From the Clinical Research Unit, Department of Dermatology, University Hospital Kiel, Schittenhelmstrasse 7, 24105 Kiel, Germany
Received for publication, September 19, 2000, and in revised form, November 15, 2000
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ABSTRACT |
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The growing public health problem of infections
caused by multiresistant Gram-positive bacteria, in particular
Staphylococcus aureus, prompted us to screen human
epithelia for endogenous S. aureus-killing factors. A novel
5-kDa, nonhemolytic antimicrobial peptide (human 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 (1). Although vertebrate epithelia are a rich source of antimicrobial proteins (2), it is a very
recent observation that human epithelia mount an innate chemical
defense by secreting antimicrobial peptides (3).
The small (3-5 kDa) cationic defensins represent an important peptide
family among antimicrobial peptides. Two subfamilies, the The first The first isolated human The second human Both human Recent investigations revealed that 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 (23). The high abundance of hBD-2 in skin (16) 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 Culture of Epithelial Cells--
Foreskin-derived keratinocytes,
airway epithelial cells, and the A549 lung epithelial cells were
prepared and cultured as described previously (17, 24). 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- 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 (25).
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 an
N-hydroxy-succinimide-activated Sepharose column
(HiTrap, 5 ml; Amersham Pharmacia Biotech) and 5 ml of an
S. aureus (clinical isolate) suspension (109
bacteria/ml) by a procedure similar to that previously described for a
P. aeruginosa affinity column (17). 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 against
S. aureus or E. coli by a radial diffusion plate
assay (26).
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 (17). Electrophoretic mobility was investigated using SDS-polyacrylamide gels
(SDS-polyacrylamide gel electrophoresis) in the presence of 8 M urea and Tricine (27) under nonreducing conditions as
described for chemokines (28). Peptides were visualized by silver
staining (27).
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 × 102
CFU 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 (29). 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 (30) 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
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 mM
isopropyl-1-thio- 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.
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 (GenBankTM
accession number AJ237673)2 encodes a 67-amino acid
precursor, and the predicted 45 amino acid-containing mature
peptide shows similarity to vertebrate epithelial
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 in
E. 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).
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 yeast
Candida albicans (Fig. 3A). Furthermore, hBD-3
kills multiresistant S. aureus and vancomycin-resistant
Enterococcus 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. aureus
shows 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.
Analysis of hBD-3 Gene Expression--
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).
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- 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.
It has been previously demonstrated that the epithelia of plants
(31), insects (32), amphibians (33), and several mammals (34) are
protected from bacterial infection by a chemical defense shield. The
recent isolation of the human epithelial peptide antibiotics hBD-1 (10)
and hBD-2 (15) and the demonstration of their expression in major
epithelia such as skin (14, 16), respiratory tract (17, 18, 35-37),
urogenital tract (11), and gut (12) 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 (39), no systematic analyses have been performed to elucidate
why healthy human skin is protected from S. aureus
infection. Our previous observation that hBD-2 is not bactericidal
toward S. aureus (15, 17) 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 number
AF189745), further supporting the idea that all human 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 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 (41), 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 (42).
Therefore, mechanisms by which hBD-3 affects S. aureus seem
to be completely different from those discussed for neutrophil
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 (16, 18)
and the epithelial bovine Several reports indicate that inactivation of antimicrobial peptide
activity in patients with cystic fibrosis may contribute to the
recurrent airway infections (49). Elevated salt concentrations in the
airway surface fluid of patients with cystic fibrosis, a matter that
has been controversially discussed (49), inactivate the antimicrobial
activity of human 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
(50)) 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 (38).
-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. aureus
revealed 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
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.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-defensins
and
-defensins, which are distinguished on the basis of the
connectivity of their six cysteine residues, and more recently the
cyclic
-defensin from macaque leukocytes (4), have been identified
in vertebrates (3). In humans two
-defensins, HD-5 and HD-6, are
produced by epithelial granulocytes of the small intestine (5, 6).
-defensin was isolated from bovine tongue (7).
