Abstract
Multidrug efflux pumps play an important role in antimicrobial resistance and pathogenicity in bacteria. Here, we report the functional characterization of the RND (resistance-nodulation- division) efflux pump, AcrAB, in Acinetobacter nosocomialis. An in silico analysis revealed that homologues of the AcrAB efflux pump, comprising AcrA and AcrB, are widely distributed among different bacterial species. Deletion of acrA and/or acrB genes led to decreased biofilm/pellicle formation and reduced antimicrobial resistance in A. nosocomialis. RNA sequencing and mRNA expression analyses showed that expression of acrA/B was downregulated in a quorum sensing (QS) regulator (anoR)-deletion mutant, indicating transcriptional activation of the acrAB operon by AnoR in A. nosocomialis. Bioassays showed that secretion of N-acyl homoserine lactones (AHLs) was unaffected in acrA and acrB deletion mutants; however, AHL secretion was limited in a deletion mutant of acrR, encoding the acrAB regulator, AcrR. An in silico analysis indicated the presence of AcrR-binding motifs in promoter regions of anoI (encoding AHL synthase) and anoR. Specific binding of AcrR was confirmed by electrophoretic mobility shift assays, which revealed that AcrR binds to positions -214 and -217 bp upstream of the translational start sites of anoI and anoR, respectively, demonstrating transcriptional regulation of these QS genes by AcrR. The current study further addresses the possibility that AcrAB is controlled by the osmotic stress regulator, OmpR, in A. nosocomialis. Our data demonstrate that the AcrAB efflux pump plays a crucial role in biofilm/pellicle formation and antimicrobial resistance in A. nosocomialis, and is under the transcriptional control of a number of regulators. In addition, the study emphasizes the interrelationship of QS and AcrAB efflux systems in A. nosocomialis.
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Alves, C. and Cunha, C. 2012. Electrophoretic mobility shift assay: analyzing protein - Nucleic acid interactions, pp. 205–229. Gel Electrophoresis-Advanced Techniques. INTECH, Rijeka, Croatia.
Baugh, S., Ekanayaka, A.S., Piddock, L.J., and Webber, M.A. 2012. Loss of or inhibition of all multidrug resistance efflux pumps of Salmonella enterica serovar Typhimurium results in impaired ability to form a biofilm. J. Antimicrob. Chemother. 67, 2409–2417.
Baugh, S., Phillips, C.R., Ekanayaka, A.S., Piddock, L.J., and Webber, M.A. 2014. Inhibition of multidrug efflux as a strategy to prevent biofilm formation. J. Antimicrob. Chemother. 69, 673–681.
Buckley, A.M., Webber, M.A., Cooles, S., Randall, L.P., La Ragione, R.M., Woodward, M.J., and Piddock, L.J. 2006. The AcrAB-TolC efflux system of Salmonella enterica serovar Typhimurium plays a role in pathogenesis. Cell. Microbiol. 8, 847–856.
Bunikis, I., Denker, K., Östberg, Y., Andersen, C., Benz, R., and Bergstrom, S. 2008. An RND-type efflux system in Borrelia burgdorferi is involved in virulence and resistance to antimicrobial compounds. PLoS Pathog. 4, e1000009.
Chan, Y.Y., Bian, H.S., Tan, T.M., Mattmann, M.E., Geske, G.D., Igarashi, J., Hatano, T., Suga, H., Blackwell, H.E., and Chua, K.L. 2007. Control of quorum sensing by a Burkholderia pseudomallei multidrug efflux pump. J. Bacteriol. 189, 4320–4324.
CLSI (Clinical and Laboratory Standards Institute). 2017. Performance standards for antimicrobial susceptibility testing. 27th edn. Clinical and Laboratory Standards Institute, Wayne, Pennsylvania, USA.
CLSI (Clinical and Laboratory Standards Institute). 2018. Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically. Clinical and Laboratory Standards Institute, Wayne, Pennsylvania, USA.
