Inhibiting the copper efflux system in microbes as a novel approach for developing antibiotics

Autoři: Aviv Meir aff001;  Veronica Lepechkin-Zilbermintz aff001;  Shirin Kahremany aff002;  Fabian Schwerdtfeger aff001;  Lada Gevorkyan-Airapetov aff001;  Anna Munder aff001;  Olga Viskind aff001;  Arie Gruzman aff001;  Sharon Ruthstein aff001
Působiště autorů: Chemistry Department, Faculty of Exact Sciences, Bar Ilan University, Ramat-Gan, Israel aff001;  Gavin Herbert Eye Institute and the Department of Ophthalmology, University of California, Irvine, California, United States of America aff002;  Faculty of Biology, Albert-Ludwigs-University Freiburg, Centre for Biological Signaling Studies (BIOSS), Freiburg, Germany aff003
Vyšlo v časopise: PLoS ONE 14(12)
Kategorie: Research Article


Five out of six people receive at least one antibiotic prescription per year. However, the ever-expanding use of antibiotics in medicine, agriculture, and food production has accelerated the evolution of antibiotic-resistant bacteria, which, in turn, made the development of novel antibiotics based on new molecular targets a priority in medicinal chemistry. One way of possibly combatting resistant bacterial infections is by inhibiting the copper transporters in prokaryotic cells. Copper is a key element within all living cells, but it can be toxic in excess. Both eukaryotic and prokaryotic cells have developed distinct copper regulation systems to prevent its toxicity. Therefore, selectively targeting the prokaryotic copper regulation system might be an initial step in developing next-generation antibiotics. One such system is the Gram-negative bacterial CusCFBA efflux system. CusB is a key protein in this system and was previously reported to play an important role in opening the channel for efflux via significant structural changes upon copper binding while also controlling the assembly and disassembly process of the entire channel. In this study, we aimed to develop novel peptide copper channel blockers, designed by in silico calculations based on the structure of CusB. Using a combination of magnetic resonance spectroscopy and various biochemical methods, we found a lead peptide that promotes copper-induced cell toxicity. Targeting copper transport in bacteria has not yet been pursued as an antibiotic mechanism of action. Thus, our study lays the foundation for discovering novel antibiotics.

Klíčová slova:

Antibiotic resistance – Antibiotics – Bacterial diseases – Crystal structure – Deer – Dimers – Electron spin resonance spectroscopy – Periplasm


1. Moreillon P, Markiewicz Z, Nachman S, Tomasz A. Two bactericidal targets for penicillin in pneumococci: autolysis-dependent and autolysis-independent killing mechanisms. Antimicrob Agents Chemother. 1990;34(1):33–9. doi: 10.1128/aac.34.1.33 1691615

2. Moon TM, D'Andrea ED, Lee CW, Soares A, Jakoncic J, Desbonnet C, et al. The structures of penicillin-binding protein 4 (PBP4) and PBP5 from Enterococci provide structural insights into beta-lactam resistance. J Biol Chem. 2018;293(48):18574–84. doi: 10.1074/jbc.RA118.006052 30355734

3. Moine P, Vallee E, Azoulay-Dupuis E, Bourget P, Bedos JP, Bauchet J, et al. In vivo efficacy of a broad-spectrum cephalosporin, ceftriaxone, against penicillin-susceptible and -resistant strains of Streptococcus pneumoniae in a mouse pneumonia model. Antimicrob Agents Chemother. 1994;38(9):1953–8. doi: 10.1128/aac.38.9.1953 7811003

4. Martin JF. Biochemistry and molecular genetics of penicillin production in Penicillium chrysogenum. Ann N Y Acad Sci. 1991;646:193–201. doi: 10.1111/j.1749-6632.1991.tb18577.x 1809189

5. Gargis AS, McLaughlin HP, Conley AB, Lascols C, Michel PA, Gee JE, et al. Analysis of Whole-Genome Sequences for the Prediction of Penicillin Resistance and beta-Lactamase Activity in Bacillus anthracis. mSystems. 2018;3(6).

