If you don't remember your password, you can reset it by entering your email address and clicking the Reset Password button. You will then receive an email that contains a secure link for resetting your password
If the address matches a valid account an email will be sent to __email__ with instructions for resetting your password
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
* 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.
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 (
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 (
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 (
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 (
), 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 (
) 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.
EXPERIMENTAL PROCEDURES
Culture of Epithelial Cells
Foreskin-derived keratinocytes, airway epithelial cells, and the A549 lung epithelial cells were prepared and cultured as described previously (
). Supernatants of the cells stimulated for 48 h with 108/ml heat-killed (65 °C, 45 min) P. aeruginosa (clinical isolate) in fetal calf serum-free medium (bacteria-to-cell ratio of 200:1) were collected for purification of antimicrobial factors. For stimulation and subsequent RNA isolation, primary keratinocytes and tracheal and bronchial cells were cultured in 6-well tissue culture plates (9.6 cm2/well, Falcon). Second passage cells were used at 70–80% confluence. After removal of growth medium and two washes with phosphate-buffered saline, cells were cultured in keratinocyte growth medium (Clonetics) lacking bovine pituitary extract for 24 h and were subsequently stimulated with recombinant TNF-α (Pepro Tech Inc.) or heat-killed (65 °C, 30 min) bacteria in 2 ml of serum-free growth medium.
Purification and Characterization of hBD-3
Pooled lesional psoriatic scales (7–50 g) or heel calluses (80–120 g) were extracted with acidic ethanolic citrate buffer as described previously (
). After diafiltration (Amicon filters; cut off, 3 kDa) of extracts (or the supernatants of cultured epithelial cells) against sodium phosphate buffer (10 mm, pH 7.4), material was applied to an S. aureus affinity column, which was prepared using anN-hydroxy-succinimide-activated Sepharose column (HiTrap, 5 ml; Amersham Pharmacia Biotech) and 5 ml of anS. aureus (clinical isolate) suspension (109bacteria/ml) by a procedure similar to that previously described for aP. aeruginosa affinity column (
). Briefly, extracts or cell culture supernatants were applied to the affinity column that had been previously equilibrated with 10 mm phosphate buffer, pH 7.4, and bound peptides were eluted with 0.1 m glycine buffer, pH 3.0, containing 1 m NaCl. After equilibration of the column with 10 mm phosphate buffer, pH 7.4, the effluent was applied to the column, and bound material was eluted as described above. This step was performed four times to increase the efficacy of the column to bind peptides. The eluates were collected and diafiltered against 0.1% trifluoroacetic acid, pH 3, for subsequent RP-HPLC.
S. aureus affinity column-bound material was then purified by a preparative wide pore RP-8-HPLC column (300 × 7 mm, C8 Nucleosil, Macherey & Nagel) that was previously equilibrated with 0.1% (v/v) trifluoroacetic acid in HPLC-grade water containing 20% (v/v) acetonitrile. Proteins were eluted with a gradient of increasing concentrations of acetonitrile containing 0.1% (v/v) trifluoroacetic acid (flow rate, 2 ml/min). Aliquots (10–30 μl) of each fraction were lyophilized, dissolved in 5 μl of 0.1% (v/v) aqueous acetic acid, and tested for antimicrobial activity againstS. aureus or E. coli by a radial diffusion plate assay (
Fractions containing antimicrobial activity against S. aureus were further purified by cation exchange HPLC followed by RP-18-HPLC as described for purification of hBD-2 (
). Electrophoretic mobility was investigated using SDS-polyacrylamide gels (SDS-polyacrylamide gel electrophoresis) in the presence of 8m urea and Tricine (
Protein sequencing was done using a pulsed liquid phase 776 automated protein sequencer (PerkinElmer Life Sciences). Electrospray ionization mass spectrometry (ESI-MS) analyses were performed in the positive ionization mode with a QTOF-II Hybrid mass spectrometer (Micromass).
Antimicrobial/Hemolytic Assay
Test organisms were incubated with hBD-3 in 100 μl of 10 mm sodium phosphate buffer (pH 7.4) containing 1% (v/v) trypticase soy broth. To investigate the salt sensitivity of hBD-3, 50 μg of hBD-3 was incubated with 1 × 105 colony-forming units (CFU) of S. aureus(ATCC 6538) in 100 μl of 10 mm sodium phosphate buffer (pH 7.4) and NaCl for 3 h at 37 °C. The antibiotic activity of hBD-3 was analyzed by plating serial dilutions of the incubation mixture and determination of the CFU the following day. The limit of detection (1 colony per plate) was equal to 1 × 102CFU per ml.
