Salt-resistanthomodimericbactenecin, a
cathelicidin-derived antimicrobial peptide
Ju Y. Lee
1
, Sung-Tae Yang
1,3
, Seung K. Lee
1
, Hyun H. Jung
1
, Song Y. Shin
2
, Kyung-Soo Hahm
2
and Jae I. Kim
1
1 Department of Life Science, BioImaging Research Center, Gwangju Institute of Science and Technology, Korea
2 Department of Bio-Materials, Graduate School and Research Center for Proteineous Materials, Chosun University, Gwangju, Korea
3 Section on Membrane Biology, Laboratory of Cellular and Molecular Biophysics, National Institute of Child Health and Human
Development, National Institutes of Health, Bethesda, MD, USA
Over the course of evolution, endogenous antimicro-
bial peptides have assumed the role of providing a first
line of defense against pathogenic infections in both
mammalian and nonmammalian species [1]. Among
these host defense peptides, the cathelicidins are char-
acterized by conserved cathelin-like domains (the
proregion) and highly variable C-terminal antimicro-
bial domains [2] that enable them to be classified into
three structural classes: amphipathic a-helical peptides,
b-hairpin peptides stabilized by disulfide bridges, and
linear Trp-rich, Pro-rich peptides [3,4]. Bactenecin, a
cathelicidin purified from the granules of bovine
neutrophils, is a b-hairpin monomer with one intra-
molecular disulfide bond, and has been shown to have
Keywords
antimicrobial peptides; bactenecin;
dimerization; peptide–membrane interaction;
salt resistance
Correspondence
J. I. Kim, Department of Life Science,
Gwangju Institute of Science and
Technology, Gwangju 500-712, Korea
Fax: +82 62 970 2484
Tel: +82 62 970 2494
E-mail: jikim@gist.ac.kr
(Received 19 July 2007, revised 28 April
2008, accepted 4 June 2008)
doi:10.1111/j.1742-4658.2008.06536.x
The cathelicidin antimicrobialpeptide bactenecin is a b-hairpin molecule
with a single disulfide bond and broad antimicrobial activity. The proform
of bactenecin exists as a dimer, however, and it has been proposed that
bactenecin is released as a dimer in vivo, although there has been little
study of the dimeric form of bactenecin. To investigate the effect of bacten-
ecin dimerization on its biological activity, we characterized the dimer’s
effect on phospholipid membranes, the kinetics of its bactericidal activity,
and its salt sensitivity. We initially synthesized two bactenecin dimers (anti-
parallel and parallel) and two monomers (b-hairpin and linear). Under oxi-
dative folding conditions, reduced linear bactenecin preferentially folded
into a dimer forming a ladder-like structure via intermolecular disulfide
bonding. As compared to the monomer, the dimer had a greater ability to
induce lysis of lipid bilayers and was more rapidly bactericidal. Interest-
ingly, the dimer retained antimicrobial activity at physiological salt concen-
trations (150 mm NaCl), although the monomer was inactivated. This salt
resistance was also seen with bactenecin dimer containing one intermole-
cular disulfide bond, and the bactenecin dimer appears to undergo multi-
meric oligomerization at high salt concentrations. Overall, dimeric
bactenecin shows potent and rapid antimicrobial activity, and resists salt-
induced inactivation under physiological conditions through condensation
and oligomerization. These characteristics shed light on the features that a
peptide would need to serve as an effective therapeutic agent.
Abbreviations
ABD, antiparallel dimer bactenecin; Acm, acetamidomethyl; CDB, C-terminal dimeric bactenecin; CFU, colony-forming unit; hRBC, human red
blood cell; KCTC, Korean Collection for Type Cultures; MIC, minimal inhibitory concentration; MTB, monomeric turn bactenecin; NDB,
N-terminal dimeric bactenecin; PDB, parallel dimer bactenecin; POPC, 1-palmitoyl-2-oleoyl-phosphatidylcholine; POPG, 1-palmitoyl-2-oleoyl-
phosphatidylglycerol; SLB, Ser-substituted linear bactenecin.
FEBS Journal 275 (2008) 3911–3920 ª 2008 The Authors Journal compilation ª 2008 FEBS 3911
antibacterial activity against both Gram-negative and
certain Gram-positive bacteria [5]. In addition, two lin-
ear variants of bactenecin, Bac2S and Bac2A, show
similar activities against Gram-negative bacteria and
stronger activities against Gram-positive bacteria [6],
and Bac2A also acts as a potent chemoattractant,
inducing chemotaxis in undifferentiated THP-1 cells
[7]. However, although bactenecin has largely been
studied as a monomeric molecule, its proform report-
edly exists as a dimer formed by intermolecular disul-
fide bridges in the C-terminal antimicrobial domain.
Moreover, it is known that the synthetic cyclic peptide
is mainly active against Gram-negative bacteria [6,8],
whereas the isolated native peptide showed activity
against both Escherichia coli and Staphylococcus aureus
[5]. This suggests that it may be necessary to recon-
sider the structure of the mature native bactenecin
in vivo [9].
Although b-hairpin bactenecin and its analogs have
been the subjects of numerous studies, little is known
about the antimicrobial activity of the dimeric form,
or the way in which it interacts with the bacterial
membrane. That said, earlier studies suggest that
dimerization of antimicrobial peptides leads to the
appearance of a more diverse spectrum of antimicro-
bial activity than is exhibited by monomers. For
instance, Tencza et al. reported that dimeric LLP1,
which is a Cys-containing peptide derived from a lenti-
virus envelope protein that spontaneously forms
disulfide-linked dimers, possesses much greater antimi-
crobial activity against S. aureus than monomeric
LLP1 [10]. In addition, disulfide-dimerized magainin 2
[(mag-N22C)
2
] induces membrane permeabilization at
lower concentrations than the monomeric form [11]. In
the case of the channel-forming peptide alamethicin,
channels formed by covalent dimers displayed lifetimes
at a particular conductance that were up to 170-fold
longer than those of monomers [12]. Consistent with
that finding, in many cases dimerization was closely
connected to enhanced antimicrobial activity mediated
by the formation of pores or channels in the lipid
membrane [13,14].