6. Hagstrand Aldman M, Skovby A, L IP. Penicillin-susceptible Staphylococcus aureus: susceptibility testing, resistance rates and outcome of infection. Infect Dis (Lond). 2017;49(6):454–60. Epub 2017/02/01. doi: 10.1080/23744235.2017.1280617 28135900.

7. Morand B, Muhlemann K. Heteroresistance to penicillin in Streptococcus pneumoniae. Proc Nat Acad Sci 2007;104(35):14098–103. doi: 10.1073/pnas.0702377104 17704255

8. Moraly J, Dahoumane R, Dubee V, Preda G, Baudel JL, Joffre J, et al. Penicillin G susceptibility in Staphylococcus aureus is not so infrequent. Minerva Anestesiol. 2018;84(1):123–4. doi: 10.23736/S0375-9393.17.12153-X 28895381

9. Martinez E, Miro JM, Almirante B, Aguado JM, Fernandez-Viladrich P, Fernandez-Guerrero ML, et al. Effect of penicillin resistance of Streptococcus pneumoniae on the presentation, prognosis, and treatment of pneumococcal endocarditis in adults. Clin Infect Dis. 2002;35(2):130–9. doi: 10.1086/341024 12087518

10. Marshall KJ, Musher DM, Watson D, Mason EO Jr., Testing of Streptococcus pneumoniae for resistance to penicillin. J Clin Microbiol. 1993;31(5):1246–50. 8501225

11. Marchese A, Ramirez M, Schito GC, Tomasz A. Molecular epidemiology of penicillin-resistant Streptococcus pneumoniae isolates recovered in Italy from 1993 to 1996. J Clin Microbiol. 1998;36(10):2944–9. 9738048

12. Braymer JJ, Giedroc DP. Recent developments in copper and zinc homeostasis in bacterial phatogenes. Curr Opin Chem Biol. 2014;19:59–66. doi: 10.1016/j.cbpa.2013.12.021 24463765

13. Fu Y, Chang F-M, J, Giedroc DP. Copper transport and trafficking at the host-bacterial pathogen interface. Acc Chem Res. 2014;47:3605–13. doi: 10.1021/ar500300n 25310275

14. Changela A, Chen K, Holschen J, Outten CE, O'Halloran TV, Mondragon A. Molecular basis of metal-ion selectivity and zeptomolar sensitivity by CueR. Science. 2003;301:1383–7. doi: 10.1126/science.1085950 12958362

15. Rensing C, Fan B, Sharma R, Mitra B, Rosen BP. CopA: An Escherichia coli Cu(I) translocating P-type ATPase. Proc Nat Acad Sci. 2000;97:652–6. doi: 10.1073/pnas.97.2.652 10639134

16. Franke S, Grass G, Rensing C, Nies DH. Molecular analysis of the copper-transporting efflux system CusCFBA of Escherchia coli. J Bacteriol. 2003;185(113):3804–12.

17. Grass G, Rensing C. CueO is a multi-copper oxidase that confers copper tolerance in escherichia coli. BioChem BioPhys Res Commun. 2001;286:902–8. doi: 10.1006/bbrc.2001.5474 11527384

18. Outten FW, Outten CE, Hale JA, O'Halloran TV. Transcriptional activation of an Escherichia coli copper efflux regulon by the chromosomal Mer homologue, CueR. J Biol Chem. 2000;275:31024–9. doi: 10.1074/jbc.M006508200 10915804

19. Robinson NJ, Winge DR. Copper Metallochaperones. Annu Rev Biochem. 2010;79:537–62. doi: 10.1146/annurev-biochem-030409-143539 20205585

20. Tottey S, Harvie DR, Robinson NJ. Understanding how cells allocate metals using metal sensors and metallochaperone. AccChem Res. 2005;38:775–83.