Coyne, S., Courvalin, P., and Périchon, B. 2011. Efflux-mediated antibiotic resistance in Acinetobacter spp. Antimicrob. Agents Chemother. 55, 947–953.
Coyne, S., Rosenfeld, N., Lambert, T., Courvalin, P., and Périchon, B. 2010. Overexpression of resistance-nodulation-cell division pump AdeFGH confers multidrug resistance in Acinetobacter baumannii. Antimicrob. Agents Chemother. 54, 4389–4393.
Damier-Piolle, L., Magnet, S., Brémont, S., Lambert, T., and Courvalin, P. 2008. AdeIJK, a resistance-nodulation-cell division pump effluxing multiple antibiotics in Acinetobacter baumannii. Antimicrob. Agents Chemother. 52, 557–562.
Evans, K., Passador, L., Srikumar, R., Tsang, E., Nezezon, J., and Poole, K. 1998. Influence of the MexAB-OprM multidrug efflux system on quorum sensing in Pseudomonas aeruginosa. J. Bacteriol. 180, 5443–5447.
Giles, S.K., Stroeher, U.H., Eijkelkamp, B.A., and Brown, M.H. 2015. Identification of genes essential for pellicle formation in Acinetobacter baumannii. BMC Microbiol. 15, 116.
Hanahan, D. 1983. Studies on transformation of Escherichia coli with plasmids. J. Mol. Biol. 166, 557–580.
Hirakata, Y., Srikumar, R., Poole, K., Gotoh, N., Suematsu, T., Kohno, S., Kamihira, S., Hancock, R.E., and Speert, D.P. 2002. Multidrug efflux systems play an important role in the invasiveness of Pseudomonas aeruginosa. J. Exp. Med. 196, 109–118.
Koronakis, V., Eswaran, J., and Hughes, C. 2004. Structure and function of TolC: the bacterial exit duct for proteins and drugs. Annu. Rev. Biochem. 73, 467–489.
Kvist, M., Hancock, V., and Klemm, P. 2008. Inactivation of efflux pumps abolishes bacterial biofilm formation. Appl. Environ. Microbiol. 74, 7376–7382.
Lee, J.O., Cho, K.S., and Kim, O.B. 2014. Overproduction of AcrR increases organic solvent tolerance mediated by modulation of SoxS regulon in Escherichia coli. Appl. Microbiol. Biotechnol. 98, 8763–8773.
Lee, H.W., Koh, Y.M., Kim, J., Lee, J.C., Lee, Y.C., Seol, S.Y., Cho, D.T., and Kim, J. 2008. Capacity of multidrug-resistant clinical isolates of Acinetobacter baumannii to form biofilm and adhere to epithelial cell surfaces. Clin. Microbiol. Infect. 14, 49–54.
Li, Y., Mima, T., Komori, Y., Morita, Y., Kuroda, T., Mizushima, T., and Tsuchiya, T. 2003. A new member of the tripartite multidrug efflux pumps, MexVW-OprM, in Pseudomonas aeruginosa. J. Antimicrob. Chemother. 52, 572–575.
Li, X.Z. and Nikaido, H. 2004. Efflux-mediated drug resistance in bacteria. Drugs 64, 159–204.
Livak, K.J. and Schmittgen, T.D. 2001. Analysis of relative gene expression data using real-time quantitative PCR and the 2−ΔΔCT method. Methods 25, 402–408.
Lynch, S.V., Dixon, L., Benoit, M.R., Brodie, E.L., Keyhan, M., Hu, P., Ackerley, D.F., Andersen, G.L., and Matin, A. 2007. Role of the rapA gene in controlling antibiotic resistance of Escherichia coli biofilms. Antimicrob. Agents Chemother. 51, 3650–3658.
Ma, D., Alberti, M., Lynch, C., Nikaido, H., and Hearst, J.E. 1996. The local repressor AcrR plays a modulating role in the regulation of acrAB genes of Escherichia coli by global stress signals. Mol. Microbiol. 19, 101–112.
