From the Department of Microbiology and Immunology, University of British Columbia, Vancouver, British Columbia V6T 1Z3, Canada
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ABSTRACT |
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Bactenecin, a 12-amino acid cationic
antimicrobial peptide from bovine neutrophils, has two cysteine
residues, which form one disulfide bond, making it a cyclic molecule.
To study the importance of the disulfide bond, a linear derivative
Bac2S was made and the reduced form (linear bactenecin) was also
included in this study. Circular dichroism spectroscopy showed that
bactenecin existed as a type I Polycationic antimicrobial peptides have been found in a variety
of sources, including humans, mammals, plants, insects, and bacteria
(1). The primary structures of these positively charged molecules are
highly diverse, yet their secondary structures share the common feature
of amphipathicity (2). Recently, a few cationic peptides with only one disulfide bond forming
a looped structure have been identified (7-11). One of them,
bactenecin (also called dodecapeptide), was found in bovine neutrophils
(12). It has 12 amino acids, including four arginine residues and two
cysteine residues and is the smallest known cationic antimicrobial
peptide. The two cysteine residues form a disulfide bond to make
bactenecin a loop molecule. Bactenecin was previously found to be
active against Escherichia coli and Staphylococcus
aureus, and strongly cytotoxic for rat embryonic neurons, fetal
rat astrocytes, and human glioblastoma (13). However, little is known
about its antimicrobial mechanism and whether it shares the common
killing mechanism of other antimicrobial peptides or if it has a
distinct mode of action due to its unique compact structure
(cf. the silk moth peptide cecropin, which is a 26-amino
acid amphipathic Bacterial Strains and Chemicals--
Bacterial strains for
antimicrobial activity testing included E. coli UB1005 and
its antibiotic supersusceptible derivative DC2 (14), Pseudomonas
aeruginosa K799 and its antibiotic-supersusceptible derivative Z61
(15), Salmonella typhimurium 14028s (16), S. aureus ATCC25923, and clinical isolates of Staphylococcus
epidermidis (clinical isolate), Enterococcus faecalis
ATCC29212, and Listeria monocytogenes (food isolate).
Polymyxin B and 1-N-phenylnaphylamine
(NPN)1 were purchased from
Sigma. 3,3-Dipropylthiacarbocyanine (DiS-C3-(5)) was from Molecular Probes (Eugene, Oregon). Dansyl-polymyxin B was synthesized as described previously (18). The lipids
1-pamitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and
1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG) were purchased from Northern Lipids Inc. (Vancouver, British Columbia, Canada).
Synthesis and Refolding of Bactenecin--
Bactenecin and
variants bac2S were synthesized by Fmoc
(N-(9-fluorenyl)methoxycarbonyl) chemistry by the Nucleic
Acid/Protein Service unit at the University of British Columbia using
an Applied Biosystems, Inc. (Foster City, CA) model 431 peptide
synthesizer. The purchased bactenecin variants were in their fully
reduced forms. After a series of trials to determine the optimal
strategy, the disulfide bond was formed by air-oxidation in 0.01 M Tris buffer at room temperature for 24 h. The
concentration of bactenecins was kept below 100 µg/ml in the
oxidation buffer to minimize the formation of multimers. A reversed
phase column Pep RPC HR5/5 (Amersham Pharmacia Biotech; Quebec, Canada)
was used to purify the disulfide-bonded bactenecins from their multimer
by-products. The column was equilibrated with 0.3% (v/v) aqueous
trifluoroacetic acid and eluted with a gradient of acetonitrile in
0.3% trifluoroacetic acid at a flow rate of 0.7 ml/min. Peptide
concentration was determined by amino acid analysis. Matrix-assisted
laser desorption/ionization (MALDI)-mass spectrometry (for native
bactenecin only) and acid-urea polyacrylamide gel electrophoresis (19)
were used to confirm that the disulfide bond was properly formed and a
pure product obtained.
Circular Dichroism--
A Jasco (Japan) J-720 spectropolarimeter
was used to measure the circular dichroism spectra (20). The data were
collected and analyzed by Jasco software. Liposomes POPC/POPG (7:3)
were prepared by the freeze-thaw method to produce multilamellar
vesicles as described previously (21), followed by extrusion through 0.1-µm double-stacked Nuclepore filters using an extruder device (Lipex Biomembranes, Vancouver, British Columbia, Canada), resulting in
unilamellar liposomes. Peptide at a final concentration of 50 µM was added to 100 µM liposomes and
incubated at room temperature for 10 min before the CD measurement.