21. Janganan TK, Bavro VN, Zhang L, Matak-Vinkovic D, Barrera NP, Venien-Bryan C, et al. Evidence for the assembly of a bacterial tripartite mutidrug pump with a stoichometry of 3:6:3. J Biol Chem. 2011;286:26900–12. doi: 10.1074/jbc.M111.246595 21610073

22. Su CC, Long F, Zimmermann MT, Rajashankar KR, Jernigan RL, Yu EW. Crystal structure of the CusBA heavy-metal efflux complex of Escherichia coli. Nature. 2011;470(7335):558–62. doi: 10.1038/nature09743 21350490

23. Su C-C, Long F, Lei H-T, Bolla JR, Do SV, Rajashankar KR, et al. Charged amino acids (R83, E567, D617, E625, R669, and K678) of CusA are required for metal ion transport in the Cus Efflux system. J Mol Biol. 2012;422:429–41. doi: 10.1016/j.jmb.2012.05.038 22683351

24. Su C-C, Yang F, Long F, Reyon D, Routh MD, Kuo DW, et al. Crystal structure of the membrane fusion protein CusB from Escherichia coli. J Mol Biol. 2009;393:342–55. doi: 10.1016/j.jmb.2009.08.029 19695261

25. Lei H-T, Bolla JR, Bishop NR, Su C-C, Yu EW. Crystal structures of CusC review conformational changes accompanying folding and transmembrane channel formation. J Mol Biol. 2014;426:403–11. doi: 10.1016/j.jmb.2013.09.042 24099674

26. Padilla-Benavides T, George Thompson AM, McEvoy MM, Arguello JM. Mechanism of ATPase-mediated Cu+ export and delivery to periplasmic chaperones: the interaction of Escherichia coli CopA and CusF. J Biol Chem. 2014;289(30):20492–501. doi: 10.1074/jbc.M114.577668 24917681

27. Chacon KN, Mealman TD, McEvoy MM, Blackburn NJ. Tracking metal ions through a Cu/Ag efflux pump assigns the functional roles of the periplasmic proteins. Proc Nat Acad Sci 2014;111(43):15373–8. doi: 10.1073/pnas.1411475111 25313055

28. Meir A, Natan A, Moskovitz Y, Ruthstein S. EPR spectroscopy identifies Met and Lys rediues that are essential for the interaction between CusB N-terminal domain and the metallochaperone CusF. Metallomics. 2015;7:1163–72. doi: 10.1039/c5mt00053j 25940871

29. Mealman TD, Bagai I, Singh P, Goodlett DR, Rensing C, Zhou H, et al. Interactions between CusF and CusB identified by NMR spectroscopy and chemical cross-linking coupled to mass spectrometry. Biochem. 2011;50:2559–66.

30. Mealman TD, Zhou M, Affandi T, Chacón KN, Aranguren ME, Blackburn NJ, et al. N-Terminal Region of CusB is sufficient for metal binding and metal transfer with the metallochaperone CusF. Biochem. 2012;51:6767–75.

31. Bagai I, Liu W, Rensing C, Blackburn NJ, McEvoy MM. Substrate-linked conformational change in the periplasmic component of a Cu(I)/Ag(I) efflux system. J Biol Chem. 2007;282:35695–702. doi: 10.1074/jbc.M703937200 17893146

32. Ucisik MN, Chakravorty DK, Merz JKM. Structure and dynamics of the N-terminal domain of the Cu(I) binding protein CusB. Biochem. 2013;52:6911–23.

33. Meir A, Abdelhai A, Moskovitz Y, Ruthstein S. EPR spectroscopy targets conformational and topological changes in the E.coli membrane fusion CusB dimer upon Cu(I) binding. Biophys J. 2017;112:2494–502. doi: 10.1016/j.bpj.2017.05.013 28636907

34. Santiago AG, Chen TY, Genova LA, Jung W, George Thompson AM, McEvoy MM, et al. Adaptor protein mediates dynamic pump assembly for bacterial metal efflux. Proc Natl Acad Sci U S A. 2017;114(26):6694–9. doi: 10.1073/pnas.1704729114 28607072