For effective use in clinical pharmacotherapy, anti-
microbial peptides need to remain active in the pres-
ence of physiological levels of salt (120–150 mm
NaCl), and structural constraints such as dimerization
or Cys-knot formation are also related to the salt sen-
sitivity of antimicrobial peptides. For instance, defen-
sins, a group of b-form antimicrobial peptides, are
generally degraded under high-salt conditions [15], but
oxidized b-defensin (Defr-1), which contains five Cys
residues that associate to produce dimers through for-
mation of various intramolecular and intermolecular
disulfide bridges, exhibits potent and broad-spectrum
antimicrobial activity that is not suppressed at high
salt concentrations [16]. In addition, the study of
protegrin-1 and rhesus theta defensin-1, which have
b-strand and cyclic structures, respectively, has shown
that structural rigidity resulting from Cys-stabilization
enables the peptides to retain activity against most
bacteria in high-salt environments [17].
It was previously reported that bactenecin is too
small to disrupt the bacterial membrane unless a multi-
mer is involved in forming pores or channels [8], and
that the native peptide may occur in both monomeric
and dimeric forms [9,18]. To test that idea, in the pres-
ent study we chemically synthesized two dimers that
adopt parallel and antiparallel conformations and two
monomers that adopt b-hairpin and linear confor-
mations, and investigated their biological activities.
Results and Discussion
Peptide folding and its characterization
To investigate the effect of dimerization on the antimi-
crobial activity of bactenecin, we designed four bacten-
ecin derivatives with differing chemical ⁄ physical
properties reflecting the interactions among their Cys
residues (Fig. 1). Under most oxidative folding condi-
tions, reduced linear bactenecin folded into a specific
form (yield, 70–80%) that trypsin digestion experi-
ments revealed to be an antiparallel dimer [antiparallel
dimer bactenecin (ADB)] (supplementary Figs S1 and
S2). Because the majority of reduced linear bactenecin
spontaneously dimerizes, even at very low oxidative
folding concentrations (e.g. 10 lm), we attempted to
synthesize monomeric turn bactenecin (MTB) by utiliz-
ing an iodine oxidation strategy often used for oxida-
tive cyclization of Cys-containing peptides having a
free Cys residue and to remove protective S-acetami-
domethyl (Acm) groups, although in this case there
was no S-Acm group [19]. Under these conditions,
dimerization was completely blocked, and MTB was
obtained with a yield of about 90%. Interestingly, we
failed to produce any parallel dimer bactenecin (PDB)
when the oxidative folding condition was applied to
unprotected ADB or MTB peptide, suggesting that
ADB is thermodynamically more favorable than PDB
in an air oxidative folding pathway. As ADB and
PDB differ only in the orientations of their two
strands with respect to one another, we suggest that
mainly unfavorable terminal charge repulsion inhibits
PDB formation. By adding one protective S-Acm to
reduced linear bactenecin (Fig. 1), we were able to
utilize an iodine oxidation strategy to synthesize PDB
Salt-resistant homodimeric bactenecin J. Y. Lee et al.
3912 FEBS Journal 275 (2008) 3911–3920 ª 2008 The Authors Journal compilation ª 2008 FEBS
and successfully harvest the dimer after a sequential
two-step reaction leading to disulfide formation (yield,
80%). The position of the Cys residue carrying the
S-Acm group was alternated because the amino acid
compositions near the two Cys residues were similar to
one another. Finally, we synthesized Ser-substituted
linear bactenecin (SLB) to investigate the structural
and ⁄ or functional role of the disulfide bond.
Conformational studies
We used CD spectroscopy to estimate the secondary
structure of the bactenecin derivatives in buffer and in
a membrane-mimicking environment achieved with the
addition of SDS (Fig. 2). Consistent with previously
reported CD spectra [8], those for MTB showed a typi-
cal type I b-turn structure with a negative band in the
vicinity of 205 nm in both environments [20]. For the
two dimers, ADB and PDB, a spectrum exhibiting a
negative band at 210 nm was observed in buffer,
whereas an ordered b-strand structure with a
maximum near 200 nm and a minimum at 220 nm was
obtained in the presence of SDS micelles. This was
well fitted to typical b-strand globular proteins, which
show a strong positive band near 200 nm and a nega-
tive band below 220 nm [21,22]. A more ordered struc-
ture indicated by the red-shift from 210 to 220 nm, as
well as the presence of a positive band at 200 nm, may
be caused not only by the interaction of b-strands
within a given dimer, but also by the interaction of
b-strands between dimers. Interestingly, SLB showed a
disordered structure in buffer but, upon interaction
with SDS micelles, the CD spectrum changed to one
similar to those of the dimers. The spectral behavior
observed for SLB suggests that linear bactenecin has a
strong propensity to form a b-structure in a membrane
environment, and may be indicative of the importance
of the dimeric structure for specific interactions with
the bacterial membrane.
Antimicrobial and hemolytic activities
The peptides’ antimicrobial activities against selected
Gram-positive and Gram-negative bacteria, as well as
their hemolytic activities, are summarized in Table 1.
As previously reported, MTB was more potent against
Gram-negative bacteria [minimal inhibitory concen-
tration (MIC) = 2–4 lm] than against Gram-positive
bacteria (MIC = 4–8 lm), and was without hemolytic
activity. ADB and PDB displayed activity similar to
that of MTB against Gram-negative bacteria, with some
hemolytic activity (10–20% hemolysis at 100 lm), but
exhibited about four times greater potency (MIC =
1–2 lm) against Gram-positive bacteria. These results
are consistent with those obtained with the isolated
native peptide, which displayed broad-spectrum antimi-
crobial activity against Gram-positive and Gram-nega-
tive bacteria, and suggests to us that it is probable that
native dimeric forms are also active in vivo.
