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Mechanisms of envelope permeability and antibiotic influx and efflux in Gram-negative bacteria

Researchers, clinicians and governments all recognize antimicrobial resistance as a serious and growing threat worldwide. New antimicrobials are urgently needed, especially for infections caused by Gram-negative bacteria, whose cell envelopes are characterized by low permeability and often contain drug efflux systems. Individual bacteria and populations control their internal concentrations of antibiotics by regulating proteins involved in membrane permeability, such as porins or efflux pumps. Robust methods to quantify and visualize intrabacterial antibiotic concentrations have identified clear correlations between efflux activity and drug diffusion and accumulation in both susceptible and resistant strains, and have also clarified how certain chemical structures can affect drug entry and residence time within the cell. In this PERSPECTIVE, we discuss the biological underpinnings of drug permeability and export using several prototypical influx and efflux systems. We also highlight how new methods for the determination of antibacterial activities enable more careful quantitation and may provide us with a way forward for capturing and correlating the modes of action and kinetics of antibiotic uptake inside bacterial cells. Together, these advances will aid efforts to generate structurally improved molecules with better access and retention within bacteria, thereby reducing the emergence and spread of resistant strains and extending the clinical use of current antibiotics.

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Figure 1: Structure of the OmpF porin of E. coli.
Figure 2: Structure and function of efflux pumps.

References

  1. Fraimow, H. S. & Tsigrelis, C. Antimicrobial resistance in the intensive care unit: mechanisms, epidemiology, and management of specific resistant pathogens. Crit. Care Clin. 27, 163–205 (2011).

    Article  CAS  PubMed  Google Scholar 

  2. Antibiotic Resistance Threats in the United States (Centers for Disease Control and Prevention, 2013).

  3. Pierluigi, V., Maddalena, G., Sara, T. & Russell, L. Treatment of MDR-Gram negative infections in the 21st century: a never ending threat for clinicians. Curr. Opin. Pharmacol. 24, 30–37 (2015).

    Article  CAS  Google Scholar 

  4. Page, M. G. & Bush, K. Discovery and development of new antibacterial agents targeting Gram-negative bacteria in the era of pandrug resistance: is the future promising? Curr. Opin. Pharmacol. 18, 91–97 (2014).

    Article  CAS  PubMed  Google Scholar 

  5. Boucher, H. W. et al. 10 x ’20 progress--development of new drugs active against gram-negative bacilli: an update from the Infectious Diseases Society of America. Clin. Infect. Dis. 56, 1685–1694 (2013).

    Article  PubMed  PubMed Central  Google Scholar 

  6. Nikaido, H. Molecular basis of bacterial outer membrane permeability revisited. Microbiol. Mol. Biol. Rev. 67, 593–656 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Zgurskaya, H. I., López, C. A. & Gnanakaran, S. Permeability barrier of Gram-negative cell envelopes and approaches to bypass it. ACS Infect. Dis. 1, 512–522 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Pagès, J.-M., James, C. E. & Winterhalter, M. The porin and the permeating antibiotic: a selective diffusion barrier in Gram-negative bacteria. Nat. Rev. Microbiol. 6, 893–903 (2008).

    Article  CAS  PubMed  Google Scholar 

  9. Delcour, A. H. Outer membrane permeability and antibiotic resistance. Biochim. Biophys. Acta 1794, 808–816 (2009).

    Article  CAS  PubMed  Google Scholar 

  10. Ruiz, N., Kahne, D. & Silhavy, T. J. Advances in understanding bacterial outer-membrane biogenesis. Nat. Rev. Micro. 4, 57–66 (2006).

    Article  CAS  Google Scholar 

  11. Li, Z. H., Plesiat, P. & Nikaido, H. The challenge of efflux-mediated antibiotic resistance in Gram-negative bacteria. Clin. Micro. Rev. 28, 337–418 (2015).

    Article  PubMed  PubMed Central  Google Scholar 

  12. Bergen, P. J. et al. Polymyxin combinations: pharmacokinetics and pharmacodynamics for rationale use. Pharmacotherapy 35, 34–42 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Vaara, M. Polymyxins and novel derivatives. Curr. Opin. Microbiol. 13, 574–581 (2010).

    Article  CAS  PubMed  Google Scholar 

  14. Urfer, M. et al. A peptidomimetic antibiotic targets outer membrane proteins and disrupts selectively the outer membrane in Escherichia coli. J. Biol. Chem. 291, 1921–1932 (2016).