35. D. S. M. E. Dassault Systèmes BIOVIA, Release 2017, San Diego: Dassault Systèmes. 2016.

36. L. Schrödinger, New York, NY Maestro, version 11.0. 2018.

37. Halgren TA, Murphy RB, Friesner RA, Beard HS, Frye LL, Pollard WT, et al. Glide: a new approach for rapid, accurate docking and scoring. 2. Enrichment factors in database screening. J Med Chem. 2004;47(7):1750–9. doi: 10.1021/jm030644s 15027866

38. Friesner RA, Banks JL, Murphy RB, Halgren TA, Klicic JJ, Mainz DT, et al. Glide: a new approach for rapid, accurate docking and scoring. 1. Method and assessment of docking accuracy. J Med Chem. 2004;47(7):1739–49. doi: 10.1021/jm0306430 15027865

39. Dixon SL, Smondyrev AM, Knoll EH, Rao SN, Shaw DE, Friesner RA. PHASE: a new engine for pharmacophore perception, 3D QSAR model development, and 3D database screening: 1. Methodology and preliminary results. J Comput Aided Mol Des. 2006;20(10–11):647–71. doi: 10.1007/s10822-006-9087-6 17124629

40. Phase vNY, NY: Schrodinger, LLC 2012.

41. Saiki RK, Gelfand DH, Stoffel S, Scharf SJ, Higuchi R, Horn GT, et al. Primer directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science. 1988;239:487–91. doi: 10.1126/science.2448875 2448875

42. Don W, Fischer SG, Kirschner MW, Laemmli UK. Peptide mapping by limited proteolysis in sodium dodecyl sulfate and analysis by gel electrophoresis. J Biol Chem. 1971;252:1102–6.

43. Peterson GL. A simplification of the protein assay method of Lowry et al. which is more generally applicable. Anal Biochem. 1997;83:346–56.

44. Jeschke G. Deeranalysis 2006: Distance measurements on nanoscopic length scales by pulse ESR. Hemminga MA, Berliner LJ, editors: Springer; 2007. 287–8 p.

45. Jeschke G. DEER Distance measurements on proteins. Annu Rev Phys Chem. 2012;63:419–46. doi: 10.1146/annurev-physchem-032511-143716 22404592

46. Kahremany S, Zhenin M, Shenberger Y, Maimoun D, Colotti G, Arad M, et al. Peptide-based development of PKA activators. New J Chem. 2018;42(23):18585–97.

47. Pinchas Zer Aviv MS, Yoni Moskovits, Olga Viskind, Amnon Albeck, Didier Vertommen, Sharon Ruthstein, et al. A New Oxopiperazin‐Based Peptidomimetic Molecule Inhibits Prostatic Acid Phosphatase Secretion and Induces Prostate Cancer Cell Apoptosis. Chemistry Select. 2016;1:4658–67.

48. Meir A, Walke G, Schwerdtfeger F, Gevorkyan-Aiapetov L, Ruthstein S. Exploring the role of the various methionine residues in the Escherichia coli CusB adapter protein. Plos One. 2019:0219337.

49. Rensing C, Grass G. Escherichia coli mechanisms of copper homeostasis in a changing environment. FEMS Microbiol Rev. 2003;27(2–3):197–213. doi: 10.1016/S0168-6445(03)00049-4 12829268

50. Mealman TD, Blackburn NJ, McEvoy MM. Metal export by CusCFBA, the periplasmic Cu(I)/Ag(I) transport system of Escherichia coli. Curr Top Membr. 2012;69:163–96. doi: 10.1016/B978-0-12-394390-3.00007-0 23046651

51. Bredeston LM, Gonzalez Flecha FL. The promiscuous phosphomonoestearase activity of Archaeoglobus fulgidus CopA, a thermophilic Cu+ transport ATPase. Biochim Biophys Acta. 2016;1858(7 Pt A):1471–8.