Ma, D., Cook, D.N., Alberti, M., Pon, N.G., Nikaido, H., and Hearst, J.E. 1993. Molecular cloning and characterization of acrA and acrE genes of Escherichia coli. J. Bacteriol. 175, 6299–6313.
Ma, D., Cook, D.N., Alberti, M., Pon, N.G., Nikaido, H., and Hearst, J.E. 1995. Genes acrA and acrB encode a stress-induced efflux system of Escherichia coli. Mol. Microbiol. 16, 45–55.
Magnet, S., Courvalin, P., and Lambert, T. 2001. Resistance-nodulation-cell division-type efflux pump involved in aminoglycoside resistance in Acinetobacter baumannii strain BM4454. Antimicrob. Agents Chemother. 45, 3375–3380.
Maseda, H., Sawada, I., Saito, K., Uchiyama, H., Nakae, T., and Nomura, N. 2004. Enhancement of the mexAB-oprM efflux pump expression by a quorum-sensing autoinducer and its cancellation by a regulator, MexT, of the mexEF-oprN efflux pump operon in Pseudomonas aeruginosa. Antimicrob. Agents Chemother. 48, 1320–1328.
Matsumura, K., Furukawa, S., Ogihara, H., and Morinaga, Y. 2011. Roles of multidrug efflux pumps on the biofilm formation of Escherichia coli K-12. Biocontrol Sci. 16, 69–72.
Morita, Y., Komori, Y., Mima, T., Kuroda, T., Mizushima, T., and Tsuchiya, T. 2001. Construction of a series of mutants lacking all of the four major mex operons for multidrug efflux pumps or possessing each one of the operons from Pseudomonas aeruginosa PAO1: MexCD-OprJ is an inducible pump. FEMS Microbiol. Lett. 202, 139–143.
Nikaido, H. 1996. Multidrug efflux pumps of Gram-negative bacteria. J. Bacteriol. 178, 5853–5859.
Oh, M.H. and Choi, C.H. 2015. Role of LuxIR homologue AnoIR in Acinetobacter nosocomialis and the effect of virstatin on the expression of anoR gene. J. Microbiol. Biotechnol. 25, 1390–1400.
Oh, M.H., Lee, J.C., Kim, J., Choi, C.H., and Han, K. 2015. Simple method for markerless gene deletion in multidrug-resistant Acinetobacter baumannii. Appl. Environ. Microbiol. 81, 3357–3368.
Park, S.Y., Lee, S.J., Oh, T.K., Oh, J.W., Koo, B.T., Yum, D.Y., and Lee, J.K. 2003. AhlD, an N-acyl homoserine lactonase in Arthrobacter sp., and predicted homologues in other bacteria. Microbiology 149, 1541–1550.
Pearson, J.P., Van Delden, C., and Iglewski, B.H. 1999. Active efflux and diffusion are involved in transport of Pseudomonas aeruginosa cell-to-cell signals. J. Bacteriol. 181, 1203–1210.
Piddock, L.J. 2006a. Clinically relevant chromosomally encoded multidrug resistance efflux pumps in bacteria. Clin. Microbiol. Rev. 19, 382–402.
Piddock, L.J. 2006b. Multidrug-resistance efflux pumps - not just for resistance. Nat. Rev. Microbiol. 4, 629–636.
Poole, K., Krebes, K., McNally, C., and Neshat, S. 1993. Multiple antibiotic resistance in Pseudomonas aeruginosa: Evidence for involvement of an efflux operon. J. Bacteriol. 175, 7363–73.
Pumbwe, L., Randall, L.P., Woodward, M.J., and Piddock, L.J. 2004. Expression of the efflux pump genes cmeB, cmeF and the porin gene porA in multiple-antibiotic-resistant Campylobacter jejuni. J. Antimicrob. Chemother. 54, 341–347.
Raczkowska, A., Trzos, J., Lewandowska, O., Nieckarz, M., and Brzostek, K. 2015. Expression of the AcrAB components of the AcrAB-TolC multidrug efflux pump of Yersinia enterocolitica is subject to dual regulation by OmpR. PLoS One 10, e0124248.
