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 residue
Trang 1cathelicidin-derived antimicrobial peptide
Ju Y Lee1, Sung-Tae Yang1,3, Seung K Lee1, Hyun H Jung1, Song Y Shin2, Kyung-Soo Hahm2 and Jae I Kim1
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 antimicrobial peptide 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 (anti-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, phosphatidylcholine; POPG, 1-palmitoyl-2-oleoyl-phosphatidylglycerol; SLB, Ser-substituted linear bactenecin.
Trang 2antibacterial 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
Trang 3and 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
H 2 N -RLCRIVVIRVCR- CO 2 H
RLCRIVVIRVCR
H 2 N -RLCRIVVIRVCR- CO 2 H
H 2 N - -CO 2 H
Acm SH
PDB ADB
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
2 h.
Trang 4rial 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
0
0 20 40
–30
–20
–40 –20 0
2 · dmol
10
15
PDB
15
20
ADB
3 (deg·c
–10 –5 0 5
0
5
10
–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 C in
10 m M sodium phosphate buffer (pH 7.4) (d) or in 30 m M SDS micelles ( ) Each peptide was used at a concentration of
25 l M
Table 1 MIC (l M ) 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 )
Bacterial strain
100
60 80
20 40
0.001 0.01 0.1 1 10
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.
Trang 5binds 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 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 abspres-ence of 150 mm NaCl In the abspres-ence 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
0
20
0
20
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.
Trang 6reinforcing 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
0
20
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.
Trang 7synthesize 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
10 20
2 ·dmol
-10 0
3 (deg·cm
30
-20
190 200 210 220 230 240 250 190 200 210 220 230 240 250
-30
30
10 20
-10 0
-20 -30
Wavelength (nm)
80 100
80 100
acteria ki 40
60
40 60
0 20
0 20
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 C in 10 m M sodium
phos-phate buffer (pH 7.4) (d) and in 30 m M SDS
micelles ( ) (C) 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.
No salt 300 m M 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.
Trang 8roacetic 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⁄ H2O⁄ 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⁄ H2O (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 47C, 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⁄ H2O (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 25C The spectra are expressed as
molar ellipticity [h] versus wavelength
Antibacterial activity
Antimicrobial activities of each peptide against six selected organisms, including three 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
37C An aliquot of the culture was then transferred to
10 mL of fresh medium and incubated for an additional 3–5 h at 37C 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· 105colony-forming units (CFU)ÆmL)1] in 96-well microtiter plates (F96 microtiter plates; Nunc, Odense, Denmark) and incubated at 37C 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⁄ Pi (35 mm phosphate buffer, 150 mm NaCl, pH 7.4), 100 lL of a 4% (v⁄ v) hRBC suspension in NaCl⁄ Piwas dispensed into sterilized 96-well plates along with 100 lL of peptide solution The plates were then incubated for 1 h at 37C 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 (%) = [(A405 nm sample ) A405 nm zero lysis)⁄ (A405 nm 100% lysis) A405 nm zero lysis)]· 100 Zero and 100% hemolysis were determined in NaCl⁄ Piand 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
Trang 9overnight 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 a peptide 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· 105CFUÆmL)1 After 0, 5, 10, 30 or 60 min of
exposure to the peptides at 37C, 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 37C
To determine the salt sensitivity of the antimicrobial
activity, peptides were incubated at 37C in 100 lL of 1%
peptone solution also containing 2· 105
CFUÆmL)1 bacte-ria and 0 or 150 mm NaCl After incubation for 3 h at
37C, 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)
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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