Skip to main content
SpringerLink
Log in

Regulation of the AcrAB efflux system by the quorum-sensing regulator AnoR in Acinetobacter nosocomialis

  • Microbial Pathogenesis and Host-Microbe Interaction
  • Published:
Journal of Microbiology Aims and scope Submit manuscript

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.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

References

  • 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.

    Article  CAS  PubMed  Google Scholar 

  • 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.

    Article  CAS  PubMed  Google Scholar 

  • 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.

    Article  CAS  PubMed  Google Scholar 

  • 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.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • 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.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • CLSI (Clinical and Laboratory Standards Institute). 2017. Performance standards for antimicrobial susceptibility testing. 27th edn. Clinical and Laboratory Standards Institute, Wayne, Pennsylvania, USA.

    Google Scholar 

  • 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.

    Google Scholar 

  • Coyne, S., Courvalin, P., and Périchon, B. 2011. Efflux-mediated antibiotic resistance in Acinetobacter spp. Antimicrob. Agents Chemother. 55, 947–953.

    Article  CAS  PubMed  Google Scholar 

  • 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.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • 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.

    Article  CAS  PubMed  Google Scholar 

  • 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.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • 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.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Hanahan, D. 1983. Studies on transformation of Escherichia coli with plasmids. J. Mol. Biol. 166, 557–580.

    Article  CAS  PubMed  Google Scholar 

  • 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.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • 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.

    Article  CAS  PubMed  Google Scholar 

  • Kvist, M., Hancock, V., and Klemm, P. 2008. Inactivation of efflux pumps abolishes bacterial biofilm formation. Appl. Environ. Microbiol. 74, 7376–7382.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • 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.

    Article  CAS  PubMed  Google Scholar 

  • 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.

    Article  CAS  PubMed  Google Scholar 

  • 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.

    Article  CAS  PubMed  Google Scholar 

  • Li, X.Z. and Nikaido, H. 2004. Efflux-mediated drug resistance in bacteria. Drugs 64, 159–204.

    Article  CAS  PubMed  Google Scholar 

  • 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.

    Article  CAS  PubMed  Google Scholar 

  • 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.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • 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.

    Article  CAS  PubMed  Google Scholar 

  • 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.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • 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.

    Article  CAS  PubMed  Google Scholar 

  • 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.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • 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.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • 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.

    Article  CAS  PubMed  Google Scholar 

  • 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.

    Article  CAS  PubMed  Google Scholar 

  • Nikaido, H. 1996. Multidrug efflux pumps of Gram-negative bacteria. J. Bacteriol. 178, 5853–5859.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • 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.

    Article  CAS  PubMed  Google Scholar 

  • 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.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • 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.

    Article  CAS  PubMed  Google Scholar 

  • 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.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Piddock, L.J. 2006a. Clinically relevant chromosomally encoded multidrug resistance efflux pumps in bacteria. Clin. Microbiol. Rev. 19, 382–402.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Piddock, L.J. 2006b. Multidrug-resistance efflux pumps - not just for resistance. Nat. Rev. Microbiol. 4, 629–636.

    Article  CAS  PubMed  Google Scholar 

  • 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.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • 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.

    Article  CAS  PubMed  Google Scholar 

  • 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.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • 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.

    Article  CAS  PubMed  Google Scholar 

  • Randall, L.P. and Woodward, M.J. 2002. The multiple antibiotic resistance (mar) locus and its significance. Res. Vet. Sci. 72, 87–93.

    Article  CAS  PubMed  Google Scholar 

  • 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.

    Article  CAS  PubMed  Google Scholar 

  • 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.

    Google Scholar 

  • 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.

    Article  CAS  PubMed  Google Scholar 

  • 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.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • 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.

    Article  CAS  Google Scholar 

  • 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.

    Article  CAS  PubMed  Google Scholar 

  • 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.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • 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.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • 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.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • 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.

    Article  CAS  Google Scholar 

  • 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.

    Article  CAS  PubMed  Google Scholar 

  • 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.

    Article  CAS  PubMed  Google Scholar 

Download references

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).

Author information

Author notes

  1. Bindu Subhadra

    Present address: Present Address: College of Veterinary Medicine, Long Island University, Brookville, New York, 11548, USA

Authors and Affiliations

  1. Department of Microbiology, Chungnam National University School of Medicine, Daejeon, 35015, Republic of Korea

    Bindu Subhadra, Surya Surendran, Bo Ra Lim, Jong Sung Yim, Dong Ho Kim, Kyungho Woo, Hwa-Jung Kim & Chul Hee Choi

  2. Department of Medical Science, Chungnam National University, Daejeon, 34134, Republic of Korea

    Bindu Subhadra, Surya Surendran, Bo Ra Lim, Jong Sung Yim, Dong Ho Kim, Kyungho Woo, Hwa-Jung Kim & Chul Hee Choi

  3. Department of Nanobiomedical Science, Dankook University, Cheonan, 31116, Republic of Korea

    Man Hwan Oh

Authors
  1. Bindu Subhadra
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Surya Surendran
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Bo Ra Lim
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Jong Sung Yim
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Dong Ho Kim
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Kyungho Woo
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Hwa-Jung Kim
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Man Hwan Oh
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Chul Hee Choi
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding authors

Correspondence to Man Hwan Oh or Chul Hee Choi.

Additional information

Conflict of Interest

The authors confirm that there are no conflicts of interest.

Supplemental material for this article may be found at http://www.springerlink.com/content/120956.

Electronic supplementary material

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

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

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s12275-020-0185-2

Keywords

Search

Navigation