    Article  CAS  PubMed  Google Scholar 

  15. Silver, L. L. Challenges of antibacterial discovery. Clin. Micro. Rev. 24, 71–109 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Nikaido, H. & Pagès, J.-M. Broad-specificity efflux pumps and their role in multidrug resistance of Gram-negative bacteria. FEMS Microbiol. Rev. 36, 340–363 (2012).

    Article  CAS  PubMed  Google Scholar 

  17. Cowan, S. W. et al. Crystal structures explain functional properties of two E. coli porins. Nature 358, 727–733 (1992).

    Article  CAS  PubMed  Google Scholar 

  18. Baslé, A., Rummel, G., Storici, P., Rosenbusch, J. P. & Schirmer, T. Crystal structure of osmoporin OmpC from E. coli at 2.0 A. J. Mol. Biol. 362, 933–942 (2006).

  19. Yoshimura, F. & Nikaido, H. Diffusion of beta-lactam antibiotics through the porin channels of Escherichia coli K-12. Antimicrob. Agents Chemother. 27, 84–92 (1985).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Nikaido, H. & Rosenberg, E. Y. Porin channels in Escherichia coli: studies with liposomes reconstituted from purified proteins. J. Bacteriol. 153, 241–252 (1983).

    CAS  PubMed  PubMed Central  Google Scholar 

  21. Zimmermann, W. & Rosselet, A. Function of the outer membrane of Escherichia coli as a permeability barrier to beta-lactam antibiotics. Antimicrob. Agents Chemother. 12, 368–372 (1977).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Nikaido, H., Rosenberg, E. Y. & Foulds, J. Porin channels in Escherichia coli: studies with beta-lactams in intact cells. J. Bacteriol. 153, 232–240 (1983).

    CAS  PubMed  PubMed Central  Google Scholar 

  23. Nikaido, H. & Normark, S. Sensitivity of Escherichia coli to various beta-lactams is determined by the interplay of outer membrane permeability and degradation by periplasmic beta-lactamases: a quantitative predictive treatment. Mol. Microbiol. 1, 29–36 (1987).

    Article  CAS  PubMed  Google Scholar 

  24. Kojima, S. & Nikaido, H. Permeation rates of penicillins indicate that Escherichia coli porins function principally as nonspecific channels. Proc. Natl Acad. Sci. USA 110, 2629–2634 (2013).

    Article  Google Scholar 

  25. Kojima, S. & Nikaido, H. High salt concentrations increase permeability through OmpC channels of Escherichia coli. J. Biol. Chem. 289, 26464–26473 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Benz, R., Schmid, A. & Hancock, R. E. Ion selectivity of Gram-negative bacterial porins. J. Bacteriol. 162, 722–727 (1985).

    CAS  PubMed  PubMed Central  Google Scholar 

  27. Winterhalter, M. & Ceccarelli, M. Physical methods to quantify small antibiotic molecules uptake into Gram-negative bacteria. Eur. J. Pharm. Biopharm. 95, 63–67 (2015).

    Article  CAS  PubMed  Google Scholar 

  28. Nestorovich, E. M., Danelon, C., Winterhalter, M. & Bezrukov, S. M. Designed to penetrate: time-resolved interaction of single antibiotic molecules with bacterial pores. Proc. Natl Acad. Sci. USA 99, 9789–9794 (2002).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Danelon, C., Nestorovich, E. M., Winterhalter, M., Ceccarelli, M. & Bezrukov, S. M. Interaction of zwitterionic penicillins with the OmpF channel facilitates their translocation. Biophys. J. 90, 1617–1627 (2006).

    Article  CAS  PubMed  Google Scholar 

  30. Simonet, V., Malléa, M. & Pagès, J.-M. Substitutions in the eyelet region disrupt cefepime diffusion through the Escherichia coli OmpF channel. Antimicrob. Agents Chemother. 44, 311–315 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Charrel, R. N., Pagès, J.-M., De Micco, P. & Mallea, M. Prevalence of outer membrane porin alteration in beta-lactam-antibiotic-resistant Enterobacter aerogenes. Antimicrob. Agents Chemother. 40, 2854–2858 (1996).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Hernández-Allés, S. et al. Porin expression in clinical isolates of Klebsiella pneumoniae. Microbiology 145, 673–679 (1999).