For analysis of hemolytic activity, up to 500 μg of hBD-3 were incubated with 1 × 109/ml human erythrocytes at 37 °C for 3 h either in 10 mm sodium phosphate buffer (pH 7.4) containing 0.34 m sucrose or only in phosphate-buffered saline. Following incubation samples were centrifuged at 10,000 × g for 10 min, and hemolysis was determined by measuring the A450 of the supernatants using 0.1% Triton X-100 for 100% hemolysis.
Transmission Electron Microscopy of Bacteria
Approximately 108 CFU of S. aureus cells (ATCC 6538) were treated with hBD-3 (500 μg/ml) in 100 μl of sodium phosphate buffer (pH 7.4) containing 1% (v/v) trypticase soy broth for various lengths of time (30–180 min) at 37 °C. The bacteria were then centrifuged (5000 × g, 5 min), immersed in cold (4 °C) 5% phosphate-buffered glutaraldehyde (pH 7.8) for 2 h, repeatedly rinsed in cold phosphate buffer, and postfixed for a further 2 h in 4% phosphate-buffered osmic acid. The sample was dehydrated in acetone and finally embedded in Araldit (Araldit Cy212, Sigma), as described previously (
). Bacteria were examined with an EM 910 electron microscope (Zeiss).
Cloning of hBD-3 cDNA from Airway Epithelial Cells and Keratinocytes
Total RNA obtained from primary human foreskin-derived keratinocytes and tracheal epithelial cells was reverse-transcribed using standard reagents (Life Technologies, Inc.). A 3′-RACE strategy (
) was used to amplify an hBD-3-specific sequence from the cDNA. Two degenerate primers (5′-GGIATHATHAAYACIYTICARAA-3′ and 5′-CCTAARGARGARCARATHGG-3′) were designed based on hBD-3 amino acid sequence data and used as sense primers for 3′-RACE. The amplified products were subcloned and sequenced. Isolation of the full-length cDNA was achieved using a 5′-RACE system for rapid amplification of cDNA ends (Life Technologies, Inc.) according to the manufacturer's protocol.
Analysis of hBD-3 Gene Expression
Real-time RT-PCR analyses were performed in a fluorescence temperature cycler (LightCycler, Roche Molecular Biochemicals) according to the manufacturer's instructions. This technique continuously monitors the cycle-by-cycle accumulation of fluorescently labeled PCR product. Briefly, total RNA from cultured epithelial cells was isolated using TRIzol reagent (Life Technologies, Inc.), and 2 μg of total RNA was reverse-transcribed using standard reagents (Life Technologies, Inc.). The cDNA corresponding to 50 ng of RNA served as a template in a 20-μl reaction containing 4 mm MgCl2, 0.5 μm each primer, and 1× LightCycler-FastStart DNA Master SYBR Green I mix (Roche Molecular Biochemicals). Samples were loaded into capillary tubes and incubated in the fluorescence thermocycler (LightCycler) for an initial denaturing at 95 °C for 10 min, followed by 45 cycles, each cycle consisting of 95 °C for 15 s, 60 °C for 5 s, and 72 °C for 10 s. SYBR Green I fluorescence was detected at 86 °C at the end of each cycle to monitor the amount of PCR product formed during that cycle. At the end of each run, melting curve profiles were produced (cooling the sample to 65 °C for 15 s and then heating slowly at 0.2 °C/s up to 95 °C with continuous measurement of fluorescence) to confirm amplification of specific transcripts. The sequences of the hBD-3-specific intron-spanning primers were 5′-AGCCTAGCAGCTATGAGGATC-3′ (forward primer) and 5′-CTTCGGCAGCATTTTGCGCCA-3′ (reverse primer). Amplification using these primers resulted in a 206-base pair fragment. The sequences of the μ-actin primers were 5′-CTCCTTAATGTCACGCAGGATTTC-3′ (forward primer) and 5′-GTGGGGCGCCCCAGGCACCA-3′ (reverse primer), and amplification using these primers resulted in a 520-base pair fragment. Cycle-to-cycle fluorescence emission readings were monitored and analyzed using LightCycler Software (Roche Molecular Biochemicals). The software first normalizes each sample by detecting the background fluorescence present in the initial cycles. Then a fluorescence threshold at 5% of full scale is set, and the software determines the cycle number at which each sample reached this threshold. This threshold fluorescence cycle number correlates inversely to the log of the initial template concentration. Relative hBD-3 transcript levels were corrected by normalization based on the μ-actin transcript levels. The specificity of the amplification products was further verified by subjecting the amplification products to electrophoresis on a 2% agarose gel. The fragments were visualized by ethidium bromide staining, and the specificity of hBD-3-encoding PCR products was verified by sequencing.
