Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Protocol Extension
  • Published:

Assessing biofilm inhibition and immunomodulatory activity of small amounts of synthetic host defense peptides synthesized using SPOT-array technology

Nature Protocols volume 16, pages 1850–1870 (2021)Cite this article

Subjects

Abstract

Peptides are promising drug candidates because of their diversity, biocompatibility and spectrum of activities. Here, we describe a protocol for high-throughput screening of SPOT-peptide arrays to assess the antibiofilm, antimicrobial and immunomodulatory activities of synthetic peptides. It is a Protocol Extension of our previous Nature Protocols article, which describes the synthesis of SPOT-peptide arrays and assays for screening antimicrobial activity. This latest protocol allows the simultaneous assessment of hundreds of synthetic host defense peptides to define their overall activity profiles and identify candidate sequences that are suitable for further characterization and development as anti-infectives. When coupled with the SPOT-array technology for peptide synthesis, the described procedures are rapid, inexpensive and straightforward for peptide library screening. The protocols can be implemented in most microbiology or immunology research laboratories without the need for specialists. The time to complete each step ranges between 1 and 4 h with overnight pauses, and datasets related to the antibiofilm and immunomodulatory activities of a large set of peptide sequences can be generated in a few days.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Fig. 1: Diagram of the steps of the biofilm inhibition screening assay.
Fig. 2: Schematic diagram of the experimental setup to screen the immunomodulatory activity of the SPOT peptides.
Fig. 3: Biofilm inhibition activities of 96 different single amino acid substitution variants of the HDP 1018 synthesized on a SPOT array.
Fig. 4: Example results from the screening of SPOT peptides for their immunomodulatory activity.

Similar content being viewed by others

Data availability

Data presented in this protocol (dataset for Figs. 3 and 4) are available in the supporting primary research articles (refs. 26,27), and raw data are available from the corresponding author upon request.

References

  1. US Centers for Disease Control and Prevention. Antibiotic Resistance Threats in the United States, 2019. https://www.cdc.gov/drugresistance/pdf/threats-report/2019-ar-threats-report-508.pdf (2019).

  2. Brown, E. D. & Wright, G. D. Antibacterial drug discovery in the resistance era. Nature 529, 336–343 (2016).

    Article  CAS  PubMed  Google Scholar 

  3. Hancock, R. E. W. The end of an era? Nat. Rev. Drug Discov. 6, 28 (2007).

    Article  CAS  Google Scholar 

  4. MacLean, R. C. & San Millan, A. The evolution of antibiotic resistance. Science 365, 1082–1083 (2019).

    Article  CAS  PubMed  Google Scholar 

  5. O’Neill, J. Antimicrobial Resistance: Tackling a Crisis for the Health and Wealth of Nations. https://amr-review.org/sites/default/files/AMR%20Review%20Paper%20-%20Tackling%20a%20crisis%20for%20the%20health%20and%20wealth%20of%20nations_1.pdf (2014).

  6. The World Bank Group. Drug-Resistant Infections: A Threat to Our Economic Future. https://www.worldbank.org/en/topic/health/publication/drug-resistant-infections-a-threat-to-our-economic-future (2017).

  7. Rudd, K. E. et al. Global, regional, and national sepsis incidence and mortality, 1990–2017: analysis for the global burden of disease study. Lancet 395, 200–211 (2020).

    Article  PubMed  PubMed Central  Google Scholar 

  8. Harrison, J. J., Ceri, H. & Turner, R. J. Multimetal resistance and tolerance in microbial biofilms. Nat. Rev. Microbiol. 5, 928–938 (2007).

    Article  CAS  PubMed  Google Scholar 

  9. Koo, H., Allan, R. N., Howlin, R. P., Stoodley, P. & Hall-Stoodley, L. Targeting microbial biofilms: current and prospective therapeutic strategies. Nat. Rev. Microbiol. 15, 740–755 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Flemming, H. C. et al. Biofilms: an emergent form of bacterial life. Nat. Rev. Microbiol. 14, 563–575 (2016).

    Article  CAS  PubMed  Google Scholar 

  11. Mookherjee, N., Anderson, M. A., Haagsman, H. P. & Davidson, D. J. Antimicrobial host defence peptides: functions and clinical potential. Nat. Rev. Drug Discov. 19, 311–332 (2020).

