(Received for publication, February 7, 1997, and in revised form, April 2, 1997)
From the Department of Membrane Research and Biophysics, Weizmann Institute of Science, Rehovot 76100, Israel
The increase in infectious diseases and bacterial
resistance to antibiotics has resulted in intensive studies focusing on the use of linear, -helical, cytolytic peptides from insects and
mammals as potential drugs for new target sites in bacteria. Recent
studies with diastereomers of the highly potent cytolytic peptides,
pardaxin and melittin, indicate that
-helical structure is required
for mammalian cells lysis but is not necessary for antibacterial
activity. Thus, hydrophobicity and net positive charge of the
polypeptide might confer selective antibacterial lytic activity. To
test this hypothesis, a series of diastereomeric model peptides (12 amino acids long) composed of varying ratios of leucine and lysine were
synthesized, and their structure and biological function were
investigated. Peptide length and the position of
D-amino acids were such that short peptides with
stretches of only 1-3 consecutive L-amino acids that
cannot form an
-helical structure were constructed. Circular
dichroism spectroscopy showed that the peptides do not retain any
detectable secondary structure in a hydrophobic environment. This
enabled examination of the sole effect of hydrophobicity and positive
charge on activity. The data reveal that modulating hydrophobicity and
positive charge is sufficient to confer antibacterial activity and cell
selectivity. A highly hydrophobic diastereomer that permeated both
zwitterionic and negatively charged phospholipid vesicles, lysed
eukaryotic and prokaryotic cells. In contrast, a highly positively
charged diastereomer that only permeated slightly negatively charged
phospholipid vesicles had low antibacterial activity and could not lyse
eukaryotic cells. In the boundary between high hydrophobicity and high
positive charge, the diastereomers acquired selective and potent
antibacterial activity. Furthermore, they were completely resistant to
human serum inactivation, which dramatically reduces the activity of native antibacterial peptides. In addition, a strong synergistic effect
was observed at nonlethal concentrations of the peptides with the
antibiotic tetracycline on resistant bacteria. The results are
discussed in terms of proposed mechanisms of antibacterial activity, as
well as a new strategy for the design of a repertoire of short, simple,
and easily manipulated antibacterial peptides as potential drugs in the
treatment of infectious diseases.
Infectious diseases are increasing phenomena today mainly as a
result of changes in the spectrum of pathogens and the increase in
antibiotic-resistant pathogens. When antibiotics were first introduced,
the proportion of resistant versus nonresistant pathogens was less than 1%, but this percentage has dramatically increased within 12 years of their uses. Although the number of antibacterial products available today is impressive (about 150), they cover only six
different target sites and are dominated by -lactams, tetracyclines,
aminoglycosides, marcolides, sulfonamides, and quinolones (1). The
search for new target sites has resulted in an increased interest in
antibacterial peptides that could serve as potential therapeutic drugs.
Antibacterial peptides were initially discovered in invertebrates (2,
3) and subsequently in vertebrates, including humans (4). Antibacterial
peptides serve as a defense system in addition or complementary to the highly specific cell-mediated immune response. This secondary, chemical
immune system provides organisms with a repertoire of small peptides
that are synthesized promptly upon induction and that act against
invasion by occasional and obligate pathogens as well as against the
uncontrolled proliferation of commensal microorganisms (5-7). So far,
more than 100 different antibacterial peptides have been isolated and
characterized. Most of them appear to act by direct lysis of the
pathogenic cell membrane. A major group within this family are short
linear polypeptides (
40 amino acids), which are devoid of disulfide
bridges (6). These polypeptides vary considerably in chain length,
hydrophobicity, and overall charge distribution but share a common
structure upon association with lipid bilayers, namely, an amphipathic
-helical structure (8). Unlike bee venom melittin (9) and the
neurotoxin pardaxin (10, 11) that are cytotoxic to both bacteria and
mammalian cells (12, 13), antibacterial peptides are active only
against bacteria. These peptides include the cecropins isolated from
the cecropia moth (2), the magainins (3), and dermaseptins (14) isolated from frogs skin.