For determination of hBD-3 mRNA in different tissues, total RNA was isolated from human skin, larynx, pharynx, polyp, tonsil, and tongue using the TRIzol reagent (Life Technologies, Inc.). All other RNAs were obtained from CLONTECH. Real-time RT-PCR was carried out as described above.
Expression of Recombinant hBD-3 in E. coli
The cDNA encoding the 45 amino acid-containing natural form of hBD-3 was cloned into the expression vector pET-30c (Novagen), which contains an NH2-terminal His-Tag sequence allowing purification of the fusion protein by the use of a nickel affinity column. A 200-ml culture of transformed E. coli (strain BL21, Novagen) was grown to an optical density of 0.6, and expression was induced by adding 1 mmisopropyl-1-thio-μ-d-galactopyranoside. Expression was carried out for 4 h, and bacteria were harvested by centrifugation at 6000 × g for 5 min and lysed by sonication. Extracts were purified with a nickel affinity column (Novagen) according to the manufacturer's protocol (Novagen) (
). Bound material was digested with enterokinase (Invitrogen), and the released 45 amino acid-containing form of hBD-3 was further purified by micro-reversed phase (RP-18) HPLC, eluting with a retention time at 25 min, identical to that of natural hBD-3. Tricine-SDS-polyacrylamide gel electrophoresis revealed a single band migrating as natural hBD-3. The identity of recombinant hBD-3 was confirmed by NH2-terminal amino acid sequencing and by ESI-MS analyses.
Synthetic hBD-3
hBD-3 was chemically synthesized by JERINI BIO TOOLS GMBH, Berlin, Germany, according to the amino acid sequence deduced from the cDNA sequence. The material eluted in a single peak upon RP-HPLC with a retention time at 25 min, identical to that of natural hBD-3. ESI-MS analyses revealed a mass of 5154.7 Da.
RESULTS
Isolation of a Novel Human Peptide Antibiotic: hBD-3
To address the question whether human skin produces S. aureus-killing proteins, we analyzed lesional scale extracts of patients with psoriasis and heel callus extracts from healthy persons for S. aureus-killing activity. Initial experiments revealed high S. aureus-killing activity in crude psoriatic scale extracts as well as in heel stratum corneum extracts. To enrich and purify staphylocidal activity from psoriatic scale extracts, in which more staphylocidal activity was observed than in heel callus extracts, an S. aureus affinity column was used. Protein(s) with microbicidal activity directed against S. aureus were found to bind to the column. Bound proteins were then separated by preparative reversed phase C8 HPLC, and HPLC fractions were analyzed for staphylocidal activity (Fig.1A). The most prominent staphylocidal activity-containing HPLC fraction was further purified using micro-cation exchange HPLC. Staphylocidal activity eluted at high salt concentration from this column, indicating a highly basic antimicrobial peptide (Fig. 1B, arrow). Final purification of this antibiotic peptide was achieved by reversed phase C2/C18 HPLC (Fig. 1C). Tricine-SDS-urea-polyacrylamide gel electrophoretic analyses revealed a single line migrating like a 9-kDa polypeptide (Fig. 1C,inset). NH2-terminal amino acid sequence analyses gave the sequence shown in Fig. 1D(GenBankTM accession number P81534), which indicated a new human antimicrobial peptide. Using degenerated primers the complementary DNA (cDNA) was isolated from primary keratinocytes. The cDNA (GenBankTMaccession number AJ237673)
After we had submitted the protein sequence (P81534) and the cDNA sequence (AJ237673) for hBD-3, sequencing of a chromosome 8 bacterial artificial chromosome clone (GenBankTM accession number AF189745) revealed the presence of a nucleotide sequence encoding a putative μ-defensin-like protein identical to hBD-3. Thereafter, two cDNA sequences were available in the GenBankTM/EBI Data Bank that encode hBD-3 (accession numbers AF217245 and AF295370).
encodes a 67-amino acid precursor, and the predicted 45 amino acid-containing mature peptide shows similarity to vertebrate epithelial μ-defensins, in particular bovine “enteric μ -defensin” (Fig.2). Because this novel antimicrobial peptide is the third isolated human μ-defensin, it was termed human μ-defensin-3 (hBD-3). By electrospray mass spectrometry, its exact molecular mass was found to be 5154.59 Da, which is 6 Da less than the mass calculated from the deduced hBD-3 amino acid sequence (5161.20 Da), supporting the idea that hBD-3 contains three cysteine bridges and the amino acid sequence shown in Fig. 2.