It was previously reported that Bac2A, in which a
Cys residue was substituted with Ala, had somewhat
better activity against Gram-positive bacteria than
MTB [6]. Like Bac2A, SLB also showed slightly better
antimicrobial activity against Gram-positive bacteria
than MTB, with no hemolytic activity. Taken together
with the results of the CD analysis, these findings
suggest that in a membrane environment, ADB, PDB
and SLB take on a common b-structure that enables
better interaction with Gram-positive bacteria.
Dye leakage from liposomes
It is well known that dimerization can cause a signifi-
cant change in a peptide’s interaction with the bacte-
MTB
SLB
H
2
N
-RLCRIVVIRVCR-
CO
2
H
RLCRIVVIRVCR
H
2
N-RLCRIVVIRVCR-CO
2
H
H
2
N
-
-CO
2
H
Acm
SH
PDBADB
H
2
N
-RLSRIVVIRVSR
-CO
2
H
H
2
N-RLCRIVVIRVCR-CO
2
H
H
2
N-RLCRIVVIRVCR-CO
2
H
Acm
SH
Acm
H
2
N-RLCRIVVIRVCR-CO
2
H
H
2
N-RLCRIVVIRVCR-CO
2
H
H
2
N-RLCRIVVIRVCR-CO
2
H
Acm
HO
2
C
-RCVRIVVIRCLR-
NH
2
H
2
N
-RLCRIVVIRVCR-
CO
2
H
H
2
N-RLCRIVVIRVCR-CO
2
H
Fig. 1. Scheme employed for the synthesis of bactenecin and its
derivatives through formation of disulfide bridges. (A) ADB was
folded in 2
M acetic acid ⁄ H
2
O ⁄ dimethylsulfoxide (1 : 2 : 1, v ⁄ v ⁄ v)
solution for 24 h at room temperature. (B) MTB was oxidized in
acetic acid ⁄ H
2
O (4 : 1, v ⁄ v) solution, after which iodine was added
(10 equivalents to the number of disulfide bonds). (C) PDB was
prepared in two steps: air oxidation in distilled water at 47 °C was
carried out for 5 days, after which the partially oxidized peptides
were dissolved in acetic acid ⁄ H
2
O (4 : 1, v ⁄ v) solution, and iodine
was added (10 equivalents to the number of disulfide bonds) for
2h.
J. Y. Lee et al. Salt-resistanthomodimeric bactenecin
FEBS Journal 275 (2008) 3911–3920 ª 2008 The Authors Journal compilation ª 2008 FEBS 3913
rial membrane, whether or not it enhances the antimi-
crobial activity of the peptide [10–14]. To assess the
effect of dimerization on peptide-induced membrane
disruption leading to microbial cell death, we examined
the capacity of peptides to release calcein from
liposomes composed of 1-palmitoyl-2-oleoyl-phosphat-
idylglycerol (POPG) ⁄ 1-palmitoyl-2-oleoyl-phosphati-
dylcholine (POPC) (1 : 1), which served as a model of
the bacterial membrane (Fig. 3). At a molar pep-
tide ⁄ liposome ratio of 1 : 10, PDB and ADB induced
leakage in about 90% and 70% of liposomes, respec-
tively. By contrast, both MTB and SLB displayed only
weak membrane lytic activity, with about 20% of lipo-
somes showing leakage. In terms of the structure–
activity relationships, it is noteworthy that ADB, PDB
and SLB all assume a common b-structure in a mem-
brane environment, despite the significant differences
in their membrane lytic activities. In that regard, a
two-step mechanism for membrane disruption leading
to leakage has been suggested [23]. The peptide first
10
60
MTB SLB
–10
0
0
20
40
MTB SLB
–30
–20
–40
–20
0
m
2
· dmol
–1
)
–40
–60
10
15
PDB
15
20
ADB
0
3
(deg·c m
–10
–5
0
5
0
5
10
[ ] X1
–25
–20
–15
–20
–15
–10
–5
190 200 210 220 230 240 250
190 200 210 220 230 240 250
Wavelength (nm)
Fig. 2. CD spectra for MTB, SLB, ADB and
PDB. Spectra were recorded at 25 °Cin
10 m
M sodium phosphate buffer (pH 7.4)
(d)orin30m
M SDS micelles ( ). Each
peptide was used at a concentration of
25 l
M.
Table 1. MIC (lM) values and hemolytic activities of the peptides.
Results indicate the ranges of three independent experiments,
each performed in triplicate. The hemolytic activity was determined
using 100 l
M peptide, and the results represent the means of
duplicate measurements from three independent assays.
MIC (l
M)
MTB SLB ADB PDB
Bacterial strain
S. aureus 4–8 2–4 1–2 1–2
S. epidermidis 2–4 2 1–2 2
B. subtilis 4 2 1–2 2
E. coli 1–2 2–4 1–2 2–4
P. aeruginosa 2–4 4–8 2–4 4
Sa. typhimurium 4–8 4–8 2–4 4
% Hemolysis 0 0 10 20
100
60
80
20
40
0.001 0.01 0.1 1 10
Calcein release (%)
0
[Peptide]/[Lipid]
Fig. 3. Calcein release from liposomes was measured as a function
of the molar peptide ⁄ lipid ratio. Peptide concentrations were 5 l
M
for POPC ⁄ POPG (1 : 1) liposomes. Fluorescence from liposomes
lysed with Triton X-100 was used as an indicator of 100% leakage.
s, MTB; d, SLB; ,, ADB; ., PDB. Results represent the means
of three independent experiments.
