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.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

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.

References

  1. 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)

  2. 2

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

  3. 3

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

  4. 4

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

  5. 5

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

  6. 6

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

  7. 7

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

  8. 8

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

  9. 9

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

  10. 10

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

  11. 11

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

  12. 12

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

  13. 13

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

  14. 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)

  15. 15

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

  16. 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)

  17. 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)

  18. 18

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

  19. 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)

  20. 20

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

  21. 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)

  22. 22

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

  23. 23

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

  24. 24

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

  25. 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)

  26. 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)

  27. 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)

  28. 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)

  29. 29

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

  30. 30

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

  31. 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. 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

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.

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

Further reading

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.