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Manhong Wu and Robert E. W. Hancock
and Cytoplasmic Membrane
OuterCationic Peptide Bactenecin with the
Interaction of the Cyclic Antimicrobial
STRUCTURE:
PROTEIN CHEMISTRY AND
doi: 10.1074/jbc.274.1.29
1999, 274:29-35.J. Biol. Chem.
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Interaction of the Cyclic Antimicrobial Cationic Peptide Bactenecin
with the Outer and Cytoplasmic Membrane*
(Received for publication, April 24, 1998, and in revised form, July 19, 1998)
Manhong Wu‡ and Robert E. W. Hancock§
From the Department of Microbiology and Immunology, University of British Columbia, Vancouver,
British Columbia V6T 1Z3, Canada
Bactenecin, a 12-amino acid cationic antimicrobial
peptide from bovine neutrophils, has two cysteine resi-
dues, 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 bactene-
cin existed as a type I
b
-turn structure regardless of its
environment, while the reduced form and linear bacte-
necin 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 Salmo-
nella typhimurium than its linear derivative and re-
duced 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 lipopolysaccha-
ride and improved ability to permeabilize the outer
membrane seemed to account for its better antimicro-
bial activity against Gram-negative bacteria. The inter-
action of bactenecin with the cytoplasmic membrane
was determined by its ability to dissipate the membrane
potential by using the fluorescence probe 3,3-dipropy-
lthiacarbocyanine and an outer membrane barrier-de-
fective 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 con-
centrations of all three against this barrier-defective
mutant.
Polycationic antimicrobial peptides have been found in a
variety of sources, including humans, mammals, plants, in-
sects, and bacteria (1). The primary structures of these posi-
tively charged molecules are highly diverse, yet their secondary
structures share the common feature of amphipathicity (2).
a
-Helical peptides, including cecropins (3) and
b
-sheet pep-
tides, 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 mem-
brane have been proposed to lead to the disruption of mem-
brane structure (5), resulting in dissipation of the transmem-
brane potential (6) and eventual cell death.
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 resi-
dues form a disulfide bond to make bactenecin a loop molecule.
Bactenecin was previously found to be active against Esche-
richia coli and Staphylococcus aureus, and strongly cytotoxic
for rat embryonic neurons, fetal rat astrocytes, and human
glioblastoma (13). However, little is known about its antimi-
crobial 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
a
-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. Interest-
ingly we found a rather distinct spectrum of activity for bacte-
necin compared with its linear form.
MATERIALS AND METHODS
Bacterial Strains and Chemicals—Bacterial strains for antimicrobial
activity testing included E. coli UB1005 and its antibiotic supersuscep-
tible derivative DC2 (14), Pseudomonas aeruginosa K799 and its anti-
biotic-supersusceptible derivative Z61 (15), Salmonella typhimurium
14028s (16), S. aureus ATCC25923, and clinical isolates of Staphylococ-
cus 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-C
3
-(5)) was from Mo-
lecular 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-phos-
phoglycerol (POPG) were purchased from Northern Lipids Inc. (Van-
couver, 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
* This work was supported by the Canadian Bacterial Diseases Net-
work and Micrologix Biotech Inc. The costs of publication of this article
were defrayed in part by the payment of page charges. This 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@cmdr.ubc.ca.
1
The abbreviations used are: NPN, 1-N-phenylnaphylamine; DiS-C3-
(5), 3,3-dipropylthiacarbocyanine; dansyl, 5-dimethylaminonaphtha-
lene-1-sulfonyl; POPC, 1-pamitoyl-2-oleoyl-sn-glycero-3-phosphocho-
line; POPG, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol; MALDI,
matrix-assisted laser desorption/ionization; MIC(s), minimal inhibitory
concentration(s); LPS, lipopolysaccharide.
THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 274, No. 1, Issue of January 1, pp. 29–35, 1999
© 1999 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.