Salt-resistant homodimeric bactenecin J. Y. Lee et al.
3914 FEBS Journal 275 (2008) 3911–3920 ª 2008 The Authors Journal compilation ª 2008 FEBS
binds to the membrane (the membrane affinity of the
peptide), after which it elicits membrane disruption
(the membrane-perturbing activity). It is thus likely
that even though all three peptides exhibit a similar
structural transition upon binding to membrane sur-
faces, only the dimers show enhanced membrane lytic
activity, perhaps due to induction of oligomerization
of the dimeric peptides by the hydrophobic membrane
environment.
Kinetics of the bactericidal activity
To further study the antibacterial activity of ADB and
PDB, the kinetics of their bactericidal activity against
both Gram-positive (S. aureus; Fig. 4A) and Gram-
negative (E. coli; Fig. 4B) bacteria were investigated,
with magainin 2 serving as a control. The time needed
for PDB and ADB to induce 100% cell death was as
little as 5 min for Gram-positive bacteria, and both
peptides showed the same kinetics. About 30 min or
more were needed to kill 100% of Gram-negative bac-
teria, with PDB acting more rapidly than ADB. The
kinetics of MTB’s bactericidal activity were similar to
those of ADB for Gram-negative bacteria, but were
very slow for Gram-positive bacteria, with about 20%
of cells remaining viable even after exposure for
60 min. SLB acted almost as rapidly as ADB or PDB
against Gram-positive bacteria, but acted more slowly
than the other three peptides against Gram-negative
bacteria. Although, overall, PDB and ADB showed
only slightly greater antimicrobial activity than MTB
and SLB, we suggest that the capacity of the dimers to
kill bacteria quickly enough to prevent replication
gives them a greater ability to control bacterial expan-
sion, thereby reducing the likelihood that resistance
will develop [24]. In other words, the rapidity with
which bacteria are killed may be an important factor
when evaluating the activity of antimicrobial peptides
in vivo and when assessing their potential for clinical
use [25].
Effect of salt
Studies of cationic antimicrobial peptides have shown
that the salt concentration can affect their activity,
even at less than physiological levels [26]. To determine
whether dimerization affects salt sensitivity, S. aureus
and E. coli were exposed to 8 lm peptide in the pres-
ence or absence of 150 mm NaCl. In the absence of
salt, all four peptides killed 100% of the bacteria. In
its presence, the two dimers exhibited generally unal-
tered activity against both Gram-positive (S. aureus;
Fig. 5A) and Gram-negative (E. coli; Fig. 5B) bacteria.
By contrast, the antimicrobial activity of MTB against
S. aureus was completely lost in the presence of
150 mm NaCl, and the activity against E. coli was
reduced by > 60%. Although in a membrane environ-
ment SLB showed a potency and CD pattern that were
similar to those of the dimers, in the presence of
150 mm NaCl, it killed only about 75% of S. aureus
and was completely inactive against E. coli. It thus
appears that Cys-derived dimerization enables the
peptides to retain potent bactericidal activity in the
presence of physiological levels of salt.
Similarly, it was previously reported that the antimi-
crobial activity of the guinea pig 11 kDa polypeptide,
which is a homodimer joined by intermolecular disul-
fide bonds, was unaffected by the presence of NaCl,
whereas the activities of the guinea pig 5 kDa peptide
and various defensins, which all contain intramolecular
disulfide bonds, were inactivated by NaCl [27].
Together, these results strongly suggest that intermo-
lecular disulfide connections contribute greatly to
retention of a peptide’s antibacterial activity at high
salt concentrations.
Peptide oligomerization
A high ionic strength may reduce the electrostatic
interaction between cationic peptides and anionic lipid
head groups through counterion screening, thereby
80
100
A B
80
100
40
60
40
60
CFU·mL
–1
(%)
0
20
0
20
0 10 20 30 40 50 60
0 10 20 30 40 50 60
Time (min)
Fig. 4. Kinetics of the bactericidal activity of
the four bactenecin derivatives against
S. aureus (A) and E. coli (B). Bacteria treated
with the respective peptides (8 l
M) were
diluted at the indicated times and then pla-
ted on LB agar. The CFUs were then
counted after 24 h of incubation at 37 °C.
s, MTB; d, SLB; ,, ADB; ., PDB;
, mag-
ainin 2. Results represent the means of two
independent experiments.
J. Y. Lee et al. Salt-resistanthomodimeric bactenecin
FEBS Journal 275 (2008) 3911–3920 ª 2008 The Authors Journal compilation ª 2008 FEBS 3915
reinforcing the membrane surface region [23,28]. In the
case of defensin, it was reported that the number of
positive charges in the molecule was directly propor-
tional to its ability to retain antimicrobial activity at
higher salt concentrations [28]. Consistent with that
relationship, the bactenecin dimers used in the present
study have twice as many positively charged residues
as bactenecin monomer.
On the other hand, the salt tolerance of dimeric bac-
tenecins may reflect, to some degree, the structural
rigidity afforded by the two intermolecular disulfide
bonds. To test that idea, we substituted the Cys resi-
due at position 11 or 3 of bactenecin with a Ser and
synthesized two PDB derivatives containing a single
disulfide bond: N-terminal dimeric bactenecin (NDB)
and C-terminal dimeric bactenecin (CDB) (Fig. 6A). In
the presence of SDS micelles, the CD spectra of both
NDB and CDB showed b-structure patterns, similar to
those of ADB and PDB, but in buffer solution NDB
showed a b-structure with reduced molar ellipticity,
whereas CDB showed a random-like conformation
(Fig. 6B). In addition, both NDB and CDB exhibited
unexpectedly lower antimicrobial potency in the
absence of salt (MIC = 32 and 16 lm for S. aureus
and E. coli, respectively), and just 30–40% of the activ-
ity of MTB. In the presence of 150 mm NaCl, how-
ever, NDB and CDB almost completely killed the
tested Gram-positive and Gram-negative bacteria, and
showed a potency similar to that of ADB and PDB,
which have two disulfide bonds (Fig. 6C). It is note-
worthy that, in the absence of salt, both NDB and
CDB exhibited much less antibacterial activity than
MTB, SLB, ADB or PDB, and they displayed very
different potencies in the presence or absence of salt.
