Abstract
Acriflavine resistance protein B (AcrB) serves as prototype for multidrug resistance (MDR) efflux transporters of resistance nodulation division (RND) superfamily. AcrB has been proven as potential drug target with many synthetic and natural inhibitors have been identified such as those belonging to pyranopyridine, naphthamide and pimozide classes. The plant derived alkaloid inhibitors represented by reserpine has been found to inhibit both ATP binding cassette and major facilitator efflux transporters. In this study we report the reserpine induced inhibition of RND transporter AcrB. The preliminary docking analysis hints that reserpine shares its binding site with ciprofloxacin, a known substrate of AcrB and could possibly act as competitive inhibitor. For in vitro validation, AcrB from Salmonellatyphi was cloned under the control of tac promoter and resulting vector was introduced into E. coli C41(DE3). Under autoinduced conditions, cells overexpressing AcrB transporter were subjected to combined dose of ciprofloxacin and reserpine. The combined exposure resulted in enhanced ciprofloxacin-induced growth inhibition of cells expressing AcrB transporter as compared to control cells transformed with vector of backbone sequence. Time kill analysis further confirmed these findings. To the best of our knowledge, this is first study to show that exposure to reserpine induces inhibition of AcrB. The assay developed in this study allows simple and reproducible detection of substrate/inhibitor effects upon AcrB and related efflux transporters.
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18 October 2019
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REFERENCES
Seeger M.A., Diederichs K., Eicher T., Brandstatter L., Schiefner A., Verrey F., Pos K.M. 2008. The AcrB efflux pump: Conformational cycling and peristalsis lead to multidrug resistance. Curr. Drug Targets. 9, 729–749.
Seeger M.A., Schiefner A., Eicher T., Verrey F., Diederichs K., Pos K.M. 2006. Structural asymmetry of AcrB trimer suggests a peristaltic pump mechanism. Science 313, 1295–1298.
Shaheen A., Iqbal M., Mirza O., Rahman M. 2017. Structural biology meets drug resistance: An overview on multidrug resistance transporters. J. Indian Inst. Sci. 97, 165–175.
Opperman T.J., Kwasny S.M., Kim H.S., Nguyen S.T., Houseweart C., D’Souza S., Walker G.C., Peet N.P., Nikaido H., Bowlin T.L. 2014. Characterization of a novel pyranopyridine inhibitor of the AcrAB efflux pump of Escherichia coli. Antimicrob. Agents Chemother. 58, 722–733.
Wang Y., Mowla R., Ji S., Guo L., De Barros Lopes M.A., Jin C., Song D., Ma S., Venter H. 2018. Design, synthesis and biological activity evaluation of novel 4-subtituted 2-naphthamide derivatives as AcrB inhibitors. Eur. J. Med. Chem. 143, 699–709.
Wang-Kan X., Blair J.M.A., Chirullo B., Betts J., La Ragione R.M., Ivens A., Ricci V., Opperman T.J., Piddock L.J.V. 2017. Lack of AcrB efflux function confers loss of virulence on Salmonella enterica serovar Typhimurium. MBio. 8 (4). pii: e00968-17. https://doi.org/10.1128/mBio.00968-17
Vargiu A.V., Nikaido H. 2012. Multidrug binding properties of the AcrB efflux pump characterized by molecular dynamics simulations. Proc. Natl. Acad. Sci. U. S. A. 109, 20637–20642.
Takatsuka Y., Chen C., Nikaido H. 2010. Mechanism of recognition of compounds of diverse structures by the multidrug efflux pump AcrB of Escherichia coli. Proc. Natl. Acad. Sci. U. S. A. 107, 6559–6565.
Aparna V., Dineshkumar K., Mohanalakshmi N., Velmurugan D., Hopper W. 2014. Identification of natural compound inhibitors for multidrug efflux pumps of Escherichia coli and Pseudomonas aeruginosa using in silico high-throughput virtual screening and in vitro validation. PLoS One. 9, e101840.
Pakzad I., Zayyen Karin M., Taherikalani M., Boustanshenas M., Lari A.R. 2013. Contribution of AcrAB efflux pump to ciprofloxacin resistance in Klebsiella pneumoniae isolated from burn patients. GMS Hyg. Infect. Control. 8 (2), Doc15. https://doi.org/10.3205/dgkh000215
Das D., Xu Q.S., Lee J.Y., Ankoudinova I., Huang C., Lou Y., DeGiovanni A., Kim R., Kim S.H. 2007. Crystal structure of the multidrug efflux transporter AcrB at 3.1A resolution reveals the N-terminal region with conserved amino acids. J. Struct. Biol. 158, 494–502.
Berman H.M., Westbrook J., Feng Z., Gilliland G., Bhat T.N., Weissig H., Shindyalov I.N., Bourne P.E. 2000. The Protein Data Bank. Nucleic Acids Res. 28, 235–242.
Murakami S., Nakashima R., Yamashita E., Matsumoto T., Yamaguchi A. 2006. Crystal structures of a multidrug transporter reveal a functionally rotating mechanism. Nature 443, 173–179.
