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Combating multidrug-resistant Gram-negative bacteria with structurally nanoengineered antimicrobial peptide polymers

Nature Microbiology volume 1, Article number: 16162 (2016) Cite this article

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Abstract

With the recent emergence of reports on resistant Gram-negative ‘superbugs’, infections caused by multidrug-resistant (MDR) Gram-negative bacteria have been named as one of the most urgent global health threats due to the lack of effective and biocompatible drugs. Here, we show that a class of antimicrobial agents, termed ‘structurally nanoengineered antimicrobial peptide polymers’ (SNAPPs) exhibit sub-μM activity against all Gram-negative bacteria tested, including ESKAPE and colistin-resistant and MDR (CMDR) pathogens, while demonstrating low toxicity. SNAPPs are highly effective in combating CMDR Acinetobacter baumannii infections in vivo, the first example of a synthetic antimicrobial polymer with CMDR Gram-negative pathogen efficacy. Furthermore, we did not observe any resistance acquisition by A. baumannii (including the CMDR strain) to SNAPPs. Comprehensive analyses using a range of microscopy and (bio)assay techniques revealed that the antimicrobial activity of SNAPPs proceeds via a multimodal mechanism of bacterial cell death by outer membrane destabilization, unregulated ion movement across the cytoplasmic membrane and induction of the apoptotic-like death pathway, possibly accounting for why we did not observe resistance to SNAPPs in CMDR bacteria. Overall, SNAPPs show great promise as low-cost and effective antimicrobial agents and may represent a weapon in combating the growing threat of MDR Gram-negative bacteria.

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Figure 1: Synthesis of SNAPPs.
Figure 2: In vivo efficacy of SNAPP S16 in a mouse peritonitis model.
Figure 3: Optical Microscope eXperimental 3D-SIM images of E. coli before and after treatment with AF488-tagged SNAPP S16 in MHB.
Figure 4: Morphological studies of E. coli before and after treatment with S16 in MHB.
Figure 5: A comparison between the antimicrobial mechanism(s) of typical membrane-disrupting cationic AMPs and the possible mechanism of SNAPPs against Gram-negative bacteria.

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References

  1. World Health Organization. Antimicrobial Resistance: Global Report on Surveillance 2014 (WHO, 2014).

  2. Rice, L. B. Federal funding for the study of antimicrobial resistance in nosocomial pathogens: no ESKAPE. J. Infect. Dis. 197, 1079–1081 (2008).

    Article  PubMed  Google Scholar 

  3. Rice, L. B. Progress and challenges in implementing the research on ESKAPE pathogens. Infect. Control Hosp. Epidemiol. 31, S7–S10 (2010).

    Article  PubMed  Google Scholar 

  4. Taubes, G. The bacteria fight back. Science 321, 356–361 (2008).

    Article  CAS  PubMed  Google Scholar 

  5. Xu, Z., Flavin, M. T. & Flavin, J. Combating multidrug-resistant Gram-negative bacterial infections. Exp. Opin. Invest. Drugs 23, 163–182 (2014).

    Article  CAS  Google Scholar 

  6. Lee, J. H., Jeong, S. H., Cha, S. & Lee, S. H. A lack of drugs for antibiotic-resistant Gram-negative bacteria. Nat. Rev. Drug Discov. 6, 29–40 (2007). doi:10.1038/nrd2201-c1.

    Article  Google Scholar 

  7. Le Moual, H. & Gruenheid, S. Resistance to antimicrobial peptides in Gram-negative bacteria. FEMS Microbiol. Lett. 330, 81–89 (2012).

    Article  PubMed  Google Scholar 

  8. Brogden, K. A. Antimicrobial peptides: pore formers or metabolic inhibitors in bacteria? Nat. Rev. Microbiol. 3, 238–250 (2005).

    Article  CAS  PubMed  Google Scholar 

  9. Kohanski, M. A., Dwyer, D. J. & Collins, J. J. How antibiotics kill bacteria: from targets to networks. Nat. Rev. Microbiol. 8, 423–435 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Zasloff, M. Antimicrobial peptides of multicellular organisms. Nature 415, 389–395 (2002).

    Article  CAS  PubMed  Google Scholar 

  11. Melo, M. N., Dugourd, D. & Castanho, M. A. Omiganan pentahydrochloride in the front line of clinical applications of antimicrobial peptides. Recent Pat. Antiinfect. Drug Discov. 1, 201–207 (2006).