Antimicrobial Activity--
The minimal inhibitory concentration
of peptides was determined by a modified 2-fold microtiter broth
dilution method modified from that of Steinberg et al. (22).
Using the classical method (23), higher concentrations of peptides tend
to precipitate in the LB broth, thus the concentrations of peptides in
the sequential wells are not accurate. Also the peptides stick to the
most readily available (tissue-culture treated polystyrene) 96-well
microtiter plates. Therefore the 2× series of dilutions was performed
in Eppendoff tubes (polypropylene) before mixing with LB broth. Serial of 2-fold dilutions of peptides ranging from 640 to 1.25 µg/ml were
made in 0.2% bovine serum albumin, 0.01% acetic acid buffer in the
Eppendoff tubes. Ten µl of each concentration was added to each
corresponding well of a 96-well microtiter plate (polypropylene cluster; Costar Corp., Cambridge, MA). Bacteria were grown overnight and diluted 10 Dansyl-Polymyxin B Displacement Assay--
E. coli
UB1005 LPS was prepared according to the phenol-chloroform-petroleum
ether extraction method (24). The dansyl-polymyxin B displacement assay
(25) was used to determine the relative binding affinity of peptides
for LPS.
Membrane Permeabilization Assays--
The ability of peptides to
permeabilize the outer membrane was determined by the NPN assay of Loh
et al. (26). Cytoplasmic membrane permeabilization was
determined by using the membrane potential sensitive cyanine dye
DiS-C3-(5) (27). The mutant E. coli DC2 with
increased outer membrane permeability was used so that
DiS-C3-(5) could reach the cytoplasmic membrane. Fresh LB
medium was inoculated with an overnight culture, grown at 37 °C, and
mid-logarithmic phase cells (A600 = 0.5-0.6)
were collected. The cells were washed with buffer (5 mM
HEPES, pH 7.2, 5 mM glucose) once, then resuspended in the
same buffer to an A600 of 0.05. The cell
suspension was incubated with 0.4 µM
DiS-C3-(5) until DiS-C3-(5) uptake was maximal
(as indicated by a stable reduction in fluorescence due to fluorescence
quenching as the dye became concentrated in the cell by the membrane
potential), and 100 mM KCl was added to equilibrate the
cytoplasmic and external K+ concentration. One ml of cell
culture was placed in a 1-cm cuvette, and the desired concentration of
tested peptide was added. The fluorescence reading was monitored by
using a Perkin-Elmer model 650-10S fluorescence spectrophotometer
(Perkin-Elmer Corp.), with an excitation wavelength of 622 nm and an
emission wavelength of 670 nm. The maximal increase of fluorescence due
to the disruption of the cytoplasmic membrane by certain concentration
of cationic peptide was recorded. A blank with only cells and the dye
was used to subtract the background. Control
experiments2 titrating with
valinomycin and K+ showed that the increase in fluorescence
was directly proportional to the membrane potential and that a buffer
concentration of 100 mM KCl prevented any effects of the
high internal K+ concentration and corresponding opposing
chemical gradient.
Bactenecin and Its Linear Derivative--
The amino acid sequence
of bactenecin and its linear derivative are shown in Table
I. The linear derivative (Lin-Bac2S) with two cysteine residues replaced by two serine residues, was made to
determine the importance of the disulfide bond in bactenecin's antimicrobial activity. The reduced form of bactenecin was also included in this study as a linear version of bactenecin. The identity
of these peptides was confirmed by MALDI mass spectrometry. The MALDI
data showed the molecular mass of the reduced bactenecin as 1486 ± 1 dalton and oxidized bactenecin as 1484 ± 1 dalton, in
agreement with formation of one disulfide bond in the latter. Linear
reduced bactenecin did not reform its disulfide bonds spontaneously within the lifetime of these experiments, as confirmed by its gel
electrophoretic mobility (which was altered by disulfide bond formation).
Circular Dichroism--
CD spectrometry (Fig.