    Article  PubMed  Google Scholar 

  33. Lavigne, J. P. et al. An adaptive response of Enterobacter aerogenes to imipenem: regulation of porin balance in clinical isolates. Int. J. Antimicrob. Agents 41, 130–136 (2013).

    Article  CAS  PubMed  Google Scholar 

  34. Philippe, N. et al. In vivo evolution of bacterial resistance in two cases of Enterobacter aerogenes infections during treatment with imipenem. PLoS ONE 10, e0138828 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Low, A. S., MacKenzie, F. M., Gould, I. M. & Booth, I. R. Protected environments allow parallel evolution of a bacterial pathogen in a patient subjected to long-term antibiotic therapy. Mol. Microbiol. 42, 619–630 (2001).

    Article  CAS  PubMed  Google Scholar 

  36. Dé, E. et al. A new mechanism of antibiotic resistance in Enterobacteriaceae induced by a structural modification of the major porin. Mol. Microbiol. 41, 189–198 (2001).

    Article  PubMed  Google Scholar 

  37. Thiolas, A., Bornet, C., Davin-Régli, A., Pagés, J.-M. & Bollet, C. Resistance to imipenem, cefepime, and cefpirome associated with mutation in Omp36 osmoporin of Enterobacter aerogenes. Biochem. Biophys. Res. Commun. 317, 851–856 (2004).

    Article  CAS  PubMed  Google Scholar 

  38. Giraud, A. et al. Dissecting the genetic components of adaptation of Escherichia coli to the mouse gut. PLoS Genet. 4, e2 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Lou, H. et al. Altered antibiotic transport in OmpC mutants isolated from a series of clinical strains of multi-drug resistant E. coli. PLoS ONE 6, e25825 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Bajaj, H. et al. Molecular basis of filtering carbapenems by porins from β-lactam-resistant clinical strains of Escherichia coli. J. Biol. Chem. 291, 2837–2847 (2016).

    Article  CAS  PubMed  Google Scholar 

  41. James, C. E. et al. How beta-lactam antibiotics enter bacteria: a dialogue with the porins. PLoS ONE 4, e5453 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Ziervogel, B. K. & Roux, B. The binding of antibiotics in OmpF porin. Structure 21, 76–87 (2013).

    Article  CAS  PubMed  Google Scholar 

  43. Pagés, J.-M., Peslier, S., Keating, T. A., Lavigne, J. P. & Nichols, W. W. The role of the outer membrane and porins in the susceptibility of β-lactamase-producing Enterobacteriaceae to ceftazidime-avibactam. Antimicrob. Agents Chemother. 60, 1349–1359 (2015).

    Article  CAS  PubMed  Google Scholar 

  44. Du, D., van Veen, H. W., Murakami, S., Pos, K. M. & Luisi, B. F. Structure, mechanism and cooperation of bacterial multidrug transporters. Curr. Opin. Struct. Biol. 33, 76–91 (2015).

    Article  CAS  PubMed  Google Scholar 

  45. Shuster, Y., Steiner-Mordoch, S., Alon-Cudkowicz, N. & Schuldiner, S. A. Transporter interactome is essential for the acquisition of antimicrobial resistance to antibiotics. PLoS ONE 11, e0152917 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Yamaguchi, A., Nakashima, R. & Sakurai, K. Structural basis of RND-type multidrug exporters. Front. Microbiol. 6, 327 (2015).

    Article  PubMed  PubMed Central  Google Scholar 

  47. Pos, K. M. Drug transport mechanism of the AcrB efflux pump. Biochim. Biophys. Acta 1794, 782–793 (2009).

    Article  CAS  PubMed  Google Scholar 

  48. Nikaido, H. & Takatsuka, Y. Mechanisms of RND multidrug efflux pumps. Biochim. Biophys. Acta 1794, 769–781 (2009).

    Article  CAS  PubMed  Google Scholar 

  49. Murakami, S., Nakashima, R., Yamashita, E. & Yamaguchi, A. Crystal structure of bacterial multidrug efflux transporter AcrB. Nature 419, 587–593 (2002).

    Article  CAS  PubMed  Google Scholar 

  50. Murakami, S., Nakashima, R., Yamashita, E., Matsumoto, T. & Yamaguchi, A. Crystal structures of a multidrug transporter reveal a functionally rotating mechanism. Nature 443, 173–179 (2006).

    Article  CAS  PubMed  Google Scholar 

  51. Seeger, M. A. et al. Structural asymmetry of AcrB trimer suggests a peristaltic pump mechanism. Science 313, 1295–1298 (2006).

