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Isolation and Characterization of Human μ-Defensin-3, a Novel Human Inducible Peptide Antibiotic*

  • Jürgen Harder
    Affiliations
    Clinical Research Unit, Department of Dermatology, University Hospital Kiel, Schittenhelmstrasse 7, 24105 Kiel, Germany
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  • Joachim Bartels
    Affiliations
    Clinical Research Unit, Department of Dermatology, University Hospital Kiel, Schittenhelmstrasse 7, 24105 Kiel, Germany
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  • Enno Christophers
    Affiliations
    Clinical Research Unit, Department of Dermatology, University Hospital Kiel, Schittenhelmstrasse 7, 24105 Kiel, Germany
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  • Jens-Michael Schröder
    Correspondence
    Supported by the Deutsche Forschungsgemeinschaft. To whom correspondence should be addressed. Tel.: 49-431-5971536; Fax: 49-431-5971611; E-mail: [email protected]
    Affiliations
    Clinical Research Unit, Department of Dermatology, University Hospital Kiel, Schittenhelmstrasse 7, 24105 Kiel, Germany
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  • Author Footnotes
    * This work was supported in part by a CERIES award (to J.-M. S.) and by Deutsche Mukoviszidose e.V.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.The nucleotide and protein sequences reported in this paper have been submitted to the GenBankTM/EBI Data Bank with accession numbers and , respectively.
Open AccessPublished:February 23, 2001DOI:https://doi.org/10.1074/jbc.M008557200
      The growing public health problem of infections caused by multiresistant Gram-positive bacteria, in particularStaphylococcus aureus, prompted us to screen human epithelia for endogenous S. aureus-killing factors. A novel 5-kDa, nonhemolytic antimicrobial peptide (human μ-defensin-3, hBD-3) was isolated from human lesional psoriatic scales and cloned from keratinocytes. hBD-3 demonstrated a salt-insensitive broad spectrum of potent antimicrobial activity against many potentially pathogenic microbes including multiresistant S. aureus and vancomycin-resistant Enterococcus faecium. Ultrastructural analyses of hBD-3-treated S. aureusrevealed signs of cell wall perforation. Recombinant hBD-3 (expressed as a His-Tag-fusion protein in Escherichia coli) and chemically synthesized hBD-3 were indistinguishable from naturally occurring peptide with respect to their antimicrobial activity and biochemical properties. Investigation of different tissues revealed skin and tonsils to be major hBD-3 mRNA-expressing tissues. Molecular cloning and biochemical analyses of antimicrobial peptides in cell culture supernatants revealed keratinocytes and airway epithelial cells as cellular sources of hBD-3. Tumor necrosis factor α and contact with bacteria were found to induce hBD-3 mRNA expression. hBD-3 therefore might be important in the innate epithelial defense of infections by various microorganisms seen in skin and lung, such as cystic fibrosis.P81534AJ237673
      hBD
      human μ-defensin
      TNF-α
      tumor necrosis factor α
      RP
      reversed phase
      HPLC
      high performance liquid chromatography
      Tricine
      N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine
      ESI-MS
      electrospray ionization mass spectrometry
      CFU
      colony-forming units
      RACE
      rapid amplification of cDNA ends
      PCR
      polymerase chain reaction
      RT-PCR
      reverse transcriptase-PCR
      Epithelia of macroorganisms represent the first barrier against invading microorganisms. However, despite constant exposure to these microbial threats, invasive infections and pathological disorders are rather rare and usually locally limited.
      Previous studies have demonstrated that plants and invertebrates produce a set of antimicrobial proteins that are highly effective at killing a wide variety of microorganisms (
      • Boman H.G.
      ). Although vertebrate epithelia are a rich source of antimicrobial proteins (
      • Boman H.G.
      ), it is a very recent observation that human epithelia mount an innate chemical defense by secreting antimicrobial peptides (
      • Schröder J.M.
      ).