Soparkar K., Kinana A.D., Weeks J.W., Morrison K.D., Nikaido H., Misra R. 2015. Reversal of the drug binding pocket defects of the AcrB multidrug efflux pump protein of Escherichia coli. J. Bacteriol. 197, 3255–3264.
Zeng B., Wang H., Zou L., Zhang A., Yang X., Guan Z. 2010. Evaluation and target validation of indole derivatives as inhibitors of the AcrAB-TolC efflux pump. Biosci. Biotechnol. Biochem. 74, 2237–2241.
Li B., Yao Q., Pan X.C., Wang N., Zhang R., Li J., Ding G., Liu X., Wu C., Ran D., Zheng J., Zhou H. 2011. Artesunate enhances the antibacterial effect of beta-lactam antibiotics against Escherichia coli by increasing antibiotic accumulation via inhibition of the multidrug efflux pump system AcrAB-TolC. J. Antimicrob. Chemother. 66, 769–777.
Vargiu A.V., Ruggerone P., Opperman T.J., Nguyen S.T., Nikaido H. 2014. Molecular mechanism of MBX2319 inhibition of Escherichia coli AcrB multidrug efflux pump and comparison with other inhibitors. Antimicrob. Agents Chemother. 58, 6224–6234.
Bohnert J.A., Schuster S., Kern W.V. 2013. Pimozide inhibits the AcrAB-TolC efflux pump in Escherichia coli. Open Microbiol. J. 7, 83–86.
Hemaiswarya S., Kruthiventi A.K., Doble M. 2008. Synergism between natural products and antibiotics against infectious diseases. Phytomedicine. 15, 639–652.
Stermitz F.R., Tawara-Matsuda J., Lorenz P., Mueller P., Zenewicz L., Lewis K. 2000. 5'-Methoxyhydnocarpin-D and pheophorbide A: Berberis species components that potentiate berberine growth inhibition of resistant Staphylococcus aureus. J. Nat. Prod. 63, 1146–1149.
Stermitz F.R., Beeson T.D., Mueller P.J., Hsiang J., Lewis K. 2001. Staphylococcus aureus MDR efflux pump inhibitors from a Berberis and a Mahonia (sensu strictu) species. Biochem. Syst. Ecol. 29, 793–798.
Castaneda-Gomez J., Figueroa-Gonzalez G., Jacobo N., Pereda-Miranda R. 2013. Purgin II, a resin glycoside ester-type dimer and inhibitor of multidrug efflux pumps from Ipomoea purga. J. Nat. Prod. 76, 64–71.
Shiu W.K., Malkinson J.P., Rahman M.M., Curry J., Stapleton P., Gunaratnam M., Neidle S., Mushtaq S., Warner M., Livermore D.M., Evangelopoulos D., Basavannacharya C., Bhakta S., Schindler B.D., Seo S.M., et al. 2013. A new plant-derived antibacterial is an inhibitor of efflux pumps in Staphylococcus aureus. Int. J. Antimicrob. Agents. 42, 513–518.
Schmitz F.J., Fluit A.C., Luckefahr M., Engler B., Hofmann B., Verhoef J., Heinz H.P., Hadding U., Jones M.E. 1998. The effect of reserpine, an inhibitor of multidrug efflux pumps, on the in vitro activities of ciprofloxacin, sparfloxacin and moxifloxacin against clinical isolates of Staphylococcus aureus. J. Antimicrob. Chemother. 42, 807–810.
Holler J.G., Christensen S.B., Slotved H.C., Rasmussen H.B., Guzman A., Olsen C.E., Petersen B., Molgaard P. 2012. Novel inhibitory activity of the Staphylococcus aureus NorA efflux pump by a kaempferol rhamnoside isolated from Persea lingue Nees. J. Antimicrob. Chemother. 67, 1138–1144.
Fiamegos Y.C., Kastritis P.L., Exarchou V., Han H., Bonvin A.M., Vervoort J., Lewis K., Hamblin M.R., Tegos G.P. 2011. Antimicrobial and efflux pump inhibitory activity of caffeoylquinic acids from Artemisia absinthium against Gram-positive pathogenic bacteria. PLoS One. 6, e18127.
Neyfakh A.A., Bidnenko V.E., Chen L.B. 1991. Efflux-mediated multidrug resistance in Bacillus subtilis: Similarities and dissimilarities with the mammalian system. Proc. Natl. Acad. Sci. U. S. A. 88, 4781–4785.
Gibbons S., Udo E.E. 2000. The effect of reserpine, a modulator of multidrug efflux pumps, on the in vitro activity of tetracycline against clinical isolates of methicillin resistant Staphylococcus aureus (MRSA) possessing the tet(K) determinant. Phytother. Res. 14, 139–140.
Neyfakh A.A., Borsch C.M., Kaatz G.W. 1993. Fluoroquinolone resistance protein NorA of Staphylococcus aureus is a multidrug efflux transporter. Antimicrob. Agents Chemother. 37, 128–129.