Figure 1Identification and purification of hBD-3.S. aureus affinity column-bound proteins of lesional psoriatic scale extracts were separated by RP-8-HPLC (A), and the fraction containing high titer antimicrobial activity (arrow) was purified to homogeneity by micro-cation exchange HPLC (B) followed by analytical C2/C18 RP-HPLC (C). Tricine-SDS-urea-polyacrylamide gel electrophoresis of the resulting peak and silver staining revealed a single band migrating as a 9-kDa peptide (C, inset). NH2-terminal amino acid sequence of 25 residues (single letter code; X, not identified) revealed a novel human antimicrobial peptide (D) (GenBankTM/EBI Data Bank accession numberP81534).
Figure 2Peptide sequence of hBD-3. The deduced amino acid sequence (single-letter code) of the native hBD-3 peptide based on the complementary DNA sequence obtained from human keratinocytes and tracheal epithelia cells is shown. For comparison, amino acid sequences of the human μ-defensins hBD-1 and hBD-2, bovine epithelial μ-defensins TAP, LAP, and EBD bovine neutrophil μ-defensin BNBD-12, as well as the μ-defensin consensus sequence (including the putative disulfide bridges) are aligned. (The dashes in the μ-defensin sequences represent gaps due to the alignment.) The complete cDNA sequence of hBD-3 has been submitted to the GenBankTM/EBI Data Bank with accession number AJ237673.
We were able to isolate 88 μg of pure hBD-3 from 7 g of psoriatic scales and 15 μg of hBD-3 from 112 g of human skin-derived stratum corneum. The recovery of hBD-3 peptide after three HPLC purification steps was found to be very low. Losses were estimated to be in the range of 80–95% of the quantity originally present.
hBD-3 peptide could be expressed as a His-Tag fusion protein inE. coli. Cleavage of the fusion protein, which was found to be weakly active against E. coli, with enterokinase and subsequent RP-HPLC analysis led to a single peptide having a molecular mass of 5154.2 Da by ESI-MS, which is exactly the mass calculated for full-length hBD-3. Antimicrobial activity against E. coli(Fig. 3B) and S. aureus was found to be equivalent to that seen for natural hBD-3. Synthetic hBD-3 gave a single peak by RP-HPLC at the same retention time as natural hBD-3 and gave a molecular mass of 5154.7 Da upon ESI-MS analyses. Interestingly, synthetic hBD-3 also demonstrated the same antimicrobial activity as natural hBD-3 (Fig. 3B).
Figure 3Antimicrobial/hemolytic activity of hBD-3. For analysis of antimicrobial activity, hBD-3 was incubated for 3 h at 37 °C in 100 μl of 10 mm sodium phosphate buffer (pH 7.4) containing 1% trypticase soy broth and the indicated concentrations of hBD-3. To determine the number of CFU, serial dilutions were plated, and colony counts were performed the following day. For analysis of hemolytic activity, hBD-3 was incubated at 37 °C for 3 h with 1 × 109/ml human erythrocytes either in 10 mm sodium phosphate buffer (pH 7.4) containing 0.34 m sucrose (closed boxes) or only in phosphate-buffered saline (open boxes). Hemolysis was determined by measuring the A450 of the supernatants using 0.1% Triton X-100 for 100% hemolysis. Panel B shows that natural, recombinant, and chemically synthesized hBD-3 exhibit identical antimicrobial activity. All investigations shown in A and C were performed with synthetic hBD-3.
hBD-3 Exhibits Salt-insensitive Broad Spectrum Antimicrobial Activity and No Hemolytic Activity
Analyses of the in vitro antimicrobial properties of hBD-3 revealed antimicrobial activity against several potential pathogenic Gram-positive bacteria (S. aureus and Streptococcus pyogenes) as well as Gram-negative bacteria (P. aeruginosa and E. coli) and the yeastCandida albicans (Fig. 3A). Furthermore, hBD-3 kills multiresistant S. aureus and vancomycin-resistantEnterococcus faecium at similar low concentrations (Fig.3A). When S. aureus was treated at higher cell densities of 8 × 105 cells/ml, we observed slightly higher killing concentrations (the hBD-3 concentration necessary to kill 90% bacteria was 4 μg/ml) than we found when 8 × 104 cells/ml S. aureus were used (the hBD-3 concentration necessary to kill 90% bacteria was 2.5 μg/ml).
S. aureus was killed by hBD-3 at low and physiologic salt concentrations (Fig. 3C). Reduced antimicrobial activity was only observed at supraphysiologic salt concentrations.