This paper is available on line at http://www.jbc.org 29
bactenecins was kept below 100
m
g/ml in the oxidation buffer to mini-
mize 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-prod-
ucts. The column was equilibrated with 0.3% (v/v) aqueous trifluoro-
acetic acid and eluted with a gradient of acetonitrile in 0.3% trifluoro-
acetic 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-
m
m double-stacked Nuclepore filters using an extruder device (Li-
pex Biomembranes, Vancouver, British Columbia, Canada), resulting
in unilamellar liposomes. Peptide at a final concentration of 50
m
M was
added to 100
m
M 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 23 series of dilutions was performed in Eppendoff tubes (polypro-
pylene) before mixing with LB broth. Serial of 2-fold dilutions of pep-
tides ranging from 640 to 1.25
m
g/ml were made in 0.2% bovine serum
albumin, 0.01% acetic acid buffer in the Eppendoff tubes. Ten
m
l of each
concentration was added to each corresponding well of a 96-well micro-
titer plate (polypropylene cluster; Costar Corp., Cambridge, MA). Bac-
teria were grown overnight and diluted 10
25
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 phos-
phate, 2.5 g/liter sodium carbonate. One-hundred
m
l of broth containing
about 10
4
–10
5
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 inhi-
bition was observed.
Dansyl-Polymyxin B Displacement Assay—E. coli UB1005 LPS was
prepared according to the phenol-chloroform-petroleum ether extrac-
tion 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 per-
meabilize the outer membrane was determined by the NPN assay of
Loh et al. (26). Cytoplasmic membrane permeabilization was deter-
mined by using the membrane potential sensitive cyanine dye DiS-C
3
-
(5) (27). The mutant E. coli DC2 with increased outer membrane per-
meability was used so that DiS-C
3
-(5) could reach the cytoplasmic
membrane. Fresh LB medium was inoculated with an overnight cul-
ture, grown at 37 °C, and mid-logarithmic phase cells (A
600
5 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 A
600
of 0.05. The cell suspension was incubated with 0.4
m
M DiS-C
3
-(5) until
DiS-C
3
-(5) uptake was maximal (as indicated by a stable reduction in
fluorescence due to fluorescence quenching as the dye became concen-
trated in the cell by the membrane potential), and 100 mM KCl was
added to equilibrate the cytoplasmic and external K
1
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 wave-
length of 622 nm and an emission wavelength of 670 nm. The maximal
increase of fluorescence due to the disruption of the cytoplasmic mem-
brane by certain concentration of cationic peptide was recorded. A blank
with only cells and the dye was used to subtract the background.
Control experiments
2
titrating with valinomycin and K
1
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
1
concentration and corresponding oppos-
ing chemical gradient.
RESULTS
Bactenecin and Its Linear Derivative—The amino acid se-
quence 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 deter-
mine the importance of the disulfide bond in bactenecin’s an-
timicrobial 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 spec-
trometry. The MALDI data showed the molecular mass of the
reduced bactenecin as 1486 6 1 dalton and oxidized bactenecin
as 1484 6 1 dalton, in agreement with formation of one disul-
fide 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 mo-
bility (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
m
M 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 elliptic-
ity near 205 nm, typical of that seen for a type I
b
-turn struc-
ture (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 m
M 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
a
-helical structure (Fig. 1B),
whereas and in the presence of liposomes or 10 m
M SDS (a
membrane-mimicking detergent), a
b
-sheet structure was evi-
dent (Fig. 1, C and D).
Antimicrobial Activity—The MIC of bactenecin and its deriv-
atives 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 5 64
m
g/ml) against the Gram-positive bacte-
2
M. Wu, E. Maier, R. Benz, and R. E. W. Hancock, submitted for
publication.