Finally, we compared the multimeric state of
bactenecin derivatives by carrying out an electrophore-
sis experiment on Tricine–acrylamide gel. Four deriva-
tives, SLB (linear bactenecin), MTB (bactenecin having
one intramolecular disulfide bond), NDB (bactenecin
having one intermolecular disulfide bond) and PDB
(bactenecin having two intermolecular disulfide bonds)
were selected and exposed to an environment con-
taining a high concentration of salt (300 mm NaCl).
As shown in Fig. 7, the two monomers (SLB and
MTB) migrated with apparent molecular masses of
1.5 kDa, whereas the two dimers (NDB and PDB)
migrated with apparent molecular masses of
6.5 kDa or more in both the presence and the
absence of salt. In addition, both SLB and MTB
showed somewhat fainter bands in the presence of salt,
but PDB exhibited a strong band whether salt was
present or not. This suggests that both SLB and MTB
are monomers and that PDB is a dimer in both the
presence and the absence of salt. Interestingly, NDB
showed a weak band at around 6.5 kDa in the
absence of salt, but a strong band at around
14.2 kDa at the presence of salt, implying that NDB
undergoes multimeric oligomerization in the presence
of 300 mm NaCl. Thus, although the same amount of
each peptide was loaded, these peptides exhibited
significantly different band densities and mobilities on
a Tricine–acrylamide gel, which provides a clue as to
why bactenecin dimers retain their potent antibacterial
activity at high salt concentrations.
In conclusion, our findings are noteworthy in part
because they confirm the potential importance of
dimeric forms of antimicrobial peptides in vivo, and
because the ladder-like structure of homodimeric
antimicrobial peptides makes them relatively easy to
100
100
A
B
80
80
40
60
40
60
0
20
% Bacteria killed % Bacteria killed
0
20
MTB
S
LB ADB PDB
MTB SLB ADB PDB
Fig. 5. Salt sensitivity of the antimicrobial activity of the four bac-
tenecin derivatives against S. aureus (A) and E. coli (B). To deter-
mine the effect of salt on the antimicrobial activity of the peptides,
each peptide (8 l
M) was incubated with bacteria for 3 h in the
absence (gray bars) or presence (black bars) of 150 m
M NaCl, after
which 50 lL aliquots of the suspension were plated on LB agar for
colony counts. Results represent the means of two independent
experiments.
Salt-resistant homodimeric bactenecin J. Y. Lee et al.
3916 FEBS Journal 275 (2008) 3911–3920 ª 2008 The Authors Journal compilation ª 2008 FEBS
synthesize. Although the two dimers studied, ADB and
PDB, had similar activities, synthesis of PDB was com-
plex. By contrast, ADB is easily folded under most
folding conditions. Interestingly, bactenecin dimers
undergo multimeric oligomerization at high salt
concentrations. Further studies on the structural
changes in PDB and NDB that occur at the mem-
brane are in progress so as to better understand the
mechanism by which each dimer interacts with the
membrane.
Experimental procedures
Peptide synthesis, disulfide formation and
characterization
All peptides were synthesized using the solid-phase peptide
synthesis method performed manually with Fmoc chemis-
try. The peptides were cleaved from the resin using trifluo-
H
2
N-RLCRIVVIRVSR-CO
2
H
H
2
N-RLCRIVVIRVSR-CO
2
H
H
2
N-RLCRIVVIRVSR-CO
2
H
NDB
B
A
C
H
2
N-RLSRIVVIRVCR-CO
2
H
H
2
N-RLSRIVVIRVCR-CO
2
H
H
2
N-RLSRIVVIRVCR-CO
2
H
CDB
30
NDB CDB
10
20
m
2
·dmol
–1
)
NDB CDB
-10
0
0
3
(deg·cm
30
-20
[ ] X 10
190 200 210 220 230 240 250
190 200 210 220 230 240 25
0
-30
30
10
20
-10
0
-20
-30
Wavelength (nm)
lled
80
100
80
100
S. aureus E. coli
acteria ki
40
60
40
60
% Ba
0
20
0
20
MTB NDB CDB
MTB NDB
C
DB
0
Fig. 6. Synthesis, secondary structure and
salt sensitivity of NDB and CDB, two
bactenecin derivatives containing a single
disulfide bond. (A) NDB and CDB were
completely folded in 2
M acetic
acid ⁄ H
2
O ⁄ dimethylsulfoxide (1 : 2 : 1,
v ⁄ v ⁄ v) solution for 36 h with gentle stirring
at room temperature. (B) CD spectra were
recorded at 25 °Cin10m
M sodium phos-
phate buffer (pH 7.4) (d) and in 30 m
M SDS
micelles (
). (C) Each peptide (8 lM) was
incubated with bacteria for 3 h in the
absence (gray bars) or presence (black bars)
of 150 m
M NaCl, after which 50 lL aliquots
of the suspension were plated on LB agar
for colony counts. Results represent the
means of two independent experiments.
No salt 300 mM salt
MK
NDB SLB PDB MTB NDB SLB PDB MTB
26.6
17
14.2
6.5
3.5
Fig. 7. Coomassie-stained 15% Tricine gel of bactenecin and its
derivatives without salt and with 300 m
M NaCl. Fifteen micrograms
of each peptide were loaded. Mass markers in kDa are shown on
the left.