    Article  CAS  PubMed  Google Scholar 

  12. Hancock, R. E. W. & Sahl, H. Antimicrobial and host-defense peptides as new anti-infective therapeutic strategies. Nat. Biotechnol. 24, 1551–1557 (2006).

    Article  CAS  PubMed  Google Scholar 

  13. Zhou, C. et al. High potency and broad-spectrum antimicrobial peptides synthesized via ring-opening polymerization of α-amino acid-N-carboxyanhydrides. Biomacromolecules 11, 60–67 (2010).

    Article  CAS  PubMed  Google Scholar 

  14. Engler, A. C. et al. Effects of side group functionality and molecular weight on the activity of synthetic antimicrobial polypeptides. Biomacromolecules 12, 1666–1674 (2011).

    Article  CAS  PubMed  Google Scholar 

  15. Sulistio, A., Blencowe, A., Widjaya, A., Zhang, X. & Qiao, G. G. Development of functional amino acid-based star polymers. Polym. Chem. 3, 224–234 (2012).

    Article  CAS  Google Scholar 

  16. Sulistio, A., Widjaya, A., Blencowe, A., Zhang, X. & Qiao, G. G. Star polymers composed entirely of amino acid building blocks: a route towards stereospecific, biodegradable and hierarchically functionalized stars. Chem. Commun. 47, 1151–1153 (2011).

    Article  CAS  Google Scholar 

  17. Wu, W., Wang, W. & Li, J. Star polymers: advances in biomedical applications. Prog. Polym. Sci. 46, 55–85 (2015).

    Article  CAS  Google Scholar 

  18. Lam, S. J. et al. Peptide-based star polymers as potential siRNA carriers. Aust. J. Chem. 67, 592–597 (2014).

    Article  CAS  Google Scholar 

  19. Byrne, M. et al. Molecular weight and architectural dependence of well-defined star-shaped poly(lysine) as a gene delivery vector. Biomater. Sci. 1, 1223–1234 (2013).

    Article  CAS  PubMed  Google Scholar 

  20. Sulistio, A. et al. Folic acid conjugated amino acid-based star polymers for active targeting of cancer cells. Biomacromolecules 12, 3469–3477 (2011).

    Article  CAS  PubMed  Google Scholar 

  21. Nederberg, F. et al. Biodegradable nanostructures with selective lysis of microbial membranes. Nat. Chem. 3, 409–414 (2011).

    Article  CAS  PubMed  Google Scholar 

  22. Liu, L. et al. Self-assembled cationic peptide nanoparticles as an efficient antimicrobial agent. Nat. Nanotech. 4, 457–463 (2009).

    Article  CAS  Google Scholar 

  23. Ng, V. W. L., Ke, X., Lee, A. L. Z., Hedrick, J. L. & Yang, Y.-Y. Synergistic co-delivery of membrane-disrupting polymers with commercial antibiotics against highly opportunistic bacteria. Adv. Mater. 25, 6730–6736 (2013).

    Article  CAS  PubMed  Google Scholar 

  24. Campos, M. A. et al. Capsule polysaccharide mediates bacterial resistance to antimicrobial peptides. Infect. Immun. 72, 7107–7114 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Llobet, E., Tomas, J. M. & Bengoechea, J. A. Capsule polysaccharide is a bacterial decoy for antimicrobial peptides. Microbiology 154, 3877–3886 (2008).

    Article  CAS  PubMed  Google Scholar 

  26. Melo, M. N., Ferre, R. & Castanho, M. A. R. B. Antimicrobial peptides: linking partition, activity and high membrane-bound concentrations. Nat. Rev. Microbiol. 7, 245–250 (2009).

    Article  CAS  PubMed  Google Scholar 

  27. Choi, H., Chakraborty, S., Liu, R., Gellman, S. H. & Weisshaar, J. C. Medium effects on minimum inhibitory concentrations of nylon-3 polymers against E. coli. PLoS ONE 9, e104500 (2014).

    Article  PubMed  PubMed Central  Google Scholar 

  28. Davis, S. D. Activity of gentamicin, tobramycin, polymyxin B, and colistimethate in mouse protection tests with Pseudomonas aeruginosa. Antimicrob. Agents Chemother. 8, 50–53 (1975).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Velkov, T. et al. Teaching ‘old’ polymyxins new tricks: new-generation lipopeptides targeting Gram-negative ‘superbugs’. ACS Chem. Biol. 9, 1172–1177 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Li, W. et al. Proline-rich antimicrobial peptides: potential therapeutics against antibiotic-resistant bacteria. Amino Acids 46, 2287–2294 (2014).