    Article  CAS  PubMed  Google Scholar 

  52. Sennhauser, G. et al. Drug export pathway of multidrug exporter AcrB revealed by DARPin inhibitors. PLoS Biol. 5, e7 (2007).

    Article  CAS  PubMed  Google Scholar 

  53. Sennhauser, G., Bukowska, M. A., Briand, C. & Grütter, M. G. Crystal structure of the multidrug exporter MexB from Pseudomonas aeruginosa. J. Mol. Biol. 389, 134–145 (2009).

    Article  CAS  PubMed  Google Scholar 

  54. Bolla, J. R. et al. Crystal structure of the Neisseria gonorrhoeae MtrD inner membrane multidrug efflux pump. PLoS ONE 9, e97903 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  55. Eicher, T. et al. Transport of drugs by the multidrug transporter AcrB involves an access and a deep binding pocket that are separated by a switch-loop. Proc. Natl Acad. Sci. USA 109, 5687–5692 (2012).

    Article  PubMed  PubMed Central  Google Scholar 

  56. Eicher, T. et al. Coupling of remote alternating-access transport mechanisms for protons and substrates in the multidrug efflux pump AcrB. eLife 3, e03145 (2014).

    Article  CAS  PubMed Central  Google Scholar 

  57. Nakashima, R., Sakurai, K., Yamasaki, S., Nishino, K. & Yamaguchi, A. Structures of the multidrug exporter AcrB reveal a proximal multisite drug-binding pocket. Nature 480, 565–569 (2011).

    Article  CAS  PubMed  Google Scholar 

  58. Nakashima, R. et al. Structural basis for the inhibition of bacterial multidrug exporters. Nature 500, 102–106 (2013).

    Article  CAS  PubMed  Google Scholar 

  59. Sjuts, H. et al. Molecular basis for inhibition of AcrB multidrug efflux pump by novel and powerful pyranopyridine derivatives. Proc. Natl Acad. Sci. USA 113, 3509–3514 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  60. Du, D. et al. Structure of the AcrAB–TolC multidrug efflux pump. Nature 509, 512–515 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  61. Kim, J. S. et al. Structure of the tripartite multidrug efflux pump AcrAB-TolC suggests an alternative assembly mode. Mol. Cells 38, 180–186 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  62. Daury, L. et al. Tripartite assembly of RND multidrug efflux pumps. Nat. Commun. 7, 10731 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  63. Kühlbrandt, W. Cryo-EM enters a new era. eLife 3, e03678 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  64. Verchére, A., Dezi, M., Adrien, V., Broutin, I. & Picard, M. In vitro transport activity of the fully assembled MexAB-OprM efflux pump from Pseudomonas aeruginosa. Nat. Commun. 6, 6890 (2015).

    Article  CAS  PubMed  Google Scholar 

  65. Chapman, J. S. & Georgopapadakou, N. H. Fluorometric assay for fleroxacin uptake by bacterial cells. Antimicrob. Agents Chemother. 33, 27–29 (1989).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  66. June, C. M. et al. A fluorescent carbapenem for structure function studies of penicillin-binding proteins, β-lactamases, and β-lactam sensors. Anal. Biochem. 463, 70–74 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  67. Zhou, Y. et al. Thinking outside the “bug„: a unique assay to measure intracellular drug penetration in Gram-negative bacteria. Anal. Chem. 87, 3579–3584 (2015).

    Article  CAS  PubMed  Google Scholar 

  68. Kinana, A. D., Vargiu, A. V., May, T. & Nikaido, H. Aminoacyl β-naphthylamides as substrates and modulators of AcrB multidrug efflux pump. Proc. Natl Acad. Sci. USA 113, 1405–1410 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  69. Davis, T. D., Gerry, C. J. & Tan, D. S. General platform for systematic quantitative evaluation of small-molecule permeability in bacteria. ACS Chem. Biol. 9, 2535–2544 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  70. Kaščáková, S., Maigre, L., Chevalier, J., Réfrégiers, M. & Pagés, J.-M. Antibiotic transport in resistant bacteria: synchrotron UV fluorescence microscopy to determine antibiotic accumulation with single cell resolution. PLoS ONE 7, e38624 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  71. Cinquin, B. et al. Microspectrometric insights on the uptake of antibiotics at the single bacterial cell level. Sci. Rep. 5, 17968 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  72. Pu, Y. et al. Enhanced efflux activity facilitates drug tolerance in dormant bacterial cells. Mol. Cells 62, 284–294 (2016).