1A) showed that linear,
reduced bactenecin and linear Bac2S were present in 10 mM
sodium phosphate buffer as unordered structures, which had a strong
negative ellipticity near 200 nm. The CD spectrum of native bactenecin
(Fig. 1A) demonstrated a negative ellipticity near 205 nm,
typical of that seen for a type I Antimicrobial Activity--
The MIC of bactenecin and its
derivatives against a range of bacteria was determined (Table
II) by using a modified broth dilution
method. Bactenecin was active against all Gram-negative bacteria
tested. It was relatively inactive (MIC = 64 µg/ml) against the
Gram-positive bacterium S. aureus, in contrast to a previous report (12). The linear variant Lin-Bac2S and reduced bactenecin (Lin-Bac) were inactive against wild type Gram-negative bacteria. P. aeruginosa Z61 and E. coli DC2 are outer
membrane barrier-defective mutants, that have more permeable outer
membranes than their parent strains, allowing potentially easier access
of the peptides to the cytoplasmic membrane. All three bactenecins
exhibited equivalent activity against these two mutants. Linearization
by reduction or changing cysteine to serines dramatically changed the
antimicrobial activity for two Gram-positive species S. epidermidis and Enterococcus facaelis. For other
antimicrobial peptides with disulfide bonds, reduction of these
disulfides generally results in complete loss of antimicrobial activity
(30-32). In contrast, accompanying the linearization was a shift in
spectrum of activity from Gram-negative selective to Gram-positive
selective, which corresponded to the substantially different structures
adopted in liposomes.
The Binding of Bactenecins to Purified E. coli UB1005--
The MIC
results indicated that the interaction with the outer membrane might be
critical in the explaining the difference in antimicrobial activity
against Gram-negative bacteria among three bactenecin forms. The first
step of cationic peptide antimicrobial action has been shown to involve
the binding of the cationic peptide to the negatively charged surface
of the target cells (1). In Gram-negative bacteria, this initial
interaction occurs between the cationic peptides and the negatively
charged LPS in the outer membrane (20, 33, 34). Such binding can be
quantified using the dansyl-polymyxin B displacement assay.
Dansyl-polymyxin B is a fluorescently tagged cationic lipopeptide,
which is nonfluorescent in free solution, but fluoresces strongly when
it binds to LPS. When the peptides bind to LPS, they displace
dansyl-polymyxin B, resulting in decreased fluorescence, which can be
assessed as a function of peptide concentration (Fig.
2). Bactenecin was a relatively weak LPS
binder compared with polymyxin B and similar to the peptide indolicidin
(13 amino acids with a net charge of +2; Ref. 20), but it was still
better than Mg2+, the native divalent cation associated
with LPS. Most importantly, it seemed that native cyclic bactenecin
bound to LPS far better than its linear derivatives, which partially
explained the difference in activities against Gram-negative
bacteria.
Effect on Outer Membrane Permeability--
Antimicrobial peptides
bind to LPS, displacing the native divalent cations. Due to their bulky
nature they disrupt the outer membrane and self-promote their own
uptake across the outer membrane (33, 34). In order to determine
whether better binding ability resulted in better outer membrane
permeabilization, a NPN assay was performed. NPN is a neutral
hydrophobic probe that is excluded by an intact outer membrane, but is
taken up into the membrane interior of an outer membrane that is
disrupted by antimicrobial peptide action. NPN fluoresces weakly in
free solution but strongly when it enters the membrane. Fig.
3 showed that polymyxin B permeabilized the outer membrane to a 50% of maximal increase in fluorescence arbitrary units at 0.4 µg/ml, while bactenecin, Lin-Bac2S, and linear
bactenecin caused half-maximal permeabilization at 0.8, 2, and 4.5 µg/ml, respectively. Bactenecin was thus better than the linearized
derivatives at permeabilizing the outer membrane of E. coli
UB1005.
Effect on the Inner Membrane Potential Gradient--
It has been
proposed that the antibacterial target of cationic peptides is at the
cytoplasmic membrane. Cationic peptides are generally able to interact
electrostatically with the negatively charged headgroups of bacterial
phospholipids and then insert into the cytoplasmic membrane, forming
channels or pores that are proposed to lead to the leakage of cell
contents and cell death. However there is very little data for peptides
pertaining to measurement of the disruption of the cytoplasmic membrane
permeability barrier, despite ample evidence that membrane disruption
can occur in model membrane systems (35). Although, some authors have utilized measurements of the accessibility of a normally
membrane-impermeable substrate to cytoplasmic Structure-Activity Relationships--
A series of peptides related
to bactenecin were made (Table I) in an attempt to decipher important
features of these peptides contributing to antimicrobial activity.