J. Y. Lee et al. Salt-resistanthomodimeric bactenecin
FEBS Journal 275 (2008) 3911–3920 ª 2008 The Authors Journal compilation ª 2008 FEBS 3917
roacetic acid containing various scavengers and purified by
preparative RP-HPLC (Shimadzu, Tokyo, Japan). The pur-
ity of peptides was verified by analytical RP-HPLC, and
correct peptide masses were confirmed by MALDI-
TOF MS (Shimadzu).
Dissolving reduced linear bactenecin to a concentration
of 1 mm in buffer solution containing 2 m acetic
acid ⁄ H
2
O ⁄ dimethylsulfoxide (1 : 2 : 1) at room temperature
for 24 h with gentle stirring effectively yielded ADB. MTB
exhibiting a b-hairpin conformation was oxidized in acetic
acid ⁄ H
2
O (4 : 1), and this was followed by addition of
iodine (10 equivalents to the number of disulfide bonds). A
two-step method for disulfide bond formation was used to
prepare PDB. Briefly, partially protected peptides were
joined using Fmoc solid-phase chemistry on Wang resin.
The free thiol groups of the peptides were bonded by air
oxidation in distilled water at 47 °C, while the course of the
reaction was monitored using HPLC. Peptides linked by
single disulfide bonds were obtained after 5 days at a yield
of > 90%. The second procedure was initiated by dissolv-
ing the peptide in acetic acid ⁄ H
2
O (4 : 1) and adding iodine
(10 equivalents to the number of disulfide bonds), after
which stirring was continued for an additional 2 h to effect
removal of the Acm groups and conversion to PDB with a
yield of 80%. ADB and PDB were confirmed by enzymatic
digestion with trypsin (supplementary Figs S1 and S2). A
linear peptide SLB, in which Cys was substituted with Ser,
was also synthesized.
Trypsin digestion
A trypsin digestion was carried out to distinguish between
PDB and ADB (supplementary Fig. S1). Samples of PDB
(100 lg) and ADB (100 lg) were dissolved in 0.2 mL of
50 mm Tris ⁄ HCl buffer (pH 8), after which modified tryp-
sin (5 lg) was added to a final protease ⁄ protein ratio of
1 : 20 (w ⁄ w), and the mixture was incubated at 37 °C for
6 h. Analytical RP-HPLC analysis of the reaction mixture
was then carried out (supplementary Fig. S2), and
MALDI-TOF MS was used to analyze the mass of each
peptide.
CD analysis
The CD spectra of the peptides were recorded using a
Jasco J-710 CD spectrophotometer (Jasco, Tokyo, Japan)
with a 1 mm path-length cell. Wavelengths were measured
from 190 to 250 nm (bandwidth, 1 nm; step resolution,
0.1 nm; speed, 50 nmÆmin
)1
; response time, 0.5 s). The col-
lected CD spectra for the peptides were averaged over 16
scans in 0.5 mm POPC ⁄ POPG (1 : 1) liposomes and over
four scans in 10 mm sodium phosphate buffer (pH 7.4) or
30 mm SDS micelles at 25 °C. The spectra are expressed as
molar ellipticity [h] versus wavelength.
Antibacterial activity
Antimicrobial activities of each peptide against six selected
organisms, including three Gram-positive and three Gram-
negative bacteria, were determined using broth microdilu-
tion assays [29]. Six organisms obtained from the Korean
Collection for Type Cultures (KCTC), Korea Research
Institute of Bioscience and Biotechnology (Taejon, Korea)
were used for the assays. The Gram-negative bacteria were
E. coli KCTC 1682, Salmonella typhimurium KCTC 1926,
and Pseudomonas aeruginosa KCTC 1637. The three
Gram-positive bacteria were Bacillus subtilis KCTC 3068,
Staphylococcus epidermidis KCTC 1917, and S. aureus
KCTC 1621. Briefly, single colonies of bacteria were inocu-
lated into medium (LB broth) and cultured overnight at
37 °C. An aliquot of the culture was then transferred to
10 mL of fresh medium and incubated for an additional
3–5 h at 37 °C until mid-logarithmic phase. A two-fold
dilution series of peptides in 1% peptone was prepared,
after which serial dilutions (100 lL) were added to 100 lL
of cells [2 · 10
5
colony-forming units (CFU)ÆmL
)1
]in
96-well microtiter plates (F96 microtiter plates; Nunc,
Odense, Denmark) and incubated at 37 °C for 16 h. The low-
est concentration of peptide that completely inhibited growth
was defined as the MIC. MIC values were acquired as aver-
age or triplicate measurements in three independent assays.
Hemolytic activity
The hemolytic activities of the peptides were determined
using human red blood cells (hRBCs). After washing of
fresh hRBCs three times with NaCl ⁄ P
i
(35 mm phosphate
buffer, 150 mm NaCl, pH 7.4), 100 lL of a 4% (v ⁄ v)
hRBC suspension in NaCl ⁄ P
i
was dispensed into sterilized
96-well plates along with 100 lL of peptide solution. The
plates were then incubated for 1 h at 37 °C and centrifuged
for 5 min at 1000 g. Aliquots (100 lL) of supernatant were
transferred to 96-well plates, and hemoglobin release was
monitored on the basis of the absorbance at 414 nm using
an ELISA plate reader (Molecular Devices, Sunnyvale, CA,
USA). Percentage hemolysis was calculated using the
following formula: hemolysis (%) = [(A
405 nm
sample
) A
405 nm
zero lysis) ⁄ (A
405 nm
100% lysis ) A
405 nm
zero
lysis)] · 100. Zero and 100% hemolysis were determined in
NaCl ⁄ P
i
and 0.1% Triton X-100, respectively. The recorded
hemolysis (%) was the average of duplicate measurements
in three independent assays.