    Article  CAS  PubMed  Google Scholar 

  31. Hilchie, A. L., Wuerth, K. & Hancock, R. E. W. Immune modulation by multifaceted cationic host defense (antimicrobial) peptides. Nat. Chem. Biol. 9, 761–768 (2013).

    Article  CAS  PubMed  Google Scholar 

  32. Lee, H. J. et al. Synergistic activity of colistin and rifampin combination against multidrug-resistant Acinetobacter baumannii in an in vitro pharmacokinetic/pharmacodynamic model. Antimicrob. Agents Chemother. 57, 3738–3745 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Benincasa, M., Pacor, S., Gennaro, R. & Scocchi, M. Rapid and reliable detection of antimicrobial peptide penetration into Gram-negative bacteria based on fluorescence quenching. Antimicrob. Agents Chemother. 53, 3501–3504 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Alves, C. S. et al. Escherichia coli cell surface perturbation and disruption induced by antimicrobial peptides BP100 and pepR. J. Biol. Chem. 285, 27536–27544 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Li, W. et al. Multimerization of a proline-rich antimicrobial peptide, Chex-Arg20, alters its mechanism of interaction with the Escherichia coli membrane. Chem. Biol. 22, 1250–1258 (2015).

    Article  CAS  PubMed  Google Scholar 

  36. Fjell, C. D., Hiss, J. A., Hancock, R. E. W. & Schneider, G. Designing antimicrobial peptides: form follows function. Nat. Rev. Drug Discov. 11, 37–51 (2012).

    Article  CAS  Google Scholar 

  37. Freire, J. M. et al. Monitoring antibacterial permeabilization in real time using time-resolved flow cytometry. Biochim. Biophys. Acta. 1848, 554–560 (2015).

    Article  CAS  PubMed  Google Scholar 

  38. Morein, S., Andersson, A., Rilfors, L. & Lindblom, G. Wild-type Escherichia coli cell regulate the membrane lipid composition in a ‘window’ between gel and non-lamellar structures. J. Biol. Chem. 271, 6801–6809 (1996).

    Article  CAS  PubMed  Google Scholar 

  39. Elie, C. R., Hebert, A., Charbonneau, M., Haiun, A. & Schmitzer, A. R. Benzimidazolium-based synthetic chloride and calcium transporters in bacterial membranes. Org. Biomol. Chem. 11, 923–928 (2013).

    Article  CAS  PubMed  Google Scholar 

  40. Erental, A., Sharon, I. & Engelberg-Kulka, H. Two programmed cell death systems in Escherichia coli: apoptotic-like death is inhibited by the mazEF-mediated death pathway. PLoS Biol. 10, e1001281 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Bayles, K. W. Bacterial programmed cell death: making sense of a paradox. Nat. Rev. Microbiol. 12, 63–69 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Erental, A., Kalderon, Z., Saada, A., Smith, Y. & Engelberg-Kulka, H. Apoptosis-like death, an extreme SOS response in Escherichia coli. mBio 5, e01426–14 (2014).

    Article  PubMed  PubMed Central  Google Scholar 

  43. Henriques, S. T., Melo, M. N. & Castanho, M. A. R. B. Cell-penetrating peptides and antimicrobial peptides: how different are they? Biochem. J. 399, 1–7 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Henriques, S. T., Melo, M. N. & Castanho, M. A. R. B. How to address CPP and AMP translocation? Methods to detect and quantify peptide internalization in vitro and in vivo. Mol. Membr. Biol. 24, 173–184 (2007).

    Article  CAS  PubMed  Google Scholar 

  45. Hancock, R. E. W. & Chapple, D. S. Peptide antibiotics. Antimicrob. Agents Chemother. 43, 1317–1323 (1999).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Sato, H. & Feix, J. B. Peptide–membrane interactions and mechanisms of membrane destruction by amphipathic α-helical antimicrobial peptides. Biochim. Biophys. Acta Biomembranes 1758, 1245–1256 (2006).

    Article  CAS  Google Scholar 

  47. Morones-Ramirez, J. R., Winkler, J. A., Spina, C. S. & Collins, J. J. Silver enhances antibiotic activity against Gram-negative bacteria. Sci. Transl. Med. 5, 190ra81 (2013).