    Article  CAS  PubMed  Google Scholar 

  12. Hancock, R. E. W., Haney, E. F. & Gill, E. E. The immunology of host defence peptides: beyond antimicrobial activity. Nat. Rev. Immunol. 16, 321–334 (2016).

    Article  CAS  PubMed  Google Scholar 

  13. Etayash, H., Azmi, S., Dangeti, R. & Kaur, K. Peptide bacteriocins—structure activity relationships. Curr. Top. Med. Chem. 16, 220–241 (2015).

    Article  PubMed  CAS  Google Scholar 

  14. Zhang, R. et al. Structure-function relationships of antimicrobial peptides and proteins with respect to contact molecules on pathogen surfaces. Curr. Top. Med. Chem. 16, 89–98 (2016).

    Article  CAS  PubMed  Google Scholar 

  15. de la Fuente-Nunez, C., Reffuveille, F., Haney, E. F., Straus, S. K. & Hancock, R. E. W. Broad-spectrum anti-biofilm peptide that targets a cellular stress response. PLoS Pathog. 10, e1004152 (2014).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  16. de la Fuente-Nunez, C., Cardoso, M. H., de Souza Candido, E., Franco, O. L. & Hancock, R. E. W. Synthetic antibiofilm peptides. Biochim. Biophys. Acta 1858, 1061–1069 (2016).

    Article  PubMed  CAS  Google Scholar 

  17. Hancock, R. E. W., Nijnik, A. & Philpott, D. J. Modulating immunity as a therapy for bacterial infections. Nat. Rev. Microbiol. 10, 243–254 (2012).

    Article  CAS  PubMed  Google Scholar 

  18. 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 

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

    Article  CAS  PubMed  Google Scholar 

  20. Yeaman, M. R. & Yount, N. Y. Unifying themes in host defence effector polypeptides. Nat. Rev. Microbiol. 5, 727–740 (2007).

    Article  CAS  PubMed  Google Scholar 

  21. Etayash, H., Pletzer, D., Kumar, P., Straus, S. K. & Hancock, R. E. W. Cyclic derivative of host-defense peptide IDR-1018 improves proteolytic stability, suppresses inflammation, and enhances in vivo activity. J. Med. Chem 63, 9228–9236 (2020).

    Article  CAS  PubMed  Google Scholar 

  22. Mookherjee, N. et al. Intracellular receptor for human host defense peptide LL-37 in monocytes. J. Immunol. 183, 2688–2696 (2009).

    Article  CAS  PubMed  Google Scholar 

  23. Yu, H. B. et al. Sequestosome-1/p62 is the key intracellular target of innate defense regulator peptide. J. Biol. Chem. 284, 36007–36011 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Choi, K. Y., Chow, L. N. & Mookherjee, N. Cationic host defence peptides: multifaceted role in immune modulation and inflammation. J. Innate Immun. 4, 361–370 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  25. Haney, E. F., Straus, S. K. & Hancock, R. E. W. Reassessing the host defense peptide landscape. Front. Chem. 7, 43 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Haney, E. F. et al. Computer-aided discovery of peptides that specifically attack bacterial biofilms. Sci. Rep. 8, 1871 (2018).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  27. Haney, E. F., Mansour, S. C., Hilchie, A. L., de la Fuente-Nunez, C. & Hancock, R. E. W. High throughput screening methods for assessing antibiofilm and immunomodulatory activities of synthetic peptides. Peptides 71, 276–285 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Haney, E. F., Barbosa, S. C., Baquir, B. & Hancock, R. E. W. Influence of non-natural cationic amino acids on the biological activity profile of innate defense regulator peptides. J. Med. Chem. 62, 10294–10304 (2019).

    Article  CAS  PubMed  Google Scholar 

  29. Hilpert, K., Winkler, D. F. & Hancock, R. E. W. Peptide arrays on cellulose support: SPOT synthesis, a time and cost efficient method for synthesis of large numbers of peptides in a parallel and addressable fashion. Nat. Protoc. 2, 1333–1349 (2007).