Included in this series were peptides that differed in charge due
either to amidation of the carboxyl terminus (Lin-Bac2S-NH2
and BacP-NH2) or addition of arginines to the
NH2 and COOH terminus (Lin-BacR and cyclic BacR), contained
an added proline residue in the bactenecin ring to promote cyclization
(BacP), or contained a substitution of three lysines for arginines
(BacP3K). In total, four linear peptides (denoted Lin-Bac for clarity)
and five cyclic peptides were investigated. Antimicrobial activity was
assessed for the bacteria studied above in addition to two
Gram-positive pathogens, S. aureus ATCC25923 and a food
isolate of L. monocytogenes (Table
III). The latter Gram-positive bacterium
was reasonably susceptible to cyclic bactenecin; however, the linear
bactenecin and Lin-Bac2S were 8-fold more active (but not particularly
active against S. aureus).
Among the linear peptides, an increase in positive charge tended to
result in increased activity against Gram-negative bacteria for both
Lin-Bac2S-NH2 (+4) and Lin-BacR (+5). However neither of
these peptides had activities (except against E. coli)
equivalent to that of cyclic bactenecin. The increase in positive
charge of the peptides also tended to result in an increase in activity against the Gram-positive bacteria (cf. BacR
versus Bac, Lin-Bac2S-NH2 versus
Lin-Bac2S, BacP-NH2 versus BacP). Clearly
amidation of the carboxyl terminus was very favorable to antimicrobial
activity against both Gram-negative and Gram-positive bacteria.
Overall, good Gram-positive activity tended to require the peptides to be linear, although the cyclic peptide BacP-NH2 had
reasonable activities against the Gram-positive bacteria S. aureus and L. monocytogenes.
The substitution of an arginine with a proline residue in the ring
structure in BacP (and moving the arginine in place of the leucine
residue at position 2) resulted in a loss of activity against all
bacteria except E. coli. This indicated that the
three-dimensional structure of the peptide was important, since the net
charge was identical to that of bactenecin, the overall hydrophobicity
very similar, and the substitution of Arg for Leu in position 2 was not
detrimental in BacR. Interestingly, the further substitution of three
arginines for three lysine residues in BacP3K resulted in a very weakly
active peptide, suggesting that these two basic residues may not be
equally effective in promoting bactenecin activity.
The Bactenecin belongs to a group of cationic peptides with only one
disulfide bond. In this study, it was shown that bactenecin was active
against the wild type Gram-negative bacteria E. coli, P. aeruginosa, and S. typhimurium, whereas the
linear derivative and reduced form were virtually inactive against
these bacteria but had gained activity against certain Gram-positive
bacteria. For other disulfide-bonded peptides such as the Furthermore, cyclic bactenecin behaved in a fundamentally different
fashion to the linear bactenecins and the In contrast, it would appear that the linear bactenecins are acting in
the same way on the cytoplasmic membrane of bacteria as do other larger
peptides. The linear variants dissipated the cytoplasmic membrane
potential at the MIC and showed partial activity on membranes (data not
shown), even at concentrations as low as 0.125 µg/ml (less than 1%
of the MIC). However, while it is straightforward to imagine how 28-mer
peptides like CEMA might be able to span a biological membrane to form
a channel by a barrel-stave mechanism (1, 5, 6), it is not so simple to
understand how a 12-mer peptide containing 50% polar residues could
span such a membrane.
Both the cyclic and linear versions of bactenecin, as well as Bac2S,
were equally active against outer membrane permeability defective
mutants of E. coli and P. aeruginosa. This
observation indicated that the disulfide bond was important for
interaction with the outer membrane as confirmed here. Bactenecin had a
better binding ability for LPS and also permeabilized the outer
membrane better, explaining its better activity versus wild
type Gram-negative bacteria. Computer modeling of bactenecin with
InsightII software (Biosym Technologies Inc., San Diego, CA) indicated
that bactenecin was a loop molecule with a hydrophobic ring and a
positively charged face constructed from the COOH- and
NH2-terminal portions of the molecule (2). Such a
conformation, which was consistent with the CD spectral studies (Fig.