Rahmati, S., Yang, S., Davidson, A.L., and Zechiedrich, E.L. 2002. Control of the AcrAB multidrug efflux pump by quorum-sensing regulator SdiA. Mol. Microbiol. 43, 677–685.
Randall, L.P. and Woodward, M.J. 2002. The multiple antibiotic resistance (mar) locus and its significance. Res. Vet. Sci. 72, 87–93.
Rosenberg, E.Y., Bertenthal, D., Nilles, M.L., Bertrand, K.P., and Nikaido, H. 2003. Bile salts and fatty acids induce the expression of Escherichia coli AcrAB multidrug efflux pump through their interaction with Rob regulatory protein. Mol. Microbiol. 48, 1609–1619.
Sambrook, J., Fritsch, E.F., and Maniatis, T. 1989. Molecular cloning: a laboratory manual. 2nd edn. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, USA.
Schlisselberg, D.B., Kler, E., Kisluk, G., Shachar, D., and Yaron, S. 2015. Biofilm formation ability of Salmonella enterica serovar Typhimurium acrAB mutants. Int. J. Antimicrob. Agents 46, 456–459.
Schneiders, T., Amyes, S.G., and Levy, S.B. 2003. Role of AcrR and RamA in fluoroquinolone resistance in clinical Klebsiella pneumoniae isolates from Singapore. Antimicrob. Agents Chemother. 47, 2831–2837.
Simon, R., Priefer, U., and Pühler, A. 1983. A broad host range mobilization system for in vivo genetic engineering transposon mutagenesis in Gram negative bacteria. Nat. Biotechnol. 1, 784–791.
Studier, F.W. and Moffatt, B.A. 1986. Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes. J. Mol. Biol. 189, 113–130.
Su, C.C., Rutherford, D.J., and Yu, E.W. 2007. Characterization of the multidrug efflux regulator AcrR from Escherichia coli. Biochem. Biophys. Res. Commun. 361, 85–90.
Subhadra, B., Kim, J., Kim, D.H., Woo, K., Oh, M.H., and Choi, C.H. 2018. Local repressor AcrR regulates AcrAB efflux pump required for biofilm formation and virulence in Acinetobacter nosocomialis. Front. Cell. Infect. Microbiol. 8, 270.
Tamura, K., Stecher, G., Peterson, D., Filipski, A., and Kumar, S. 2013. MEGA6: Molecular evolutionary genetics analysis version 6.0. Mol. Biol. Evol. 30, 2725–2729.
Tipton, K.A., Farokhyfar, M., and Rather, P.N. 2017. Multiple roles for a novel RND-type efflux system in Acinetobacter baumannii AB5075. Microbiologyopen 6, e00418.
Viveiros, M., Martins, A., Paixão, L., Rodrigues, L., Martins, M., Couto, I., Fähnrich, E., Kern, W.V., and Amaral, L. 2008. Demonstration of intrinsic efflux activity of Escherichia coli K-12 AG100 by an automated ethidium bromide method. Int. J. Antimicrob. Agents 31, 458–462.
Zhang, L., Murphy, P.J., Kerr, A., and Tate, M.E. 1993. Agrobacterium conjugation and gene regulation by N-acyl-L-homoserine lactones. Nature 362, 446–448.
Acknowledgments
This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) (NRF-2019R1F1A1043436, NRF-2019M3E5D1A-02068575, NRF-2017R1A5A2015385, NRF-2014R1A6A10-29617) and by a grant of the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health & Welfare, Republic of Korea (grant number: HI17C1657).
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Subhadra, B., Surendran, S., Lim, B.R. et al. Regulation of the AcrAB efflux system by the quorum-sensing regulator AnoR in Acinetobacter nosocomialis. J Microbiol. 58, 507–518 (2020). https://doi.org/10.1007/s12275-020-0185-2
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DOI: https://doi.org/10.1007/s12275-020-0185-2