    Article  CAS  PubMed  Google Scholar 

  30. Rabin, N. et al. Agents that inhibit bacterial biofilm formation. Future Med. Chem. 7, 647–671 (2015).

    Article  CAS  PubMed  Google Scholar 

  31. Haney, E. F., Trimble, M. J., Cheng, J. T., Valle, Q. & Hancock, R. E. W. Critical assessment of methods to quantify biofilm growth and evaluate antibiofilm activity of host defence peptides. Biomolecules 8, 29 (2018).

    Article  PubMed Central  CAS  Google Scholar 

  32. Münzker, L., Oddo, A. & Hansen, P. R. Chemical synthesis of antimicrobial peptides. Methods Mol. Biol. 1548, 35–49 (2017).

    Article  PubMed  CAS  Google Scholar 

  33. Kimmerlin, T. & Seebach, D. ‘100 years of peptide synthesis’: ligation methods for peptide and protein synthesis with applications to beta-peptide assemblies. J. Pept. Res. 65, 229–260 (2005).

    Article  CAS  PubMed  Google Scholar 

  34. Fields, G. B. Introduction to peptide synthesis. Curr. Protoc. Mol. Biol. Ch. 11, Unit 11.15 (2002).

  35. Süssmuth, R. D. & Mainz, A. Nonribosomal peptide synthesis-principles and prospects. Angew Chem. Int. Ed. Engl. 56, 3770–3821 (2017).

    Article  PubMed  CAS  Google Scholar 

  36. Jaradat, D. M. M. Thirteen decades of peptide synthesis: key developments in solid phase peptide synthesis and amide bond formation utilized in peptide ligation. Amino Acids 50, 39–68 (2018).

    Article  CAS  PubMed  Google Scholar 

  37. Stawikowski, M. & Fields, G. B. Introduction to peptide synthesis. Curr. Protoc. Protein Sci. Ch. 18, Unit 18.11 (2012).

  38. Coin, I., Beyermann, M. & Bienert, M. Solid-phase peptide synthesis: from standard procedures to the synthesis of difficult sequences. Nat. Protoc. 2, 3247–3256 (2007).

    Article  CAS  PubMed  Google Scholar 

  39. Behrendt, R., White, P. & Offer, J. Advances in Fmoc solid-phase peptide synthesis. J. Pept. Sci. 22, 4–27 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Mäde, V., Els-Heindl, S. & Beck-Sickinger, A. G. Automated solid-phase peptide synthesis to obtain therapeutic peptides. J. Org. Chem. 10, 1197–1212 (2014).

    Google Scholar 

  41. Merrifield, R. B. Solid-phase peptide synthesis. Adv. Enzymol. Relat. Areas Mol. Biol. 32, 221–296 (1969).

    CAS  PubMed  Google Scholar 

  42. da Silva, A. Jr. et al. Avian anticoccidial activity of a novel membrane-interactive peptide selected from phage display libraries. Mol. Biochem. Parasitol. 120, 53–60 (2002).

    Article  PubMed  Google Scholar 

  43. Pini, A. et al. Antimicrobial activity of novel dendrimeric peptides obtained by phage display selection and rational modification. Antimicrob. Agents Chemother. 49, 2665–2672 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Ashby, M., Petkova, A., Gani, J., Mikut, R. & Hilpert, K. Use of peptide libraries for identification and optimization of novel antimicrobial peptides. Curr. Top Med. Chem. 17, 537–553 (2017).

    Article  CAS  PubMed  Google Scholar 

  45. Winkler, D. F., Hilpert, K., Brandt, O. & Hancock, R. E. W. Synthesis of peptide arrays using SPOT-technology and the CelluSpots-method. Methods Mol. Biol. 570, 157–174 (2009).

    Article  CAS  PubMed  Google Scholar 

  46. Winkler, D. F., Andresen, H. & Hilpert, K. SPOT synthesis as a tool to study protein-protein interactions. Methods Mol. Biol. 723, 105–127 (2011).

    Article  CAS  PubMed  Google Scholar 

  47. Kaur, K., Ahmed, S., Soudy, R. & Azmi, S. Screening peptide array library for the identification of cancer cell-binding peptides. Methods Mol. Biol. 1248, 239–247 (2015).