2) which indicated that bactenecin existed as a rigid The original report of the isolation of bactenecin suggested it
was active against both E. coli and S. aureus
(12), whereas we demonstrated that cyclic bactenecin has very little
activity against the latter bacterium. Therefore, we are tempted to
speculate that Romeo et al. (12) were working with a mixture
of linear and cyclic bactenecin or that their preparations were partly
or completely amidated (since amidation of two of our peptides improved activity against S. aureus by 4-8-fold). Unfortunately,
despite two attempts to synthesize amidated bactenecin, we were unable to obtain a preparation sufficiently pure enough to permit
identification of the desired product.
Our studies of structure activity relationships revealed certain
factors that were important in the activity of the linear and cyclic
bactenecins against bacteria. The most obvious correlations observed
were the improvement in activity against Gram-negative bacteria with
cyclization (due to disulfide bond formation) and with increased
positive charge. In addition while cyclization tended to decrease
activity against Gram-positive bacteria, while increasing the positive
charge by addition of two arginines or by amidation of the
COOH-terminal carboxyl, led to an improvement in activity against
Gram-positive bacteria. Despite the small size of these peptides, we
observed MICs against important bacterial pathogens that are equal to
or better than much larger peptides, and we suggest that these peptides
offer a potentially fruitful basis for isolation of antibiotic peptides
for clinical use.
-turn structure regardless of its
environment, while the reduced form and linear bactenecin adopted
different conformations according to the lipophilicity of the
environment. Bactenecin was more active against the Gram-negative wild
type bacteria Escherichia coli, Pseudomonas
aeruginosa, and Salmonella typhimurium than its
linear derivative and reduced form, while all three peptides were
equally active against the outer membrane barrier-defective mutants of
the first two bacteria. Only the two linear peptides showed activity
against the Gram-positive bacteria Staphylococcus
epidermidis and Enterococcus facaelis. Bactenecin
interacted well with the outer membrane and its higher affinity for
E. coli UB1005 lipopolysaccharide and improved ability to
permeabilize the outer membrane seemed to account for its better antimicrobial activity against Gram-negative bacteria. The interaction of bactenecin with the cytoplasmic membrane was determined by its
ability to dissipate the membrane potential by using the fluorescence probe 3,3-dipropylthiacarbocyanine and an outer membrane
barrier-defective mutant E. coli DC2. It was shown that the
linear derivative and reduced form were able to dissipate the membrane
potential at much lower concentrations than bactenecin despite the
similar minimal inhibitory concentrations of all three against this
barrier-defective mutant.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
REFERENCES
-Helical peptides, including cecropins (3)
and
-sheet peptides, including defensins (4), have been studied
extensively. It has been proposed (1, 2) that cationic peptides first
interact with bacteria by binding to their negatively charged surfaces,
and for Gram-negative bacteria they act as outer membrane
permeabilizers. Their interactions with the cytoplasmic membrane have
been proposed to lead to the disruption of membrane structure (5),
resulting in dissipation of the transmembrane potential (6) and
eventual cell death.
-helix). Its small size and only single disulfide
bond also makes bactenecin an interesting candidate for research and
drug development. The aim of this study was to investigate how
bactenecin interacts with and kills microoganisms. Interestingly we
found a rather distinct spectrum of activity for bactenecin compared
with its linear form.
MATERIALS AND METHODS
5 into fresh LB broth or Todd Hewitt broth
for Streptococcus. LB medium contained 10 g/liter tryptone
and 5 g/liter yeast extract, with no salt. Todd Hewitt contained 500 g/liter beef heart infusion, 20 g/liter bacto-neopeptone, 2 g/liter
bacto-dextrose, 2 g/liter sodium chloride, 0.4 g/liter disodium
phosphate, 2.5 g/liter sodium carbonate. One-hundred µl of broth
containing about 104-105 colony-forming
units/ml of tested bacteria was added to each well. The plate was
incubated at 37 °C overnight. The MIC was taken as the concentration
at which greater than 90% of growth inhibition was observed.
RESULTS
Amino acid sequences of bactenecin and its derivatives
-turn structure (29) and
resembling oxyribonuclease and nuclease, which are short polypeptides
with a disulfide bond (28). In 60% TFE buffer, in the presence of
liposomes and 10 mM SDS, the native bactenecin retained a
similar structure (Fig. 1, B-D). However, the
reduced form and Lin-Bac2S exhibited clearly distinct structures from
those observed in the aqueous solution. In 60% trifluoroethanol
(considered a helix-inducing solvent), these two peptides tended to
form an
-helical structure (Fig. 1B), whereas and in the
presence of liposomes or 10 mM SDS (a membrane-mimicking
detergent), a
-sheet structure was evident (Fig. 1, C and
D).