    Article  CAS  Google Scholar 

  73. Burns, A. R. et al. A predictive model for drug bioaccumulation and bioactivity in Caenorhabditis elegans. Nat. Chem. Biol. 6, 549–557 (2010).

    Article  CAS  PubMed  Google Scholar 

  74. Brown, A. R. et al. A mass spectrometry-based assay for improved quantitative measurements of efflux pump inhibition. PLoS ONE 10, e0124814 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  75. Bolla, J. M. et al. Strategies for bypassing the membrane barrier in multidrug resistant Gram-negative bacteria. FEBS Lett. 585, 1682–1690 (2011).

    Article  CAS  PubMed  Google Scholar 

  76. Bergen, P. J. et al. Polymyxin combinations: pharmacokinetics and pharmacodynamics for rationale use. Pharmacotherapy 35, 34–42 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  77. de Carvalho, C. C. C. R. & Fernandes, P. Siderophores as “Trojan Horses„: tackling multidrug resistance? Front. Microbiol. 5, 290 (2014).

    Article  PubMed  PubMed Central  Google Scholar 

  78. Mislin, G. L. & Schalk, I. J. Siderophore-dependent iron uptake systems as gates for antibiotic Trojan horse strategies against Pseudomonas aeruginosa. Metallomics 6, 408–420 (2014).

    Article  CAS  PubMed  Google Scholar 

  79. Dreier, J. & Ruggerone, P. Interaction of antibacterial compounds with RND efflux pumps in Pseudomonas aeruginosa. Front. Microbiol. 6, 660 (2015).

    Article  PubMed  PubMed Central  Google Scholar 

  80. Lomovskaya, O. & Bostian, K. A. Practical applications and feasibility of efflux pump inhibitors in the clinic -a vision for applied use. Biochem. Pharmacol. 71, 910–918 (2006).

    Article  CAS  PubMed  Google Scholar 

  81. Pagés, J.-M., Amaral L. & Fanning, S. An original deal for new molecule: reversal of efflux pump activity, a rational strategy to combat Gram-negative resistant bacteria. Curr. Med. Chem. 18, 2969–2980 (2011).

    Article  PubMed  Google Scholar 

  82. Sánchez-Romero, M. A. & Casadesús, J. Contribution of phenotypic heterogeneity to adaptive antibiotic resistance. Proc. Natl Acad. Sci. USA 111, 355–360 (2014).

    Article  CAS  PubMed  Google Scholar 

  83. Opperman, T. J. & Nguyen S. T. Recent advances toward a molecular mechanism of efflux pump inhibition. Front. Microbiol. 6, 421 (2015).

    Article  PubMed  PubMed Central  Google Scholar 

  84. Dörr, T., Vuli, C., M. & Lewis, K. Ciprofloxacin causes persister formation by inducing the TisB toxin in Escherichia coli. PLoS Biol. 8, e1000317 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  85. Maisonneuve, E. & Gerdes, K. Molecular mechanisms underlying bacterial persisters. Cell 157, 539–548 (2014).

    Article  CAS  PubMed  Google Scholar 

  86. Nagano, K. & Nikaido, H. Kinetic behavior of the major multidrug efflux pump AcrB of Escherichia coli. Proc. Natl Acad. Sci. USA 106, 5854–5858 (2009).

    Article  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

We thank all the partners of IMI-Translocation consortium, especially R. A. Stavenger, for their fruitful discussions and suggestions. The research leading to the results discussed here was conducted as part of the translocation consortium (www.translocation.eu) and has received support from the Innovative Medicines Initiative Joint Undertaking under grant agreement no. 115525, resources which are composed of financial contribution from the European Union's seventh framework program (FP/2007-2013) and EFPIA companies in kind contributions.

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All authors, M.M., M.R., K.M.P. and J.-M.P., have contributed to the preparation of the manuscript. M.M. and J.-M.P. have corrected and improved the manuscript with the input from the referees and editor.

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Correspondence to Jean-Marie Pagès.

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Masi, M., Réfregiers, M., Pos, K. et al. Mechanisms of envelope permeability and antibiotic influx and efflux in Gram-negative bacteria. Nat Microbiol 2, 17001 (2017). https://doi.org/10.1038/nmicrobiol.2017.1

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