    Article  CAS  PubMed  Google Scholar 

  48. Soudy, R., Ahmed, S. & Kaur, K. NGR peptide ligands for targeting CD13/APN identified through peptide array screening resemble fibronectin sequences. ACS Comb. Sci. 14, 590–599 (2012).

    Article  CAS  PubMed  Google Scholar 

  49. Bluhm, M. E., Knappe, D. & Hoffmann, R. Structure-activity relationship study using peptide arrays to optimize Api137 for an increased antimicrobial activity against Pseudomonas aeruginosa. Eur. J. Med. Chem. 103, 574–582 (2015).

    Article  CAS  PubMed  Google Scholar 

  50. Knappe, D. et al. Optimization of oncocin for antibacterial activity using a SPOT synthesis approach: extending the pathogen spectrum to Staphylococcus aureus. Amino Acids 48, 269–280 (2016).

    Article  CAS  PubMed  Google Scholar 

  51. Ommen, P., Zobek, N. & Meyer, R. L. Quantification of biofilm biomass by staining: non-toxic safranin can replace the popular crystal violet. J. Microbiol. Methods 141, 87–89 (2017).

    Article  CAS  PubMed  Google Scholar 

  52. Toté, K., Vanden Berghe, D., Maes, L. & Cos, P. A new colorimetric microtitre model for the detection of Staphylococcus aureus biofilms. Lett. Appl. Microbiol. 46, 249–254 (2008).

    Article  PubMed  Google Scholar 

  53. Stiefel, P. et al. Is biofilm removal properly assessed? Comparison of different quantification methods in a 96-well plate system. Appl. Microbiol. Biotechnol. 100, 4135–4145 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  54. Harrison, J. J. et al. Microtiter susceptibility testing of microbes growing on peg lids: a miniaturized biofilm model for high-throughput screening. Nat. Protoc. 5, 1236–1254 (2010).

    Article  CAS  PubMed  Google Scholar 

  55. Amsen, D., de Visser, K. E. & Town, T. Approaches to determine expression of inflammatory cytokines. Methods Mol. Biol. 511, 107–142 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  56. Huang, X. et al. Effect of arginine on the growth and biofilm formation of oral bacteria. Arch. Oral Biol. 82, 256–262 (2017).

    Article  CAS  PubMed  Google Scholar 

  57. Chen, P., Abercrombie, J. J., Jeffrey, N. R. & Leung, K. P. An improved medium for growing Staphylococcus aureus biofilm. J. Microbiol. Methods 90, 115–118 (2012).

    Article  CAS  PubMed  Google Scholar 

  58. Bowdish, D. M. et al. Impact of LL-37 on anti-infective immunity. J. Leukoc. Biol. 77, 451–459 (2005).

    Article  CAS  PubMed  Google Scholar 

  59. Tomita, T. et al. Effect of ions on antibacterial activity of human beta defensin 2. Microbiol. Immunol. 44, 749–754 (2000).

    Article  CAS  PubMed  Google Scholar 

  60. Hammond, A., Dertien, J., Colmer-Hamood, J. A., Griswold, J. A. & Hamood, A. N. Serum inhibits P. aeruginosa biofilm formation on plastic surfaces and intravenous catheters. J. Surg. Res. 159, 735–746 (2010).

    Article  CAS  PubMed  Google Scholar 

  61. Wiegand, I., Hilpert, K. & Hancock, R. E. W. Agar and broth dilution methods to determine the minimal inhibitory concentration (MIC) of antimicrobial substances. Nat. Protoc. 3, 163–175 (2008).

    Article  CAS  PubMed  Google Scholar 

  62. Metcalf, T. U. et al. Global analyses revealed age-related alterations in innate immune responses after stimulation of pathogen recognition receptors. Aging Cell 14, 421–432 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  63. Ter Horst, R. et al. Host and environmental factors influencing individual human cytokine responses. Cell 167, 1111–1124.e13 (2016).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  64. Li, Y. et al. A functional genomics approach to understand variation in cytokine production in humans. Cell 167, 1099–1110.e14 (2016).