52. Almeida AA, Lopes CM, Silva AM, Barrado E. Trace elements in human milk: correlation with blood levels, inter-element correlations and changes in concentration during the first month of lactation. J Trace Elem Med Biol. 2008;22(3):196–205. doi: 10.1016/j.jtemb.2008.03.007 18755395

53. Ferrini O, Gambaro CG. Studies on water-salts equilibrium in blood and tissue; flame spectrophotometric studies on distribution of water and salts in the blood and muscles in normal rabbit, guinea pig and rat. Arch Maragliano Patol Clin. 1952;7(6):1085–110. 13041458

54. Marx R, Hiller C. Salts in blood and fibrinolysis in human serum. Klin Wochenschr. 1952;30(3–4):71–3. doi: 10.1007/bf01479708 14939657

55. Shehata TE, Marr AG. Effect of nutrient concentration on the growth of Escherichia coli. Journal of bacteriology. 1971;107(1):210–6. 4935320

56. Shiloach J, Kaufman J, Guillard AS, Fass R. Effect of glucose supply strategy on acetate accumulation, growth, and recombinant protein production by Escherichia coli BL21 (lambdaDE3) and Escherichia coli JM109. Biotechnol Bioeng. 1996;49(4):421–8. doi: 10.1002/(SICI)1097-0290(19960220)49:4<421::AID-BIT9>3.0.CO;2-R 18623597

57. Aitha M, Moritz L, Sahu ID, Sanyurah O, Roche Z, McCarrick RM, et al. Conformational dynamics of metallo-b-lactamase CcrA during catalysis investigated by using DEER spectroscopy. J Biol Inorg Chem. 2015;20:585–94. doi: 10.1007/s00775-015-1244-8 25827593

58. Joseph B, Morkhov VM, Yulikov M, Jeschke G, Bordignon E. Conformational cycle of the vitamin B12ABC importer in liposomes detected by Double Electron-Electron Resonance (DEER). J Biol Chem. 2014;289:3176–85. doi: 10.1074/jbc.M113.512178 24362024

59. Sahu ID, McCarrick RM, Troxel KR, Zhang R, Smith HJ, Dunagan MM, et al. DEER EPR measurements for membrane protein structures via bifunctional spin labels for lipodisp nanoparticles. Biochem. 2013;52:6627–32.

60. Klare JP. Site-directed spin labeling EPR spectroscopy in protein research. Biol Chem. 2013;394:1281–300. doi: 10.1515/hsz-2013-0155 23912220

61. Freed DM, Lukasik SM, Sikora A, Mokad A, Cafiso DS. Monomeric TonB and the Ton Box are required for the formation of a high affinity transporter-TonB complex. Biochem. 2013;52:2638–48.

62. Bhatnagar J, Sircar R, Borbat PP, Freed JH, Crane BR. Self-association of the histidine kinase CheA as studied by pulsed dipolar ESR spectroscopy. Biophys J. 2012;102:2192–201. doi: 10.1016/j.bpj.2012.03.038 22824284

63. Yardeni EH, Bahrenberg T, Stein RA, Mishra S, Zomot E, Graham B, et al. Probing the solution structure of the E. coli multidrug transporter MdfA using DEER distance measurements with nitroxide and Gd(III) spin labels. Sci Rep. 2019;9(1):12528. doi: 10.1038/s41598-019-48694-0 31467343

64. Joseph B, Jaumann EA, Sikora A, Barth K, Prisner TF, Cafiso DS. In situ observation of conformational dynamics and protein ligand-substrate interactions in outer-membrane proteins with DEER/PELDOR spectroscopy. Nat Protoc. 2019;14(8):2344–69. d doi: 10.1038/s41596-019-0182-2 31278399

65. Hubbell WL, Gross A, Langen R, Lietzow MA. Recent advances in site-directed spin labeling of proteins. Curr Opin Struc Biol. 1998;8(5):649–56.

66. Hubbell WL, Lopez CJ, Altenbach C, Yang Z. Technological advances in site-directed spin labeling of proteins. Curr Opin Chem Biol. 2013;23:725–33.

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