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.

  • Letter
  • Published:

Reduction of disulphide bonds unmasks potent antimicrobial activity of human β-defensin 1

Abstract

Human epithelia are permanently challenged by bacteria and fungi, including commensal and pathogenic microbiota1,2. In the gut, the fraction of strict anaerobes increases from proximal to distal, reaching 99% of bacterial species in the colon3. At colonic mucosa, oxygen partial pressure is below 25% of airborne oxygen content, moreover microbial metabolism causes reduction to a low redox potential of −200 mV to –300 mV in the colon4. Defensins, characterized by three intramolecular disulphide-bridges, are key effector molecules of innate immunity that protect the host from infectious microbes and shape the composition of microbiota at mucosal surfaces5,6,7,8. Human β-defensin 1 (hBD-1) is one of the most prominent peptides of its class but despite ubiquitous expression by all human epithelia, comparison with other defensins suggested only minor antibiotic killing activity9,10. Whereas much is known about the activity of antimicrobial peptides in aerobic environments, data about reducing environments are limited. Herein we show that after reduction of disulphide-bridges hBD-1 becomes a potent antimicrobial peptide against the opportunistic pathogenic fungus Candida albicans and against anaerobic, Gram-positive commensals of Bifidobacterium and Lactobacillus species. Reduced hBD-1 differs structurally from oxidized hBD-1 and free cysteines in the carboxy terminus seem important for the bactericidal effect. In vitro, the thioredoxin (TRX) system11 is able to reduce hBD-1 and TRX co-localizes with reduced hBD-1 in human epithelia. Hence our study indicates that reduced hBD-1 shields the healthy epithelium against colonisation by commensal bacteria and opportunistic fungi. Accordingly, an intimate interplay between redox-regulation and innate immune defence seems crucial for an effective barrier protecting human epithelia.

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

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

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

Figure 1: hBD-1 shows antimicrobial activity under reducing conditions.
Figure 2: Reduced hBD-1 differs structurally from oxidized hBD-1.
Figure 3: Reduced but not oxidized hBD-1 has a microbicidal effect.
Figure 4: Thioredoxin (TRX) catalyses reduction of oxidized hBD-1 and co-localizes with redhBD-1 in vivo.

Similar content being viewed by others

References

  1. Round, J. L. & Mazmanian, S. K. The gut microbiota shapes intestinal immune responses during health and disease. Nature Rev. Immunol. 9, 313–323 (2009)

    Article  CAS  Google Scholar 

  2. Macpherson, A. J. & Harris, N. L. Interactions between commensal intestinal bacteria and the immune system. Nature Rev. Immunol. 4, 478–485 (2004)

    Article  CAS  Google Scholar 

  3. Hooper, L. V. et al. Molecular analysis of commensal host-microbial relationships in the intestine. Science 291, 881–884 (2001)

    Article  ADS  CAS  Google Scholar 

  4. Wilson, M. in Microbial Inhabitants of Humans Ch. 7 pp. 251–317 (Cambridge University Press, 2005)

    Google Scholar 

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

    Article  ADS  CAS  Google Scholar 

  6. Harder, J., Glaser, R. & Schroder, J. M. Human antimicrobial proteins effectors of innate immunity. J. Endotoxin Res. 13, 317–338 (2007)

    Article  CAS  Google Scholar 

  7. Bevins, C. L. Antimicrobial peptides as effector molecules of mammalian host defense. Contrib. Microbiol. 10, 106–148 (2003)

    Article  CAS  Google Scholar 

  8. Peschel, A. & Sahl, H. G. The co-evolution of host cationic antimicrobial peptides and microbial resistance. Nature Rev. Microbiol. 4, 529–536 (2006)

    Article  CAS  Google Scholar 

  9. Bensch, K. W. et al. hBD-1: a novel β-defensin from human plasma. FEBS Lett. 368, 331–335 (1995)

    Article  CAS  Google Scholar 

  10. Tollin, M. et al. Antimicrobial peptides in the first line defence of human colon mucosa. Peptides 24, 523–530 (2003)

    Article  CAS  Google Scholar 

  11. Arnér, E. S. J. & Holmgren, A. Physiological functions of thioredoxin and thioredoxin reductase. Eur. J. Biochem. 267, 6102–6109 (2000)

    Article  Google Scholar 

  12. Lehrer, R. I. et al. Ultrasensitive assays for endogenous antimicrobial polypeptides. J. Immunol. Methods 137, 167–173 (1991)

    Article  ADS  CAS  Google Scholar 

  13. Ganz, T. & Lehrer, R. I. Defensins. Curr. Opin. Immunol. 6, 584–589 (1994)

    Article  CAS  Google Scholar 

  14. Scudiero, O. et al. Novel synthetic, salt-resistant analogs of human beta-defensins 1 and 3 endowed with enhanced antimicrobial activity. Antimicrob. Agents Chemother. 54, 2312–2322 (2010)

    Article  CAS  Google Scholar 

  15. Taylor, K., Barran, P. E. & Dorin, J. R. Structure-activity relationships in beta-defensin peptides. Biopolymers 90, 1–7 (2008)

    Article  CAS  Google Scholar 

  16. Nuding, S. et al. Antibacterial activity of human defensins on anaerobic intestinal bacterial species: a major role of HBD-3. Microbes Infect. 11, 384–393 (2009)

    Article  CAS  Google Scholar 

  17. Nuding, S. et al. A flow cytometric assay to monitor antimicrobial activity of defensins and cationic tissue extracts. J. Microbiol. Methods 65, 335–345 (2006)