T
ABLE I
Amino acid sequences of bactenecin and its derivatives
Peptide classification Name Amino acid sequence
a
Size Net charge
Native Cyclic bactenecin RLCRIVVIRVCR 12 14
Linear Lin Bac
RLCRIVVIRVCR 12 14
Lin Bac2S
RLSRIVVIRVSR 12 14
Lin Bac2S-NH
2
RLSRIVVIRVSR-NH
2
12 15
Lin BacR
RRLCRIVVIRVCRR 14 16
Cyclic BacR
RRLCRIVVIRVCRR 14 16
BacP
RRCPIVVIRVCR 12 14
BacP-NH
2
RRCPIVVIRVCR-NH
2
12 15
BacP3K
KKCPIVVIRVCK 12 14
a
One-letter amino acid code. Cysteine residues linked together with disulphide bonds are underlined
Antimicrobial Mechanism of the Cyclic Cationic Peptide Bactenecin30
rium S. aureus, in contrast to a previous report (12). The linear
variant Lin-Bac2S and reduced bactenecin (Lin-Bac) were in-
active 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. Lin-
earization by reduction or changing cysteine to serines dramat-
ically 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 selec-
tive to Gram-positive selective, which corresponded to the sub-
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
m
M, 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 m
M SDS. Open circles, bactenecin; solid line, reduced bactenecin; dashed line, Lin-Bac2S.
T
ABLE II
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
Species and strains Relevant phenotype
MIC
Bactenecin (oxidized) Lin Bac Lin Bac2S
m
g/ml
E. coli UB1005 Parent of DC2 8 .64 32
E. coli DC2 Antibiotic-sensitive 2 2 2
P. aeruginosa K799 Parent of Z61 4 .64 .64
P. aeruginosa Z61 Antibiotic-sensitive 0.5 0.5 0.5
S. typhimurium Wild type 8 .64 .64
S. epidermidis Clinical isolate .64 8 8
E. faecalis Wild type .64 8 8
Antimicrobial Mechanism of the Cyclic Cationic Peptide Bactenecin 31
stantially different structures adopted in liposomes.
The Binding of Bactenecins to Purified E. coli UB1005—The
MIC results indicated that the interaction with the outer mem-
brane 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 anti-
microbial 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 fluo-
resces 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 12; Ref. 20),
but it was still better than Mg
21
, the native divalent cation
associated with LPS. Most importantly, it seemed that native
cyclic bactenecin bound to LPS far better than its linear deriv-
atives, which partially explained the difference in activities
against Gram-negative bacteria.
Effect on Outer Membrane Permeability—Antimicrobial pep-
tides 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 ex-
cluded 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
m
g/ml, while bactenecin, Lin-Bac2S, and linear bactenecin
caused half-maximal permeabilization at 0.8, 2, and 4.5
m
g/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 gener-
ally 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). Al-
though, some authors have utilized measurements of the ac-
cessibility of a normally membrane-impermeable substrate to
cytoplasmic
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-C
3
-(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 fluores-
cent probe diS-C
3
-(5), which is a caged cation, distributes be-
tween cells and medium depending on the cytoplasmic mem-
brane potential. Once it is inside the cells, it becomes
concentrated and self-quenches its own fluorescence. If pep-
tides form channels or otherwise disrupt the membrane, the
membrane potential will be dissipated, and the DiS-C
3
-(5) will
be released into the medium causing the fluorescence to in-
crease, 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
1
inside and outside the cells. Therefore
the MICs of bactenecin, reduced bactenecin, and bac2S in the
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
m
g/ml LPS to a final concentration
of 1
m
M, which saturated the binding sites on LPS, and the fluorescence
sensitivity was adjusted to 90%. The peptides and Mg
21
were titrated
in, resulting in a decrease in fluorescence due to the competitive dis-
placement 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, MgCl
2
.
FIG.3.Peptide-induced outer membrane permeabilization as-
sessed 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) bactene-
cin; open circles, polymyxin B.
Antimicrobial Mechanism of the Cyclic Cationic Peptide Bactenecin32
presence of 0.1 M KCl were determined and shown to be 8–16
m
g/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
m
g/ml) caused a rapid increase in fluorescence that was
similar to that seen for a control
a
-helical peptide CEMA at its
MIC of 1
m
g/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 mem-
brane (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
m
g/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).
Structure-Activity Relationships—A series of peptides re-
lated to bactenecin were made (Table I) in an attempt to deci-
pher important features of these peptides contributing to anti-
microbial activity. Included in this series were peptides that
differed in charge due either to amidation of the carboxyl ter-
minus (Lin-Bac2S-NH
2
and BacP-NH
2
) or addition of arginines
to the NH
2
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 in-
vestigated. 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-NH
2
(14) and Lin-BacR (15).