    Article  PubMed  PubMed Central  Google Scholar 

  48. Gullberg, E. et al. Selection of resistant bacteria at very low antibiotic concentrations. PLoS Pathogens 7, e1002158 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Siegel, R. E. Emerging Gram-negative antibiotic resistance: daunting challenges declining sensitivities, and dire consequences. Respir. Care 53, 471–479 (2008).

    PubMed  Google Scholar 

  50. Sani, M.-A. et al. Maculatin 1.1 disrupts Staphylococcus aureus lipid membranes via a pore mechanism. Antimicrob. Agents Chemother. 57, 3593–3600 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. Moritz, E. M. & Hergenrother, P. J. Toxin–antitoxin systems are ubiquitous and plasmid-encoded in vancomycin-resistant enterococci. Proc. Natl Acad. Sci. USA 104, 311–316 (2007).

    Article  CAS  PubMed  Google Scholar 

  52. Livak, K. J. & Schmittgen, T. D. Analysis of relative gene expression data using real-time quantitative PCR and the 2(−ΔΔC(T)) method. Methods 25, 402–408 (2001).

    Article  CAS  PubMed  Google Scholar 

  53. Chen, Y.-Y. et al. The outer membrane protein LptO is essential for O-deacylation of LPS and the co-ordinated secretion and attachment of A-LPS and CTD proteins in Porphyromonas gingivalis. Mol. Microbiol. 79, 1380–1401 (2011).

    Article  CAS  PubMed  Google Scholar 

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Acknowledgements

G.G.Q. acknowledges financial support from the Australian Research Council under the Future Fellowship (FT110100411) scheme. E.C.R. acknowledges financial support from the Australian Government, Department of Industry, Innovation and Science. S.J.L. acknowledges the Australian Government for providing an International Postgraduate Research Scholarship (IPRS) and an Australian Postgraduate Award (APAInt). The authors thank the Advanced Fluorescence Imaging Platform at the Materials Characterisation and Fabrication Platform (The University of Melbourne) for instrument access. The authors thank B. Hibbs for assistance with the Delta Vision OMX V4 BLAZE, S. Lowe for technical laboratory assistance and J. Li for the CMDR bacterial strains.

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Authors and Affiliations

  1. Department of Chemical & Biomolecular Engineering, Polymer Science Group, The University of Melbourne, Parkville, 3010, Victoria, Australia

    Shu J. Lam, Adrian Sulistio, Edgar H. H. Wong, Anton Blencowe & Greg G. Qiao

  2. Melbourne Dental School and The Bio21 Institute of Molecular Science and Biotechnology, Oral Health CRC, The University of Melbourne, Parkville, 3010, Victoria, Australia

    Neil M. O'Brien-Simpson, Namfon Pantarat, Yu-Yen Chen, Jason C. Lenzo, James A. Holden & Eric C. Reynolds

  3. Division of Health Sciences, School of Pharmacy and Medical Sciences, The University of South Australia, Adelaide, 5000, South Australia, Australia

    Anton Blencowe

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Contributions

G.G.Q., N.M.O'B.-S., A.B. and E.C.R. oversaw the project. S.J.L. synthesized and characterized the polymers, performed the in vitro and imaging experiments and wrote the paper, with intellectual input from E.H.H.W., A.S. and A.B. N.P. contributed to the in vitro experiments. J.C.L. contributed to the in vivo animal models and immune cell phenotyping and in vitro experiments. J.A.H. contributed to the RT-PCR and in vitro experiments. Y.-Y.C. contributed to the cryo-TEM experiments. All authors gave suggestions to improve the presentation of the paper.

Corresponding authors

Correspondence to Eric C. Reynolds or Greg G. Qiao.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Information

Supplementary Materials and Instrumentation, Supplementary Figures 1–36, legends for Supplementary Videos 1 and 2, Supplementary Tables 1–11, Supplementary References. (PDF 5572 kb)

Supplementary Video 1

A sample 3D view of an E. coli cell that demonstrates the membrane association of SNAPPs after treatment. (MOV 1499 kb)

Supplementary Video 2

A sample 3D view of an E. coli cell that demonstrates the cell internalization of SNAPPs after treatment. (MOV 1648 kb)

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Lam, S., O'Brien-Simpson, N., Pantarat, N. et al. Combating multidrug-resistant Gram-negative bacteria with structurally nanoengineered antimicrobial peptide polymers. Nat Microbiol 1, 16162 (2016). https://doi.org/10.1038/nmicrobiol.2016.162

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