    Article  CAS  Google Scholar 

  18. Holmgren, A. Thioredoxin. Annu. Rev. Biochem. 54, 237–271 (1985)

    Article  CAS  Google Scholar 

  19. Sido, B. et al. Potential role of thioredoxin in immune responses in intestinal lamina propria T lymphocytes. Eur. J. Immunol. 35, 408–417 (2005)

    Article  CAS  Google Scholar 

  20. Holmgren, A. Enzymatic reduction-oxidation of protein disulfides by thioredoxin. Methods Enzymol. 107, 295–300 (1984)

    Article  CAS  Google Scholar 

  21. Tamaki, H. et al. Human thioredoxin-1 ameliorates experimental murine colitis in association with suppressed macrophage inhibitory factor production. Gastroenterology 131, 1110–1121 (2006)

    Article  CAS  Google Scholar 

  22. Nizet, V. & Johnson, R. S. Interdependence of hypoxic and innate immune responses. Nature Rev. Immunol. 9, 609–617 (2009)

    Article  CAS  Google Scholar 

  23. Nagy, E. Anaerobic infections: update on treatment considerations. Drugs 70, 841–858 (2010)

    Article  CAS  Google Scholar 

  24. Ozturk, A., Famili, P. & Vieira, A. R. The antimicrobial peptide DEFB1 is associated with caries. J. Dent. Res. 89, 631–636 (2010)

    Article  CAS  Google Scholar 

  25. Schaefer, A. S. et al. A 3′ UTR transition within DEFB1 is associated with chronic and aggressive periodontitis. Genes Immun. 11, 45–54 (2010)

    Article  CAS  Google Scholar 

  26. Jurevic, R. J. et al. Single-nucleotide polymorphisms (SNPs) in human β-defensin 1: high-throughput SNP assays and association with Candida carriage in type I diabetics and nondiabetic controls. J. Clin. Microbiol. 41, 90–96 (2003)

    Article  CAS  Google Scholar 

  27. Kocsis, A. K. et al. Association of beta-defensin 1 single nucleotide polymorphisms with Crohn’s disease. Scand. J. Gastroenterol. 43, 299–307 (2008)

    Article  CAS  Google Scholar 

  28. Peyrin-Biroulet, L. et al. Peroxisome proliferator-activated receptor gamma activation is required for maintenance of innate antimicrobial immunity in the colon. Proc. Natl Acad. Sci. USA 107, 8772–8777 (2010)

    Article  ADS  CAS  Google Scholar 

  29. Singh, P. K. et al. Production of β-defensins by human airway epithelia. Proc. Natl Acad. Sci. USA 95, 14961–14966 (1998)

    Article  ADS  CAS  Google Scholar 

  30. Schröder, J. M. Purification of antimicrobial peptides from human skin. Methods Mol. Biol. 618, 15–30 (2010)

    Article  Google Scholar 

  31. Schroeder, B. O. & Wehkamp, J. Measurement of antimicrobial activity under reducing conditions in a modified radial diffusion assay. Protocol Exchange 10.1038/protex.2010.204 (2011)

  32. Wu, Z., Schroeder, B. O., Schroeder, J.-M. & Wehkamp, J. Production of recombinant hBD-1 in Escherichla coli and its specific polyclonal antibody in rabbits. Protocol Exchange 10.1038/protex.2010.205 (2011)

Download references

Acknowledgements

We thank M. Katajew, H. Löffler, C. Martensen-Kerl, C. Mehrens, J. Quitzau and A. Rose for technical assistance, B. Fehrenbacher for performing electron microscopy and H.-P. Kreichgauer, C. Schäfer, O. Müller, K. R. Herrlinger and M. Escher for collecting biopsies. Furthermore we thank M. Schwab for discussions and support, C. L. Bevins and J.-M. Schröder for discussions and reading of the manuscript and Ardeypharm GmbH for providing anaerobic bacterial strains and L. Zabel for providing C. albicans strains. This work was supported by Deutsche Forschungsgemeinschaft (WE 436/1-1, SCH 897/1-3 and SFB685) and the Robert-Bosch Foundation (Stuttgart, Germany). J.W. is an Emmy Noether Scholar of Deutsche Forschungsgemeinschaft.

Author information

Authors and Affiliations

Authors

Contributions

B.O.S. performed antimicrobial activity assays, HPLC analyses, MALDI-MS and TRX assays, designed and evaluated experiments, generated figures and wrote the manuscript. Z.W. generated and purified recombinant hBD-1, its 15N-labelled forms and hBD-1-variants, generated alkhBD-1-affinity columns and affinity-purified the red/alkhBD-1-antibody. S.N. performed flow cytometric analyses, S.G. performed NMR spectroscopy and analysed data, M.M. performed CD spectroscopy and analysed data together with J.Bu., J.Be. performed RT-PCR and M.S. was in charge of electron microscopy. E.F.S. and J.W. designed and evaluated experiments and wrote the manuscript. All authors were involved in data discussions and the final version of the manuscript.

Corresponding author

Correspondence to Jan Wehkamp.

Ethics declarations

Competing interests

B.O.S., S.N., E.F.S. and J.W. filed a patent application on the subject of this manuscript.

Supplementary information

Supplementary Information

The file contains Supplementary Figures 1-8 with legends, Supplementary Tables 1-2, Supplementary Methods and additional references. (PDF 1812 kb)

PowerPoint slides

Rights and permissions

Reprints and permissions

About this article

Cite this article

Schroeder, B., Wu, Z., Nuding, S. et al. Reduction of disulphide bonds unmasks potent antimicrobial activity of human β-defensin 1. Nature 469, 419–423 (2011). https://doi.org/10.1038/nature09674

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nature09674

This article is cited by

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

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