      The small (3–5 kDa) cationic defensins represent an important peptide family among antimicrobial peptides. Two subfamilies, the α-defensins and μ-defensins, which are distinguished on the basis of the connectivity of their six cysteine residues, and more recently the cyclic θ-defensin from macaque leukocytes (
      • Tang Y.Q.
      • Yuan J.
      • Osapay G.
      • Osapay K.
      • Tran D.
      • Miller C.J.
      • Ouellette A.J.
      • Selsted M.E.
      ), have been identified in vertebrates (
      • Schröder J.M.
      ). In humans two α-defensins, HD-5 and HD-6, are produced by epithelial granulocytes of the small intestine (
      • Jones D.E.
      • Bevins C.L.
      ,
      • Jones D.E.
      • Bevins C.L.
      ).
      The first μ-defensin was isolated from bovine tongue (
      • Diamond G.
      • Zasloff M.
      • Eck H.
      • Brasseur M.
      • Maloy W.L.
      • Bevins C.L.
      ). Subsequently, 13 novel μ-defensins were purified from bovine neutrophils (
      • Selsted M.E.
      • Tang Y.Q.
      • Morris W.L.
      • McGuire P.A.
      • Novotny M.J.
      • Smith W.
      • Henschen A.H.
      • Cullor J.S.
      ), and the three-dimensional structure, including the disulfide array of one of these μ-defensins, has been determined (
      • Tang Y.Q.
      • Selsted M.E.
      ).
      The first isolated human μ-defensin, human μ-defensin-1 (hBD-1),1 was purified from hemofiltrates (
      • Bensch K.W.
      • Raida M.
      • Magert H.J.
      • Schulz-Knappe P.
      • Forssmann W.G.
      ) and was later found in urine as a Gram-negative bacteria-killing antibiotic (
      • Valore E.V.
      • Park C.H.
      • Quayle A.J.
      • Wiles K.R.
      • McCray Jr., P.B.
      • Ganz T.
      ). mRNA of this antimicrobial peptide is constitutively expressed in various epithelia (
      • Bensch K.W.
      • Raida M.
      • Magert H.J.
      • Schulz-Knappe P.
      • Forssmann W.G.
      ,
      • Valore E.V.
      • Park C.H.
      • Quayle A.J.
      • Wiles K.R.
      • McCray Jr., P.B.
      • Ganz T.
      ,
      • O'Neil D.A.
      • Porter E.M.
      • Elewaut D.
      • Anderson G.M.
      • Eckmann L.
      • Ganz T.
      • Kagnoff M.F.
      ,
      • Goldman M.J.
      • Anderson G.M.
      • Stolzenberg E.D.
      • Kari U.P.
      • Zasloff M.
      • Wilson J.M.
      ,
      • Fulton C.
      • Anderson G.M.
      • Zasloff M.
      • Bull R.
      • Quinn A.G.
      ).
      The second human μ-defensin, hBD-2, was discovered in extracts of lesional scales from patients suffering from psoriasis, a noninfectious proinflammatory and hyperproliferative skin disease (
      • Harder J.
      • Bartels J.
      • Christophers E.
      • Schröder J.M.
      ,
      • Schröder J.M.
      • Harder J.
      ). hBD-2 is expressed in inflamed skin and lung and is induced in epithelial cells upon treatment with TNF-α (
      • Harder J.
      • Bartels J.
      • Christophers E.
      • Schröder J.M.
      ,
      • Harder J.
      • Meyer-Hoffert U.
      • Teran L.M.
      • Schwichtenberg L.
      • Bartels J.
      • Maune S.
      • Schröder J.M.
      ), interleukin-1μ (
      • Harder J.
      • Meyer-Hoffert U.
      • Teran L.M.
      • Schwichtenberg L.
      • Bartels J.
      • Maune S.
      • Schröder J.M.
      ,
      • Singh P.K.
      • Jia H.P.
      • Wiles K.
      • Hesselberth J.
      • Liu L.