Klemm E.J., Shakoor S., Page A.J., Qamar F.N., Judge K., Saeed D.K., Wong V.K., Dallman T.J., Nair S., Baker S., Shaheen G., Qureshi S., Yousafzai M.T., Saleem M.K., Hasan Z., et al. 2018. Emergence of an extensively drug-resistant Salmonella enterica serovar Typhi clone harboring a promiscuous plasmid encoding resistance to fluoroquinolones and third-generation cephalosporins. MBio. 9(1), pii: e00105-18. https://doi.org/10.1128/mBio.00105-18
Kumar S., Stecher G., Tamura K. 2016. MEGA7: Molecular evolutionary genetics analysis version 7.0 for bigger datasets. Mol. Biol. Evol. 33, 1870–1874.
Waterhouse A.M., Procter J.B., Martin D.M., Clamp M., Barton G.J. 2009. Jalview version 2-a multiple sequence alignment editor and analysis workbench. Bioinformatics. 25, 1189–1191.
Pettersen E.F., Goddard T.D., Huang C.C., Couch G.S., Greenblatt D.M., Meng E.C., Ferrin T.E. 2004. UCSF chimera: A visualization system for exploratory research and analysis. J. Comput. Chem. 25, 1605–1612.
Trott O., Olson A.J. 2010. AutoDock Vina: Improving the speed and accuracy of docking with a new scoring function, efficient optimization, and multithreading. J. Comput. Chem. 31, 455–461.
Shaheen A., Ismat F., Iqbal M., Haque A., De Zorzi R., Mirza O., Walz T., Rahman M. 2015. Characterization of putative multidrug resistance transporters of the major facilitator-superfamily expressed in Salmonella typhi. J. Infect. Chemother. 21, 357–362.
Miroux B., Walker J.E. 1996. Over-production of proteins in Escherichia coli: Mutant hosts that allow synthesis of some membrane proteins and globular proteins at high levels. J. Mol. Biol. 260, 289–298.
Studier F.W. 2005. Protein production by auto-induction in high density shaking cultures. Protein Expr. Purif. 41, 207–234.
Hayashi K., Nakashima R., Sakurai K., Kitagawa K., Yamasaki S., Nishino K., Yamaguchi A. 2016. AcrB-AcrA fusion proteins that act as multidrug efflux transporters. J. Bacteriol. 198, 332–342.
Ward A., Sanderson N.M., O’Reilly J., Rutherford N.G., Poolman B., Henderson P.J.F. 1999. The amplified expression, identification, purification, assay, and properties of hexahistidine-tagged bacterial membrane transport proteins. In: Membrane Transport–A Practical Approach. Oxford: Oxford Univ. Press.
Wink M. 2008. Ecological roles of alkaloids. In: Modern Alkaloids: Structure, Isolation, Synthesis, and Biology. Eds. Fattorusso E., Taglialatela-Scafati O. Wiley-VCH, vol. 1.
Roberts M.F. 2013. Enzymology of alkaloid biosynthesis. In: Alkaloids: Biochemistry, Ecology, and Medicinal Applications. Ed. Roberts M.F. Springer Science & Business Media.
Liu M., Heng J., Gao Y., Wang X. 2016. Crystal structures of MdfA complexed with acetylcholine and inhibitor reserpine. Biophys. Rep. 2, 78–85.
Beyer R., Pestova E., Millichap J.J., Stosor V., Noskin G.A., Peterson L.R. 2000. A convenient assay for estimating the possible involvement of efflux of fluoroquinolones by Streptococcus pneumoniae and Staphylococcus aureus: Evidence for diminished moxifloxacin, sparfloxacin, and trovafloxacin efflux. Antimicrob. Agents Chemother. 44, 798–801.
Abdelfatah S.A., Efferth T. 2015. Cytotoxicity of the indole alkaloid reserpine from Rauwolfia serpentina against drug-resistant tumor cells. Phytomedicine 22, 308–318.
Ahmed M., Borsch C.M., Neyfakh A.A., Schuldiner S. 1993. Mutants of the Bacillus subtilis multidrug transporter Bmr with altered sensitivity to the antihypertensive alkaloid reserpine. J. Biol. Chem. 268, 11086–11089.
Lee E.W., Huda M.N., Kuroda T., Mizushima T., Tsuchiya T. 2003. EfrAB, an ABC multidrug efflux pump in Enterococcus faecalis. Antimicrob. Agents Chemother. 47, 3733–3738.
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This research work was funded by the Higher Education Commission of Pakistan (grant no. 20-1504 to M. Rahman).
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The authors declare that they have no conflict of interest. This article does not contain any studies involving animals or human participants performed by any of the authors.
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W.A. Afridia and S. Mahbooba are both equally contributed to this work.
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Shaheen, A., Afridi, W.A., Mahboob, S. et al. Reserpine Is the New Addition into the Repertoire of AcrB Efflux Pump Inhibitors. Mol Biol 53, 596–605 (2019). https://doi.org/10.1134/S0026893319040113
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DOI: https://doi.org/10.1134/S0026893319040113