Preparation of liposomes
Large unilamellar vesicles (average diameter, 100 nm) con-
taining the fluorescent probe calcein were prepared by
extrusion [30]. Briefly, phospholipids composed of POPG ⁄
POPC (1 : 1) were dissolved in chloroform and then dried
Salt-resistant homodimeric bactenecin J. Y. Lee et al.
3918 FEBS Journal 275 (2008) 3911–3920 ª 2008 The Authors Journal compilation ª 2008 FEBS
overnight under vacuum to make a thin lipid film. The
dried film was then hydrated with Tris ⁄ HCl buffer (10 mm
Tris, 150 mm NaCl, 1 mm EDTA, pH 7.4) containing
70 mm calcein (pH adjusted to 7.4 with NaOH) and vortex-
mixed. The suspensions were subjected to five freeze–thaw
cycles and then pressure-extruded through polycarbonate
filters (LiposoFast, 0.1 lm pore size, 20 times). Vesicles
containing entrapped calcein were separated from free
calcein by gel filtration on Sephadex G-50 columns (Phar-
macia, Uppsala, Sweden) equilibrated with Tris ⁄ HCl buffer.
To prepare the small unilamellar vesicles used for CD
spectroscopy, dried lipid film was hydrated with Tris ⁄ HCl
buffer and then sonicated in an ice bath for 30 min using a
titanium-tipped sonicator. The lipid concentration was
0.5 mm.
Calcein leakage studies
As mentioned above, the fluorescent probe calcein was
encapsulated in large unilamellar vesicles at a self-quench-
ing concentration of 70 mm. For leakage experiments, the
indicated amounts of peptide were added to 3 mL of buffer
containing calcein-loaded liposomes. The fluorescence inten-
sity of the calcein released from the liposomes, which was
measured with mixing after the addition of a peptide, was
monitored at 520 nm (excited at 490 nm) in a Shima-
dzu RF-5301 spectrofluorometer. Fluorescence from
liposomes lysed with Triton X-100 (20% in Tris buffer)
was used as an indicator of 100% leakage.
Kinetics of bactericidal activity and salt
sensitivity
The kinetics of the peptides’ bactericidal activity was
assessed using E. coli KCTC 1682 and S. aureus
KCTC 1621 at apeptide concentration of 8 lm, which was
the highest MIC for any bactenecin derivative against the
strains used. The initial density of the cultures was approxi-
mately 2 · 10
5
CFUÆmL
)1
. After 0, 5, 10, 30 or 60 min of
exposure to the peptides at 37 °C, 50 lL aliquots of serial
10-fold dilutions (up to 10
)3
) of the cultures were plated
onto LB agar plates to obtain viability counts. Colonies
were counted after incubation for 24 h at 37 °C.
To determine the salt sensitivity of the antimicrobial
activity, peptides were incubated at 37 ° Cin100lLof1%
peptone solution also containing 2 · 10
5
CFUÆmL
)1
bacte-
ria and 0 or 150 mm NaCl. After incubation for 3 h at
37 °C, 50 lL of the suspension was plated on LB agar for
colony counts.
Tricine gel electrophoresis
Electrophoresis was performed with 15 lg samples of each
bactenecin derivative dissolved in 2· sample buffer
(125 mm Tris ⁄ HCl, pH 6.8, 20% glycerol, 2% mercaptoeth-
anol, 0.04% bromophenol blue, and 4% SDS). The entire
sample was loaded onto a 15% Tricine gel, after which the
gel was fixed and stained with Coomassie dye.
Acknowledgements
This study was supported by the SRC ⁄ ERC program
of MOST ⁄ KOSEF (R11-2000-083-00000-0) and the
Brain Research Center of the 21st Century Frontier
Research Program (M103KV010005-06K2201-00510).
References
1 Hoffmann JA, Kafatos FC, Janeway CA & Ezekowitz
RA (1999) Phylogenetic perspectives in innate immu-
nity. Science 284, 1313–1318.
2 Zelezetsky I, Pontillo A, Puzzi L, Antcheva N, Segat L,
Pacor S, Crovella S & Tossi A (2006) Evolution of the
primate cathelicidin. Correlation between structural
variations and antimicrobial activity. J Biol Chem 281,
19861–19871.
3 Zanetti M (2004) Cathelicidins, multifunctional peptides
of the innate immunity. J Leukoc Biol 75, 39–48.
4 Bals R & Wilson JM (2003) Cathelicidins – a family of
multifunctional antimicrobial peptides. Cell Mol Life
Sci 60, 711–720.
5 Romeo D, Skerlavaj B, Bolognesi M & Gennaro R
(1988) Structure and bactericidal activity of an antibi-
otic dodecapeptide purified from bovine neutrophils.
J Biol Chem 263, 9573–9575.
6 Wu M & Hancock REW (1999) Improved derivatives of
bactenecin, a cyclic dodecameric antimicrobial cationic
peptide. Antimicrob Agents Chemother 43, 1274–1276.
7 Bowdish DM, Davidson DJ, Scott MG & Hancock
REW (2005) Immunomodulatory activities of small host
defense peptides. Antimicrob Agents Chemother 49,
1727–1732.
8 Wu M & Hancock REW (1999) Interaction of the cyclic
antimicrobial cationic peptide bactenecin with the outer
and cytoplasmic membrane. J Biol Chem 274, 29–35.
9 Storici P, Tossi A, Lenarcic B & Romeo D (1996) Puri-
fication and structural characterization of bovine cath-
elicidins, precursors of antimicrobial peptides. Eur J
Biochem 238, 769–776.
10 Tencza SB, Creighton DJ, Yuan T, Vogel HJ, Montel-
aro RC & Mietzner TA (1999) Lentivirus-derived anti-
microbial peptides: increased potency by sequence
engineering and dimerization. J Antimicrob Chemother
44, 33–41.
11 Dempsey CE, Ueno S & Avison MB (2003) Enhanced
membrane permeabilization and antibacterial activity of
a disulfide-dimerized magainin analogue. Biochemistry
42, 402–409.