      • Conway B.A.
      • Greenberg E.P.
      • Valore E.V.
      • Welsh M.J.
      • Ganz T.
      • Tack B.F.
      • McCray Jr., P.B.
      ), and contact with mucoid forms of Pseudomonas aeruginosa bacteria (
      • Harder J.
      • Meyer-Hoffert U.
      • Teran L.M.
      • Schwichtenberg L.
      • Bartels J.
      • Maune S.
      • Schröder J.M.
      ).
      Both human μ-defensins show microbicidal activity predominantly against Gram-negative bacteria like Escherichia coli andP. aeruginosa. However, they demonstrate only low, if any, microbicidal activity against Gram-positive bacteria such asStaphylococcus aureus (
      • Schröder J.M.
      ,
      • Harder J.
      • Bartels J.
      • Christophers E.
      • Schröder J.M.
      ,
      • Zucht H.D.
      • Grabowsky J.
      • Schrader M.
      • Liepke C.
      • Jurgens M.
      • Schulz-Knappe P.
      • Forssmann W.G.
      ), a bacterium that causes infections ranging from skin abscesses to life-threatening conditions such as endocarditis and toxic shock (
      • Turnidge J.
      • Grayson M.L.
      ).
      Recent investigations revealed that α-defensins also have the ability to attract T cells (
      • Chertov O.
      • Michiel D.F.
      • Xu L.
      • Wang J.M.
      • Tani K.
      • Murphy W.J.
      • Longo D.L.
      • Taub D.D.
      • Oppenheim J.J.
      ). Very recent investigations indicate that human μ-defensins attract immature dendritic cells and memory T cells via the chemokine receptor CCR6 (
      • Yang D.
      • Chertov O.
      • Bykovskaia S.N.
      • Chen Q.
      • Buffo M.J.
      • Shogan J.
      • Anderson M.
      • Schröder J.M.
      • Wang J.M.
      • Howard O.M.
      • et al.
      ), providing a link between innate epithelial defense and adaptive immunity.
      Whereas skin infections caused by Gram-negative bacteria are rather rare, S. aureus is a major cause for skin and lung infections, in particular in atopic dermatitis (
      • Abeck D.
      • Mempel M.
      ). The high abundance of hBD-2 in skin (
      • Schröder J.M.
      • Harder J.
      ) might explain its high resistance against Gram-negative bacterial infection. In contrast, the factors that protect skin from S. aureus infection remain speculative. We therefore hypothesized that human skin produces, in addition to the Gram-negative bacteria-killing hBD-2, peptide antibiotics directed against S. aureus. In the present study, we report the discovery of a novel human epithelial broad spectrum and multiresistant bacteria-killing peptide antibiotic, which we termed human μ-defensin-3 (hBD-3) and which is inducibly expressed by various human epithelial cells.

      DISCUSSION

      It has been previously demonstrated that the epithelia of plants (
      • Broekaert W.F.
      • Cammue B.P.A.
      • DeBolle M.F.C.
      • Thevissen K.
      • De Samblanx G.W.
      • Osborn R.W.
      ), insects (
      • Hoffmann J.A.
      • Hetru C.
      ), amphibians (
      • Barra D.
      • Simmaco M.
      ), and several mammals (
      • Ganz T.
      • Lehrer R.I.
      ) are protected from bacterial infection by a chemical defense shield. The recent isolation of the human epithelial peptide antibiotics hBD-1 (
      • Bensch K.W.
      • Raida M.
      • Magert H.J.
      • Schulz-Knappe P.
      • Forssmann W.G.
      ) and hBD-2 (
      • Harder J.
      • Bartels J.
      • Christophers E.
      • Schröder J.M.
      ) and the demonstration of their expression in major epithelia such as skin (
      • Fulton C.
      • Anderson G.M.
      • Zasloff M.
      • Bull R.
      • Quinn A.G.
      ,
      • Schröder J.M.
      • Harder J.