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Fig. 1.
CD spectra of bactenecin, its linear
(reduced) form Lin Bac and a linear variant Lin bac2S in media of
various lipophilicity. The concentrations of peptides and
liposomes were 50 and 100 µM, respectively. CD
measurements were taken in 10 mM sodium phosphate buffer
(pH 7.0) in the absence (A) and the presence (B)
of POPC/POPG. C shows the spectra in the presence of 60%
(v/v) TFE, and D shows the spectra in the presence of 10 mM
SDS. Open circles, bactenecin; solid line,
reduced bactenecin; dashed line,
Lin-Bac2S.
Differential activity of native cyclic and linear bactenecins against
Gram-negative bacteria, outer membrane-altered, antibiotic
supersusceptible mutants DC2 and Z61, and selected Gram-positive
bacteria
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Fig. 2.
Binding of peptides to LPS as assessed by
their ability to displace dansyl-polymyxin B from E. coli
UB1005 LPS. Dansyl-polymyxin B was added to 1 ml of 3 µg/ml LPS to a final concentration of 1 µM, which
saturated the binding sites on LPS, and the fluorescence sensitivity
was adjusted to 90%. The peptides and Mg2+ were titrated
in, resulting in a decrease in fluorescence due to the competitive
displacement of dansyl-polymyxin from the LPS, resulting in a reduction
in fluorescence. Symbols: triangles, cyclic bactenecin;
squares, Lin-Bac2S; closed circles, linear
(reduced) bactenecin (Lin Bac), dashed line, polymyxin B;
dotted line, indolicidin (from Ref. 20);
diamonds, MgCl2.
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Fig. 3.
Peptide-induced outer membrane
permeabilization assessed by the NPN uptake in E. coli
UB1005. Mid-log phase E. coli cells were
collected and incubated with NPN in the presence of various
concentrations of native cyclic bactenecin (oxidized), linear reduced
bactenecin (Lin Bac), and Lin Bac2S. NPN was taken up into cells when
the outer membrane was disrupted by the peptides. The uptake of NPN was
measured by the increase of fluorescence. Symbols:
triangles, cyclic bactenecin; squares, Lin-Bac2S;
closed circles, linear (reduced) bactenecin; open
circles, polymyxin B.
-galactosidase, this
assay suffers from using a bulky substrate (ortho-nitrophenyl
galactoside) (36, 37). To circumvent this, we have developed an assay
involving the membrane potential-sensitive dye diS-C3-(5)
to measure the disruption of electrical potential gradients in intact
bacteria. The use of the E. coli mutant DC2 permitted us to
perform this assay in the absence of EDTA (required by previous workers
who have used similar assays in E. coli (38, 39)). The
fluorescent probe diS-C3-(5), which is a caged cation,
distributes between cells and medium depending on the cytoplasmic
membrane potential. Once it is inside the cells, it becomes
concentrated and self-quenches its own fluorescence. If peptides form
channels or otherwise disrupt the membrane, the membrane potential will
be dissipated, and the DiS-C3-(5) will be released into the
medium causing the fluorescence to increase, as can be detected by
fluorescent spectrometry. In these assays, 0.1 M KCl was
added to the buffer to balance the chemical potential of K+
inside and outside the cells. Therefore the MICs of bactenecin, reduced
bactenecin, and bac2S in the presence of 0.1 M KCl were determined and shown to be 8-16 µg/ml (i.e. 4-8-fold
higher than in low salt). Despite these similar MICs for the three
peptides versus E. coli DC2, the influence of
these peptides on the membrane potential was quite different (Fig.
4). The linear bactenecins at around
their MIC (8 µg/ml) caused a rapid increase in fluorescence that was
similar to that seen for a control
-helical peptide CEMA at its MIC
of 1 µg/ml. However despite a similar 30-s delay prior to initiation,
the kinetics were somewhat slower with CEMA causing a maximal
depolarization of the cytoplasmic membrane (increase in fluorescence)
within 2 min, whereas the linear bactenecins caused only 50% maximal
depolarization of the cytoplasmic membrane in this period of time. In
stark contrast to both the linear bactenecins and CEMA, native cyclic
bactenecin at 8 µg/ml caused a very modest depolarization within the
first 5 min (14% of that observed with reduced bactenecin, reaching a
maximum of 30% in 1 h).
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Fig. 4.