J. Y. Lee et al. Salt-resistanthomodimeric bactenecin
FEBS Journal 275 (2008) 3911–3920 ª 2008 The Authors Journal compilation ª 2008 FEBS 3919
12 You S, Peng S, Lien L, Breed J, Sansom MS & Wool-
ley GA (1996) Engineering stabilized ion channels:
covalent dimers of alamethicin. Biochemistry 35, 6225–
6232.
13 Hara T, Kodama H, Kondo M, Wakamatsu K, Takeda
A, Tachi T & Matsuzaki K (2001) Effects of peptide
dimerization on pore formation: antiparallel disulfide-
dimerized magainin 2 analogue. Biopolymers 58, 437–
446.
14 Takei J, Remenyi A, Clarke AR & Dempsey CE (1998)
Self-association of disulfide-dimerized melittin ana-
logues. Biochemistry 37, 5699–5708.
15 Singh PK, Jia HP, Wiles K, Hesselberth J, Liu L, Con-
way BA, Greenberg EP, Valore EV, Welsh MJ, Ganz T
et al. (1998) Production of beta-defensins by human
airway epithelia. Proc Natl Acad Sci USA 95, 14961–
14966.
16 Campopiano DJ, Clarke DJ, Polfer NC, Barran PE,
Langley RJ, Govan JR, Maxwell A & Dorin JR (2004)
Structure–activity relationships in defensin dimers: a
novel link between beta-defensin tertiary structure
and antimicrobial activity. J Biol Chem 279, 48671–
48679.
17 Tam JP, Wu C & Yang JL. (2000) Membranolytic
selectivity of cystine-stabilized cyclic protegrins. Eur J
Biochem 267, 3289–3300.
18 Scocchi M, Zelezetsky I, Benincasa M, Gennaro R,
Mazzoli A & Tossi A (2005) Structural aspects and bio-
logical properties of the cathelicidin PMAP-36. FEBS J
272, 4398–4406.
19 Basak A & Lotfipour F (2005) Modulating furin activ-
ity with designed mini-PDX peptides: synthesis and
in vitro kinetic evaluation. FEBS Lett 579, 4813–4821.
20 Perczel A & Hollosi M (1996) Circular Dichroism and
the Conformational Analysis of Biomolecules. Plenum
Press, New York.
21 Chang CT, Wu CS & Yang JT (1978) Circular dichroic
analysis of protein conformation: inclusion of the beta-
turns. Anal Biochem 91, 13–31.
22 Woody RW. (1995) Circular dichroism. Methods
Enzymol 246, 34–71.
23 Matsuzaki K, Harada M, Funakoshi S, Fujii N &
Miyajima K (1991) Physicochemical determinants for
the interactions of magainins 1 and 2 with acidic lipid
bilayers. Biochim Biophys Acta 1063, 162–170.
24 Hornef MW, Putsep K, Karlsson J, Refai E & Anders-
son M (2004) Increased diversity of intestinal antimicro-
bial peptides by covalent dimer formation. Nat Immunol
5, 836–843.
25 Travis SM, Anderson NN, Forsyth WR, Espiritu C,
Conway BD, Greenberg EP, McCray PB, Lehrer RI,
Welsh MJ & Tack BF (2000) Bactericidal activity of
mammalian cathelicidin-derived peptides. Infect Immun
68, 2748–2755.
26 Goldman MJ, Anderson GM, Stolzenberg ED, Kari
UP, Zasloff M & Wilson JM (1997) Human beta-defen-
sin-1 is a salt-sensitive antibiotic in lung that is inacti-
vated in cystic fibrosis. Cell 88, 553–560.
27 Yomogida S, Nagaoka I & Yamashita T (1996) Purifi-
cation of the 11- and 5-kDa antibacterial polypeptides
from guinea pig neutrophils. Arch Biochem Biophys 328,
219–226.
28 Wu Z, Hoover DM, Yang D, Boule
`
gue C, Santamaria
F, Oppenheim JJ, Lubkowski J & Lu W (2003) Engi-
neering disulfide bridges to dissect antimicrobial and
chemotactic activities of human beta-defensin 3. Proc
Natl Acad Sci USA 100, 8880.
29 Yang ST, Lee JY, Kim HJ, Eu YJ, Shin SY, Hahm KS
& Kim JI (2006) Contribution of a central proline in
model amphipathic alpha-helical peptides to self-associ-
ation, interaction with phospholipids, and antimicrobial
mode of action. FEBS J 273, 4040–4054.
30 Yang ST, Jeon JH, Kim Y, Shin SY, Hahm KS & Kim
JI (2006) Possible role of a PXXP central hinge in the
antibacterial activity and membrane interaction of
PMAP-23, a member of cathelicidin family. Biochemis-
try 45, 1775–1784.
Supplementary material
The following supplementary material is available
online:
Fig. S1. Trypsin cleavage sites and mass values of each
peptide.
Fig. S2. HPLC profiles of the peptide fragments after
trypsin digestion.
This material is available as part of the online article
from http://www.blackwell-synergy.com
Please note: Blackwell Publishing is not responsible
for the content or functionality of any supplementary
materials supplied by the authors. Any queries (other
than missing material) should be directed to the corre-
sponding author for the article.
Salt-resistant homodimeric bactenecin J. Y. Lee et al.
3920 FEBS Journal 275 (2008) 3911–3920 ª 2008 The Authors Journal compilation ª 2008 FEBS
. bacteria [5]. In addition, two lin-
ear variants of bactenecin, Bac2S and Bac 2A, show
similar activities against Gram-negative bacteria and
stronger activities. that
adopt parallel and antiparallel conformations and two
monomers that adopt b-hairpin and linear confor-
mations, and investigated their biological activities.
Results