Subsequently, 13 novel
-defensins were purified from bovine neutrophils (8), and the three-dimensional structure, including the
disulfide array of one of these
-defensins, has been determined (9).
-defensin, human
-defensin-1
(hBD-1),1 was purified from
hemofiltrates (10) and was later found in urine as a Gram-negative
bacteria-killing antibiotic (11). mRNA of this antimicrobial
peptide is constitutively expressed in various epithelia (10-14).
-defensin, hBD-2, was discovered in extracts of
lesional scales from patients suffering from psoriasis, a noninfectious
proinflammatory and hyperproliferative skin disease (15, 16). hBD-2 is
expressed in inflamed skin and lung and is induced in epithelial cells
upon treatment with TNF-
(15, 17), interleukin-1
(17, 18), and
contact with mucoid forms of Pseudomonas aeruginosa bacteria
(17).
-defensins show microbicidal activity predominantly
against Gram-negative bacteria like Escherichia coli and
P. aeruginosa. However, they demonstrate only low, if any,
microbicidal activity against Gram-positive bacteria such as
Staphylococcus aureus (3, 15, 19), a bacterium that causes
infections ranging from skin abscesses to life-threatening conditions
such as endocarditis and toxic shock (20).
-defensins also have the ability
to attract T cells (21). Very recent investigations indicate that human
-defensins attract immature dendritic cells and memory T cells via
the chemokine receptor CCR6 (22), providing a link between
innate epithelial defense and adaptive immunity.
-defensin-3 (hBD-3) and which is inducibly expressed by
various human epithelial cells.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(Pepro Tech Inc.) or heat-killed (65 °C, 30 min)
bacteria in 2 ml of serum-free growth medium.
-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
-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.
-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) (51). 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.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-defensins, in
particular bovine "enteric
-defensin" (Fig.
2). Because this novel antimicrobial
peptide is the third isolated human
-defensin, it was termed human
-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.
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Fig. 1.
Identification 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 number
P81534).
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Fig. 2.
Peptide 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 -defensins hBD-1 and
hBD-2, bovine epithelial
-defensins TAP, LAP, and
EBD bovine neutrophil
-defensin BNBD-12, as well as the
-defensin consensus sequence (including the putative disulfide
bridges) are aligned. (The dashes in the
-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.
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Fig. 3.
Antimicrobial/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.
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Fig. 4.
Morphology 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.
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Fig. 5.
Tissue 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- (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
-actin transcript
levels.
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).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-defensins are
clustered on chromosome 8 (40).
-defensins 1 and 2 are less potent peptide
antibiotics and predominantly active against Gram-negative bacteria and
yeasts (15, 17, 18).
-defensins, where lamellar mesosome-like structures at the cell
membrane level were seen in S. aureus (43). Striking
electron-dense deposits were present in the periplasmic space affixed
to the outer membrane when E. coli was investigated (44). 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
(45). 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 (46). 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.
-defensins LAP and TAP (47) and
unlike hBD-1 (48), proinflammatory cytokines such as TNF-
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 (15), airway epithelial cells (17), and
intestinal epithelium (12). Thus hBD-3 represents the second member of
the human
-defensin family where expression is regulated by
inflammatory stimuli at a transcriptional level.
-defensins (13, 18, 35), possibly by inhibiting
the binding of positively charged defensins to negatively charged
bacterial surfaces. In contrast to both known human
-defensins (18,
35), our findings indicate that the bactericidal activity of hBD-3 is
not salt-sensitive at physiologic salt concentrations, which makes this
-defensin of particular relevance in cystic fibrosis.
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ACKNOWLEDGEMENTS |
---|
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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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 P81534 and AJ237673, respectively.
Supported by the Deutsche Forschungsgemeinschaft. To whom
correspondence should be addressed. Tel.: 49-431-5971536; Fax:
49-431-5971611; E-mail: jschroeder@dermatology.uni-kiel.de.
Published, JBC Papers in Press, November 20, 2000, DOI 10.1074/jbc.M008557200
2
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 -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).
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ABBREVIATIONS |
---|
The abbreviations used are:
hBD, human
-defensin;
TNF-
, tumor necrosis factor
;
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.
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