      ), respiratory tract (
      • Harder J.
      • Meyer-Hoffert U.
      • Teran L.M.
      • Schwichtenberg L.
      • Bartels J.
      • Maune S.
      • Schröder J.M.
      ,
      • Singh P.K.
      • Jia H.P.
      • Wiles K.
      • Hesselberth J.
      • Liu L.
      • Conway B.A.
      • Greenberg E.P.
      • Valore E.V.
      • Welsh M.J.
      • Ganz T.
      • Tack B.F.
      • McCray Jr., P.B.
      ,
      • Bals R.
      • Wang X.
      • Wu Z.
      • Freeman T.
      • Bafna V.
      • Zasloff M.
      • Wilson J.M.
      ,
      • Becker M.N.
      • Diamond G.
      • Verghese M.W.
      • Randell S.H.
      ,
      • McCray Jr., P.B.
      • Bentley L.
      ), urogenital tract (
      • Valore E.V.
      • Park C.H.
      • Quayle A.J.
      • Wiles K.R.
      • McCray Jr., P.B.
      • Ganz T.
      ), and gut (
      • O'Neil D.A.
      • Porter E.M.
      • Elewaut D.
      • Anderson G.M.
      • Eckmann L.
      • Ganz T.
      • Kagnoff M.F.
      ) confirms the hypothesis that human epithelia are similarly protected.
      Although in human secretions such as tears secretory phospholipase A2 may represent one of the most potent Gram-positive bacteria-killing factors (
      • Qu X.D.
      • Lehrer R.I.
      ), no systematic analyses have been performed to elucidate why healthy human skin is protected from S. aureusinfection. Our previous observation that hBD-2 is not bactericidal toward S. aureus (
      • Harder J.
      • Bartels J.
      • Christophers E.
      • Schröder J.M.
      ,
      • Harder J.
      • Meyer-Hoffert U.
      • Teran L.M.
      • Schwichtenberg L.
      • Bartels J.
      • Maune S.
      • Schröder J.M.
      ) prompted us to investigate human skin extracts for S. aureus-killing factor(s). These analyses have led to the purification of a novel peptide antibiotic, which we identified as hBD-3. A very recent data bank search indicated that, upon sequencing of human chromosome 8 bacterial artificial chromosomes, the hBD-3 gene was identified 15,000 base pairs distant from the hBD-2 gene (GenBankTM accession numberAF189745), further supporting the idea that all human μ-defensins are clustered on chromosome 8 (
      • Harder J.
      • Siebert R.
      • Zhang Y.
      • Matthiesen P.
      • Christophers E.
      • Schlegelberger B.
      • Schröder J.M.
      ).
      Although originally purified as an S. aureus-killing peptide antibiotic, our data clearly show that hBD-3 is a broad spectrum peptide antibiotic that kills, at low micromolar concentrations, many other potential pathogenic microbes including P. aeruginosa,S. pyogenes, multiresistant S. aureus, vancomycin-resistant E. faecium, and the yeast C. albicans. The human μ-defensins 1 and 2 are less potent peptide antibiotics and predominantly active against Gram-negative bacteria and yeasts (
      • Harder J.
      • Bartels J.
      • Christophers E.
      • Schröder J.M.
      ,
      • Harder J.
      • Meyer-Hoffert U.
      • Teran L.M.
      • Schwichtenberg L.
      • Bartels J.
      • Maune S.
      • Schröder J.M.
      ,
      • Singh P.K.
      • Jia H.P.
      • Wiles K.
      • Hesselberth J.
      • Liu L.
      • Conway B.A.
      • Greenberg E.P.
      • Valore E.V.
      • Welsh M.J.
      • Ganz T.
      • Tack B.F.
      • McCray Jr., P.B.
      ).
      We were able to express a recombinant hBD-3 fusion protein in E. coli, which to our surprise could be enzymatically cleaved to generate a fully active recombinant version of hBD-3 with biochemical and biological properties indistinguishable from those of the naturally occurring hBD-3 peptide. Only a few reports describe the expression of antimicrobial peptides in bacteria (
      • Piers K.L.
