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

Journal name:
Nature
Volume:
469,
Pages:
419–423
Date published:
DOI:
doi:10.1038/nature09674
Received
Accepted
Published online

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 −200mV to –300mV 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.

At a glance

Figures

  1. hBD-1 shows antimicrobial activity under reducing conditions.
    Figure 1: hBD-1 shows antimicrobial activity under reducing conditions.

    a, b, Bifidobacterium adolescentis was incubated with up to 2mM DTT under anaerobic conditions with hBD-1 (a) or hBD-3 (b). Inhibition zones were measured and statistically evaluated using student’s t-test with *P<0.05, **P<0.01, ***P<0.001. Representative radial diffusion assays of three experiments are shown. Data are presented as means, error bars indicate standard deviation. c, Comparison of inhibition zones between hBD-1 and hBD-3 in growth medium without or with 2mM DTT. Dotted lines at 2.5mm represent base line diameter of punched wells.

  2. Reduced hBD-1 differs structurally from oxidized hBD-1.
    Figure 2: Reduced hBD-1 differs structurally from oxidized hBD-1.

    a, hBD-1 (1µg) was incubated with different concentrations of DTT, alkylated and analysed by MALDI-MS. b, hBD-1 (1µg) was incubated with different concentrations of DTT and analysed by RP-HPLC. oxhBD-1, oxidized hBD-1; redhBD-1, reduced hBD-1. c, Nuclear magnetic resonance (NMR) analysis of hBD-1. Superimposed 1H-15N-heteronuclear single quantum coherence (HSQC) spectra of oxidized (black) and reduced (red) hBD-1. δ1-15N, chemical shifts of 15N nuclei; δ2-1H, chemical shifts of protons. d, Circular dichroism spectroscopy of oxidized (black) and reduced (red) hBD-1. ΘMRW, molar ellipticity normalized to mean amino acid residue weight.

  3. Reduced but not oxidized hBD-1 has a microbicidal effect.
    Figure 3: Reduced but not oxidized hBD-1 has a microbicidal effect.

    a, b, oxhBD-1 and redhBD-1 (1.5µg) were incubated with bifidobacteria (a) and lactobacilli (3µg defensin), Bacteroides vulgatus and Escherichia coli (b). B. adol., B. adolescentis; L. acid., Lactobacillus acidophilus. c, Flow cytometric antimicrobial killing assay of C. albicans incubated with reduced (red) or oxidized (black) hBD-1. Data are presented as mean±s.e.m. of two independent experiments each done in duplicates. d, RP-HPLC of oxidized and reduced hBD-1 and alanine/serine variants. e, Flow cytometric antimicrobial killing assay of B. adolescentis incubated with reduced (red) or oxidized (black) hBD-1 for 30 (squares) or 90 (triangles) minutes. Results are presented as mean±s.e.m. of two independent experiments each done in duplicates. f, Transmission electron microscopy of B. adolescentis incubated with oxidized hBD-1, without defensin (control), reduced hBD-1 or with hBD-3. Scale bars, 200nm.

  4. Thioredoxin (TRX) catalyses reduction of oxidized hBD-1 and co-localizes with redhBD-1 in vivo.
    Figure 4: Thioredoxin (TRX) catalyses reduction of oxidized hBD-1 and co-localizes with redhBD-1 in vivo.

    a, Oxidized hBD-1 was incubated with human thioredoxin, rat thioredoxin reductase and NADPH. Incubation mixtures were analysed by RP-HPLC. b, Immunohistochemical analysis of reduced/alkylated hBD-1 (red/alkhBD-1; left), thioredoxin (TRX, centre) and pre-immune IgG fraction (right). Staining of human colon (upper row), ileum (middle row) and skin sections (bottom row) are shown. In the pre-immune IgG-treated skin (bottom row, right), brownish cells in the basal epidermal layer are melanocytes. Scale bars, 50µm.

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Author information

Affiliations

  1. Dr. Margarete Fischer-Bosch-Institute of Clinical Pharmacology, 70376 Stuttgart, Germany

    • Bjoern O. Schroeder,
    • Sabine Nuding,
    • Julia Beisner &
    • Jan Wehkamp
  2. University of Tübingen, 72076 Tübingen, Germany

    • Bjoern O. Schroeder,
    • Sabine Nuding,
    • Julia Beisner &
    • Jan Wehkamp
  3. Department of Dermatology, University Hospital Schleswig-Holstein, Campus Kiel, 24105 Kiel, Germany

    • Zhihong Wu
  4. Department 1 Protein Evolution, Max Planck Institute for Developmental Biology, 72076 Tübingen, Germany

    • Sandra Groscurth
  5. Center for Integrated Protein Science Munich, Department Chemie, Technische Universität München, 85747 Garching, Germany

    • Moritz Marcinowski &
    • Johannes Buchner
  6. Department of Dermatology, University Hospital Tübingen, 72076 Tübingen, Germany

    • Martin Schaller
  7. Department of Internal Medicine I, Robert Bosch Hospital, 70376 Stuttgart, Germany

    • Eduard F. Stange &
    • Jan Wehkamp
  8. Present address: Bruker BioSpin AG, 8117 Fällanden, Switzerland.

    • Sandra Groscurth

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.

Competing financial interests

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

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