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-NH
2
versus Lin-Bac2S, BacP-
NH
2
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-pos-
itive activity tended to require the peptides to be linear, al-
though the cyclic peptide BacP-NH
2
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. Interest-
ingly, the further substitution of three arginines for three ly-
sine residues in BacP3K resulted in a very weakly active pep-
tide, suggesting that these two basic residues may not be
equally effective in promoting bactenecin activity.
DISCUSSION
The
a
-helical and
b
-structured classes are two groups of
antimicrobial polycationic peptides that have been well stud-
ied. Although their precise antimicrobial mechanism is some-
what 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.
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
b
-sheet defensins (30), the protegrins (31), and the tachy-
plesins (32), the loss of ability to form a disulfide bond results
in a complete loss of structure and activity. Thus the observa-
tion that bactenecin, when linearized, undergoes a dramatic
shift in activity spectrum (Table II) and in structure (Fig. 2) is
unprecedented and surprising.
Furthermore, cyclic bactenecin behaved in a fundamentally
different fashion to the linear bactenecins and the
a
-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 mem-
branes. In this study, live cells of E. coli DC2, an outer mem-
brane hyperpermeable mutant, were used in conjunction with a
fluorescent dye, diS-C
3
-(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
FIG.4.Peptide-induced inner membrane permeabilization as-
sessed by the diS-C
3
-(5) assay. Mid-log phase cells were collected and
resuspended in buffer (5 m
M HEPES, 5 mM glucose) to an A
600
of 0.05.
A 0.4
m
M final concentration of diS-C
3
-(5) was incubated with cell
suspensions until no more quenching was detected, then 0.1
M KCl was
added. The desired peptide concentration (8
m
g/ml for the bactenecins
and 1
m
g/ml for CEMA) was added to a 1-cm cuvette containing 1 ml of
cell suspension. The fluorescence change (in arbitrary units) was ob-
served as a function of time. Symbols: triangles, cyclic bactenecin;
squares, Lin-Bac2S; closed circles, linear (reduced) bactenecin; open
circles, CEMA.
Antimicrobial Mechanism of the Cyclic Cationic Peptide Bactenecin 33
conclude that cyclic bactenecin kills cells in a completely dif-
ferent 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).
In contrast, it would appear that the linear bactenecins are
acting in the same way on the cytoplasmic membrane of bac-
teria 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 con-
centrations as low as 0.125
m
g/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 mem-
brane to form a channel by a barrel-stave mechanism (1, 5, 6),
it is not so simple to understand how a 12-mer peptide contain-
ing 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 permeabil-
ity defective mutants of E. coli and P. aeruginosa. This obser-
vation indicated that the disulfide bond was important for
interaction with the outer membrane as confirmed here. Bacte-
necin had a better binding ability for LPS and also permeabi-
lized 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 NH
2
-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
b
-turn loop molecule regardless of its envi-
ronment, 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 bacte-
necin 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.
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 activ-
ity 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 suffi-
ciently pure enough to permit identification of the desired
product.
Our studies of structure activity relationships revealed cer-
tain factors that were important in the activity of the linear
and cyclic bactenecins against bacteria. The most obvious cor-
relations 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 addi-
tion 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.
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MIC
Bac Lin-Bac Lin-Bac2S Lin-Bac2S-NH
2
Lin-BacR BacR BacP BacP-NH
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BacP3K
m
g/ml
E. coli 8 .64 32 2 4 2 4 2 16
P. aeruginosa 4 .64 .64 16 16 4 .32 16 .64
S. typhimurium 8 .64 .64 32 32 4 16 16 32
S. aureus 64 .64 16 4 .64 64 32 4 .64
S. epidermidis .64 8 8 1 4 8 .32 32 .64
E. facaelis .64 8 8 4 4 32 .32 .64 .64
L. monocytogenes 8 1 1 0.25 0.5 0.125 16 1 16
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Antimicrobial Mechanism of the Cyclic Cationic Peptide Bactenecin 35