      • Brown M.H.
      • Hancock R.E.
      ), a fact that reflects the difficulties of expressing bactericidal peptides in a bacterial host cell. In addition, correct folding is a general problem in proteins with a high number of cysteine bridges when expressed in bacteria. However, our observation that recombinant as well as chemically synthesized hBD-3 are indistinguishable from natural hBD-3 with respect to their antimicrobial activity and biochemical properties makes it likely that recombinant and synthetic hBD-3 show the same tertiary structure as natural hBD-3, a hypothesis that remains to be proven.
      To elucidate how S. aureus is possibly killed by hBD-3, we examined morphological changes occurring upon hBD-3 treatment of S. aureus by transmission electron microscopy. The morphological effects resemble those seen when S. aureus is treated with penicillin, an antibiotic that interferes with the cross-linking of the bacterial peptidoglycan cell wall (
      • Giesbrecht P.
      • Kersten T.
      • Maidhof H.
      • Wecke J.
      ). Therefore, mechanisms by which hBD-3 affects S. aureus seem to be completely different from those discussed for neutrophil α-defensins, where lamellar mesosome-like structures at the cell membrane level were seen in S. aureus (
      • Shimoda M.
      • Ohki K.
      • Shimamoto Y.
      • Kohashi O.
      ). Striking electron-dense deposits were present in the periplasmic space affixed to the outer membrane when E. coli was investigated (
      • Lehrer R.I.
      • Barton A.
      • Daher K.A.
      • Harwig S.S.
      • Ganz T.
      • Selsted M.E.
      ). It has been suggested that, because of their cationic and amphiphilic characteristics, antimicrobial peptides bind and insert into the cytoplasmic membrane, where they assemble into multimeric pores (
      • White S.H.
      • Wimley W.C.
      • Selsted M.E.
      ). However, a very recent investigation indicates that, at least in the case of the octamer-forming hBD-2, bactericidal activity could also result from electrostatic charge-based mechanisms of membrane permeabilization, rather than a mechanism based on formation of bilayer-spanning pores (
      • Hoover D.M.
      • Rajashankar K.R.
      • Blumenthal R.
      • Puri A.
      • Oppenheim J.J.
      • Chertov O.
      • Lubkowski J.
      ). It remains to be determined whether hBD-3 kills bacteria by a similar mechanism and how hBD-3 affects cell wall perforation in S. aureus. The identification of hBD-3 in normal stratum corneum and the isolation of hBD-3 peptide from culture supernatants revealed skin keratinocytes as a possible cellular source of hBD-3.
      Expression of hBD-3 in epithelial cells was further confirmed by the detection of hBD-3 mRNA in primary keratinocytes as well as in primary respiratory epithelial cells, where we also isolated the protein from culture supernatants. Whereas low hBD-3 mRNA expression was found in many normal epithelial tissues including that of the respiratory tract and genitourinary tract, real-time RT-PCR revealed high levels of hBD-3 mRNA expression in skin and, surprisingly, tonsils. It is interesting to speculate that microbial stimulation is possibly responsible for these findings.
      The isolation of 10- to 30-fold higher amounts of hBD-3 from psoriatic lesions, when compared with normal stratum corneum, indicated that hBD-3 is also inducible by inflammatory stimuli. Like hBD-2 (
      • Schröder J.M.
      • Harder J.
      ,
      • Singh P.K.
      • Jia H.P.
      • Wiles K.
      • Hesselberth J.
      • Liu L.
      • Conway B.A.
      • Greenberg E.P.
      • Valore E.V.
      • Welsh M.J.
      • Ganz T.
      • Tack B.F.
      • McCray Jr., P.B.
      ) and the epithelial bovine μ-defensins LAP and TAP (
      • Diamond G.
      • Bevins C.L.
      ) and unlike hBD-1 (
      • Zhao C.