    Article  CAS  PubMed  Google Scholar 

  65. Schirmer, M., Kumar, V., Netea, M. G. & Xavier, R. J. The causes and consequences of variation in human cytokine production in health. Curr. Opin. Immunol. 54, 50–58 (2018).

    Article  CAS  PubMed  Google Scholar 

  66. Wu, B. C., Lee, A. H. & Hancock, R. E. W. Mechanisms of the innate defense regulator peptide-1002 anti-inflammatory activity in a sterile inflammation mouse model. J. Immunol. 199, 3592–3603 (2017).

    Article  CAS  PubMed  Google Scholar 

  67. Haney, E. F. et al. Identification of an IDR peptide formulation candidate that prevents peptide aggregation and retains immunomodulatory activity. Pept. Sci. (Hoboken) 111, e24077 (2019).

    Article  CAS  Google Scholar 

  68. Martikainen, M. V. & Roponen, M. Cryopreservation affected the levels of immune responses of PBMCs and antigen-presenting cells. Toxicol. In Vitro 67, 104918 (2020).

    Article  CAS  PubMed  Google Scholar 

  69. Yang, J. et al. The effects of storage temperature on PBMC gene expression. BMC Immunol. 17, 6 (2016).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  70. Li, X., Zhong, Z., Liang, S., Wang, X. & Zhong, F. Effect of cryopreservation on IL-4, IFNγ and IL-6 production of porcine peripheral blood lymphocytes. Cryobiology 59, 322–326 (2009).

    Article  CAS  PubMed  Google Scholar 

  71. Darveau, R. P. & Hancock, R. E. Procedure for isolation of bacterial lipopolysaccharides from both smooth and rough Pseudomonas aeruginosa and Salmonella typhimurium strains. J. Bacteriol. 155, 831–838 (1983).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

We acknowledge the financial assistance of the Canadian Institutes of Health Research, Foundation grant FDN-154287 to R.E.W.H. for funding peptide research in our laboratory. H.E. is the recipient of a UBC Killam Fellowship and a Research Trainee Award from the Michael Smith Foundation for Health Research. R.E.W.H. is a Canada Research Chair in Health and Genomics and holds a UBC Killam Professorship.

Author information

Authors and Affiliations

  1. Centre for Microbial Diseases and Immunity Research, Department of Microbiology and Immunology, University of British Columbia, Vancouver, British Columbia, Canada

    Hashem Etayash, Evan F. Haney & Robert E. W. Hancock

Authors
  1. Hashem Etayash
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Evan F. Haney
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Robert E. W. Hancock
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

H.E. and E.F.H. wrote the protocol. H.E. drew the illustrations in Figs. 1 and 2. R.E.W.H. extensively edited the article.

Corresponding author

Correspondence to Robert E. W. Hancock.

Ethics declarations

Competing interests

E.F.H. and R.E.W.H. have filed patents related to the antibiofilm and immunomodulatory functions of synthetic HDPs. These patents have been assigned to their employer, the University of British Columbia, and licensed to ABT Innovations Inc., in which R.E.W.H. has an ownership position.

Additional information

Peer review information Nature Protocols thanks Donald Davidson, Sam Walker and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Related links

Key references using this protocol

Haney, E. F. et al. Sci. Rep. 8, 1871 (2018): https://doi.org/10.1038/s41598-018-19669-4

Haney, E. F., Mansour, S. C., Hilchie, A. L., de la Fuente-Nunez, C. & Hancock, R. E. W. Peptides 71, 276–285 (2015): https://doi.org/10.1016/j.peptides.2015.03.015

Haney, E. F., Barbosa, S. C., Baquir, B. & Hancock, R. E. W. J. Med. Chem. 62, 10294–10304 (2019): https://doi.org/10.1021/acs.jmedchem.9b01344

This protocol is an extension to: Nat. Protoc. 2, 1333–1349 (2007): https://doi.org/10.1038/nprot.2007.160

Supplementary information

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Etayash, H., Haney, E.F. & Hancock, R.E.W. Assessing biofilm inhibition and immunomodulatory activity of small amounts of synthetic host defense peptides synthesized using SPOT-array technology. Nat Protoc 16, 1850–1870 (2021). https://doi.org/10.1038/s41596-021-00500-w

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41596-021-00500-w

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing