virulence factors and toxin-coregulated pilus is initiated by tcpPH promoter on Vibrio pathogenicity island (VPI), which in turn is regulated by transcriptional factors AphA, a winged helix DNA binding protein and AphB, a LysR-type regulator. AphA facilitates the binding of AphB to the promoter. AphB has a conserved structure with a N-terminal DNA binding domain and a C-terminal regulatory domain (Kovacikova
et al. 2010; Liu et al. 2010). From the results generated in this analysis, it was predicted that resveratrol could possibly interact with T31 (Thr31 ) and showed AphB as the putative target of resveratrol in V. cholerae. In this study, we have demonstrated for the first time the antibiofilm activity of resveratrol against V. cholerae, the causative agent of cholera. As this compound acts against biofilm formation and virulence factors rather than changing the viability of the bacteria, the selective pressure for developing resistance would be less. It is a well-characterized natural compound. In this context, we propose that resveratrol is a potential molecule for the development of a novel drug which may be used alone or in combination with other antibiotics to tackle the disease, cholera. Further studies including gene expression and in vivo models are needed to explore the molecular mechanism of action in detail. Conflict of interest There is no conflict of interest among authors. Acknowledgements This study was supported by a research grant from Defence Research Development Organisation (DRDO), Ministry of Defence, Govt. of India, New Delhi. The authors are thankful to Prof. M. Radhakrishna Pillai, Director, Rajiv Gandhi Centre for Biotechnology (RGCB), for the facilities provided. Authors are thankful to Mr. Anurup K.G., RGCB, for providing with technical help in carrying out CLSM analysis. References
Fig. 4. Docking conformation of resveratrol with AphB. Residues interacting with ligand are represented with lines.
Baur, J.A., Sinclair, D.A., 2006. Therapeutic potential of resveratrol: the in vivo evidence. Nature Reviews Drug Discovery 5, 493–506. CLSI, 2010. Performance standards for antimicrobial susceptibility testing; Twentieth Informational Supplement: CLSI document M100-S20. Clinical and Laboratory Standard Institute, 940 West Valley Road, Suite 1400, Wayne Pennsylvania 19087-1898, USA. Coenye, T., Brackman, G., Rigole, P., De Witte, E., Honraet, K., Rossel, B., Nelis, H.J., 2012. Eradication of Propionibacterium acnes biofilms by plant extracts and putative identification of icariin, resveratrol and salidroside as active compounds. Phytomedicine 19, 409–412. Kierek, K., Watnick, P.I., 2003. Environmental determinants of Vibrio cholerae biofilm development. Applied and Environmental Microbiology 69, 5079–5088. Kovacikova, G., Lin, W., Skorupski, K., 2010. The LysR-type virulence activator AphB regulates the expression of genes in Vibrio cholerae in response to low pH and anaerobiosis. Journal of Bacteriology 192, 4181–4191. Kumar, P., Wilson, P.A., Bhai, R., Thomas, S., 2009. Characterization of an SXT variant Vibrio cholerae O1 Ogawa isolated from a patient in Trivandrum, India. FEMS Microbiology Letters 303, 132–136. Liu, Z., Yang, M., Peterfreund, G.L., Tsou, A.M., Selamoglu, N., Daldal, F., Zhong, Z., Kan, B., Zhu, J., 2010. Vibrio cholerae anaerobic induction of virulence gene expression
N. Augustine et al. / Phytomedicine 21 (2014) 286–289 is controlled by thiol-based switches of virulence regulator AphB. Proceedings of the National Academy of Sciences 108, 810–815. Morinaga, N., Yahiro, K., Noda, M., 2010. Resveratrol, a natural polyphenolic compound, inhibits cholera toxin-induced cyclic AMP accumulation in vero cells. Toxicon 56, 29–35. Mshana, R.N., Tadesse, G., Abate, G., MiÖrner, H.K., 1998. Use of 3-(4,5dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide for rapid detection of rifampin-resistant Mycobacterium tuberculosis. Journal of Clinical Microbiology 36, 1214–1219. O’Toole, G.A., 2011. Microtiter dish biofilm formation assay. Journal of Visualized Experiments: JoVE 47, 2437. Sambanthamoorthy, K., Sloup, R.E., Parashar, V., Smith, J.M., Kim, E.E., Semmelhack, M.F., Neiditch, M.B., Waters, C.M., 2012. Identification of small molecules that antagonize diguanylatecyclase enzymes to inhibit biofilm formation. Antimicrobial Agents and Chemotherapy 56, 5202–5211. Tamayo, R., Patimalla, B., Camilli, A., 2010. Growth in a biofilm induces a hyperinfectious phenotype in Vibrio cholerae. Infection and Immunity 78, 3560–3569. Taylor, J.L., De Silva, R.S., Kovacikova, G., Lin, W., Taylor, R.K., Skorupski, K., Kull, F.J., 2012. The crystal structure of AphB, a virulence gene activator from Vibrio
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