Peptide-induced inner membrane
permeabilization assessed by the diS-C3-(5) assay.
Mid-log phase cells were collected and resuspended in buffer (5 mM HEPES, 5 mM glucose) to an
A600 of 0.05. A 0.4 µM final
concentration of diS-C3-(5) was incubated with cell
suspensions until no more quenching was detected, then 0.1 M KCl was added. The desired peptide concentration (8 µg/ml for the bactenecins and 1 µg/ml for CEMA) was added to a 1-cm
cuvette containing 1 ml of cell suspension. The fluorescence change (in
arbitrary units) was observed as a function of time. Symbols:
triangles, cyclic bactenecin; squares, Lin-Bac2S;
closed circles, linear (reduced) bactenecin; open
circles, CEMA.
Structure-activity relationships amongst cyclic and linear bactenecins
DISCUSSION
-helical and
-structured classes are two groups of
antimicrobial polycationic peptides that have been well studied. Although their precise antimicrobial mechanism is somewhat unclear, it
has been proposed that the outer and the cytoplasmic membranes of
Gram-negative bacteria are their primary and final targets respectively
(1). They have been proposed to kill bacteria by first
electrostatically interacting with the surface of the bacterial
cytoplasmic membrane (after self-promoted uptake across the outer
membrane for Gram-negative bacteria). Then under the influence of a
membrane potential, they are proposed to insert into the membrane and
form channels to leak internal constituents. However much of this
mechanism is based on data from model membrane studies.
-sheet
defensins (30), the protegrins (31), and the tachyplesins (32), the loss of ability to form a disulfide bond results in a complete loss of
structure and activity. Thus the observation that bactenecin, when
linearized, undergoes a dramatic shift in activity spectrum (Table II)
and in structure (Fig. 2) is unprecedented and surprising.
-helical 28-amino acid
peptide CEMA (34), with respect to cytoplasmic membrane permeabilization (depolarization of the membrane potential gradient). Previous studies used artificial liposomes to study the interaction of
antimicrobial peptides with membranes. In this study, live cells of
E. coli DC2, an outer membrane hyperpermeable mutant, were
used in conjunction with a fluorescent dye, diS-C3-(5),
which was released from cells when the membrane potential is disrupted, leading to fluorescence dequenching. Despite their equivalent MIC value
against E. coli DC2, the pattern of interaction of
bactenecin and its linear variants with the cytoplasmic membrane was
quite different. Whereas CEMA and linear bactenecin and Lin-Bac2S
caused rapid depolarization of the membrane, cyclic bactenecin caused only a slow and minor change in membrane potential. Thus we conclude that cyclic bactenecin kills cells in a completely different way to the
other antimicrobial peptides, which have been proposed to act on the
cytoplasmic membrane of bacteria. Although the actual mechanism of
killing was not investigated in this study, we propose that bactenecin
is able to cross the cytoplasmic membrane of Gram-negative bacteria and
act on a target inside cells (e.g. negatively charged nucleic acids).
-turn loop
molecule regardless of its environment, may make bactenecin a more
amphipathic molecule than the unstructured linear and reduced forms,
which exist in solution as random structures. This could explain why
bactenecin interacted better with the negatively charged LPS than its
linear and reduced form. It is also worth mentioning that bactenecin
would also be too small to span the membrane and form pores or channels
unless a multimer is involved.
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FOOTNOTES |
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* This work was supported by the Canadian Bacterial Diseases Network and Micrologix Biotech Inc.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.
Recipient of a British Columbia Science Council Graduate Research
Engineering and Technology studentship.
§ Medical Research Council Distinguished Scientist Award. To whom correspondence should be addressed. Tel.: 604-822-2682; Fax: 604-822-6041; E-mail: bob{at}cmdr.ubc.ca.
2 M. Wu, E. Maier, R. Benz, and R. E. W. Hancock, submitted for publication.
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ABBREVIATIONS |
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The abbreviations used are: NPN, 1-N-phenylnaphylamine; DiS-C3-(5), 3,3-dipropylthiacarbocyanine; dansyl, 5-dimethylaminonaphthalene-1-sulfonyl; POPC, 1-pamitoyl-2-oleoyl-sn-glycero-3-phosphocholine; POPG, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol; MALDI, matrix-assisted laser desorption/ionization; MIC(s), minimal inhibitory concentration(s); LPS, lipopolysaccharide..
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REFERENCES |
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