Because several cationic antimicrobial peptides have been reported to exhibit cytotoxic activity against eukaryotic cells, hBD-3 was also assayed for hemolytic activity against human erythrocytes. No significant hemolytic activity (<0.5%) was observed using concentrations of hBD-3 up to 500 μg/ml at physiologic salt concentrations. However, significant hemolytic activity was seen at high hBD-3 concentrations in 10 mm sodium phosphate buffer containing 0.34 m sucrose (Fig. 3A).
Ultrastructure of hBD-3-killed S. aureus
To develop an insight into the mechanisms by which S. aureus is possibly killed by hBD-3, we examined the morphological changes of S. aureus exposed to hBD-3 by transmission electron microscopy. As shown in Fig. 4, S. aureusshows signs of perforation of the peripheral cell wall, with explosion-like liberation of the plasma membrane within 30 min. After 2 h most cells undergo bacteriolysis with different degrees of cellular disintegration.
Figure 4Morphology of hBD-3-treated S. aureus. Transmission electron micrographs of S. aureus (108 cells/ml) incubated in 10 mm phosphate buffer for 2 h (A) or treated with synthetic hBD-3 (500 μg/ml) for 30 min (B) or 2 h (C and D) are shown. Bars represent 0.1μm.
To investigate the tissue distribution of hBD-3 mRNA expression, we analyzed mRNA obtained from various body sites by real-time RT-PCR. Low or no hBD-3 mRNA expression was seen in most of the analyzed organs including the respiratory tract, gastrointestinal tract, and genitourinary tract, whereas strong expression was detected in skin and tonsils (Fig.5A).
Figure 5Tissue expression of hBD-3 mRNA. Low hBD-3 mRNA expression (analyzed by real-time RT-PCR) was detected in many tissues (A). Normal skin and tonsils showed the highest hBD-3 transcript level. (n.d., not detected.) hBD-3 mRNA is expressed in cultivated human primary keratinocytes (B) or primary tracheal epithelial cells (C) and is up-regulated by treatment of the cells with heat-inactivated bacteria (108 cells/ml) or TNF-α (10 ng/ml) for 6 h. The mucoid clinical isolate of P. aeruginosa proved to be the strongest inducer of hBD-3. Bars represent the relative hBD-3 transcript levels normalized to μ-actin transcript levels.
To investigate the cellular origin of hBD-3, we first analyzed cultured primary keratinocytes as well as respiratory tract epithelial cells for hBD-3 mRNA expression. As shown in Fig. 5B, primary keratinocytes express hBD-3 mRNA at a low level. Similarly, we found hBD-3 mRNA expression at a low level in primary tracheal (Fig. 5C), nasal, and bronchial airway epithelial cells.
We next assessed whether inflammatory stimuli up-regulate the expression of the hBD-3 gene in epithelial cells. TNF-α induced hBD-3 gene expression in primary keratinocytes (Fig. 5B) as well as in primary tracheal epithelial cells (Fig. 5C) at physiologically relevant concentrations. In addition, the contact of keratinocytes or primary tracheal epithelial cells with heat-inactivated Gram-negative and Gram-positive bacteria like P. aeruginosa and S. aureus, respectively, induced hBD-3 mRNA (Fig. 5, B and C).
hBD-3 Peptide Is Produced by Keratinocytes and Lung Epithelial Cells
We then investigated whether epithelial cells produce hBD-3 peptide. Biochemical analyses of culture supernatants of primary keratinocytes as well as of A549 lung epithelial cells previously pretreated with P. aeruginosa led to the isolation of a peptide antibiotic showing identical biochemical properties, including the NH2-terminal sequence, as seen for the skin-derived hBD-3 (data not shown). We were able to purify ∼10 μg of hBD-3 from the supernatants of both 109 primary keratinocytes and 109 A549 cells, indicating that skin keratinocytes as well as epithelial cells of the respiratory tract represent cellular sources for hBD-3.
DISCUSSION
It has been previously demonstrated that the epithelia of plants (
), 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 (
) 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 (
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 (
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 (
), 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 (
). 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 (
). 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 (
). 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 (
). 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 (
), 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 (
). 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 (
), possibly by inhibiting the binding of positively charged defensins to negatively charged bacterial surfaces. In contrast to both known human μ-defensins (
), 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 (
)) 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 (
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.
46316-3&id=gr1.jpg){kind=link}
46316-3&id=gr2.jpg){kind=link}
46316-3&id=gr3.jpg){kind=link}
46316-3&id=gr4.jpg){kind=link}
46316-3&id=gr5.jpg){kind=link}