      • Wang I.
      • Lehrer R.I.
      ), proinflammatory cytokines such as TNF-α induce hBD-3 in primary epithelial cells at physiologically relevant concentrations. Furthermore the contact of epithelial cells with bacteria induces hBD-3 gene expression, a finding that is known for hBD-2 in keratinocytes (
      • Harder J.
      • Bartels J.
      • Christophers E.
      • Schröder J.M.
      ), airway epithelial cells (
      • Harder J.
      • Meyer-Hoffert U.
      • Teran L.M.
      • Schwichtenberg L.
      • Bartels J.
      • Maune S.
      • Schröder J.M.
      ), and intestinal epithelium (
      • O'Neil D.A.
      • Porter E.M.
      • Elewaut D.
      • Anderson G.M.
      • Eckmann L.
      • Ganz T.
      • Kagnoff M.F.
      ). Thus hBD-3 represents the second member of the human μ-defensin family where expression is regulated by inflammatory stimuli at a transcriptional level.
      Several reports indicate that inactivation of antimicrobial peptide activity in patients with cystic fibrosis may contribute to the recurrent airway infections (). Elevated salt concentrations in the airway surface fluid of patients with cystic fibrosis, a matter that has been controversially discussed (), inactivate the antimicrobial activity of human μ-defensins (
      • Goldman M.J.
      • Anderson G.M.
      • Stolzenberg E.D.
      • Kari U.P.
      • Zasloff M.
      • Wilson J.M.
      ,
      • Singh P.K.
      • Jia H.P.
      • Wiles K.
      • Hesselberth J.
      • Liu L.
      • Conway B.A.
      • Greenberg E.P.
      • Valore E.V.
      • Welsh M.J.
      • Ganz T.
      • Tack B.F.
      • McCray Jr., P.B.
      ,
      • Bals R.
      • Wang X.
      • Wu Z.
      • Freeman T.
      • Bafna V.
      • Zasloff M.
      • Wilson J.M.
      ), possibly by inhibiting the binding of positively charged defensins to negatively charged bacterial surfaces. In contrast to both known human μ-defensins (
      • Singh P.K.
      • Jia H.P.
      • Wiles K.
      • Hesselberth J.
      • Liu L.
      • Conway B.A.
      • Greenberg E.P.
      • Valore E.V.
      • Welsh M.J.
      • Ganz T.
      • Tack B.F.
      • McCray Jr., P.B.
      ,
      • Bals R.
      • Wang X.
      • Wu Z.
      • Freeman T.
      • Bafna V.
      • Zasloff M.
      • Wilson J.M.
      ), our findings indicate that the bactericidal activity of hBD-3 is not salt-sensitive at physiologic salt concentrations, which makes this μ-defensin of particular relevance in cystic fibrosis.
      In summary, the discovery of a novel human epithelial broad-spectrum antimicrobial peptide confirms the hypothesis that antimicrobial peptides represent an integral part in the innate immunity of human epithelia (as is found in organisms lacking an adaptive immune system (
      • Hoffmann J.A.
      • Kafatos F.C.
      • Janeway C.A.
      • Ezekowitz R.A.
      )) that complements the adaptive cellular immune system and offers an immediate host response against infectious agents. Finally, the discovery of this human inducible, epithelial antimicrobial peptide may prove to be a vital advance in dealing with skin and respiratory tract infections and in the development of novel strategies for antimicrobial therapy, i.e. by artificial stimulation of epithelial peptide antibiotic synthesis, as recently shown with the amino acid 1-isoleucin (
      • Fehlbaum P.
      • Rao M.
      • Zasloff M.
      • Anderson G.M.
      ).

      Acknowledgments

      We thank J. Quitzau, M. Brandt, R. Rohde, and C. Gerbrecht-Gliessmann for excellent technical assistance. We also thank Dr. Y. Acil and G. Otto for their help with real-time PCR and use of the LightCycler.

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