Numerous studies conducted on various native antibacterial peptides
tend to emphasize the importance of an amphipathic -helical structure and a net positive charge for cytolytic activity. The positive charge facilitates peptides interaction with the negatively charged membranes (15-18) found in higher concentrations in the pathogenic cell membrane as compared with normal eukaryotic cells, and
the amphipathic
-helical structure is essential for lytic activity
(18, 19). Studies using model antibacterial peptides focused on chain
length and amino acid composition, as well as amphipathic structure
(20-24). Most of these studies simultaneously examined at least three
parameters, making it difficult to distinguish between the effect of
each individual parameter on the overall biological activity.
Nevertheless, these studies proved that an amphipathic
-helix and a
net positive charge are required for antibacterial activity.
In previous studies, D-amino acids were incorporated into
the cytolytic peptides pardaxin (25) and melittin (26). The resulting
diastereomers did not retain their -helical structure, which caused
abrogation of their cytotoxic effects on mammalian cells. However, the
diastereomers retained high antibacterial activity. These results
suggest that hydrophobicity and a net positive charge confer selective
antibacterial activity to nonselective cytolytic peptides and that
amphipathic
-helical structure is not required. However, the
diastereomers of pardaxin and melittin contained long stretchs of
L-amino acids (14-17 amino acids long), which raises the
possibility that the low residual helicity could be sufficient for
membrane binding and destabilization.
To examine whether modulating hydrophobicity and the net positive
charge of linear cytotoxic polypeptides is sufficient to confer
selective antibacterial activity, we chose to investigate diastereomers
of short model peptides (12 amino acids long), composed of varying
ratios of leucine and lysine and one third of their sequence composed
of D-amino acids. Peptide length and the position of
D-amino acids were such that short peptides with very short consecutive stretches of 1-3 L-amino acids that cannot
form an -helical structure were constructed. The diastereomers were
evaluated with regard to 1) their cytotoxicity against bacteria and
human erythrocytes, 2) their structure, and (3) their ability to interact and perturb the morphology of the bacterial wall and model
phospholipid membranes. The data show that modulating hydrophobicity and positive charge is sufficient to confer antibacterial activity and
cytolytic selectivity. Furthermore, the resulting antibacterial peptides act synergistically at nonlethal concentrations with antibacterial drugs such as tetracycline, and they are totally resistant to human serum inactivation that dramatically reduces the
activity of native antibacterial peptides. The results are discussed in
light of proposed mechanisms of antibacterial activity, as well as a
new strategy for the design of a repertoire of short, simple, and
easily manipulated antibacterial peptides as potential drugs in the
treatment of infectious diseases.
Butyloxycarbonyl-(amino
acid)-(phenylacetamido)methylresin was purchased from Applied
Biosystems (Foster City, CA), and butyloxycarbonyl amino acids were
obtained from Peninsula Laboratories (Belmont, CA). Other reagents used
for peptide synthesis included trifluoroacetic acid (Sigma),
N,N-diisopropylethylamine (Aldrich, distilled
over ninhydrin), dicyclohexylcarbodiimide (Fluka),
1-hydroxybenzotriazole (Pierce), and dimethylformamide (peptide
synthesis grade, Biolab). Egg phosphatidylcholine
(PC)1 was purchased from Lipid Products
(South Nutfield, UK). Egg phosphatidylglycerol (PG) and
phosphatidylethanolamine (PE) (Type V, from Escherichia coli) were purchased from Sigma. Cholesterol (extra pure) was supplied by Merck (Darmstadt, Germany) and recrystallized twice from
ethanol. 3,3-diethylthio-dicarbocyanine iodide (diS-C2-5) was obtained from Molecular Probes (Eugene, OR). All other reagents were of analytical grade. Buffers were prepared in double
glass-distilled water.
Peptides were synthesized by a solid phase method on butyloxycarbonyl-(amino acid)-(phenylacetamido) methyl resin (0.05 meq) (27). The resin-bound peptides were cleaved from the resins by hydrogen fluoride and after hydrogen fluoride evaporation extracted with dry ether. These crude peptide preparations contained one major peak, as revealed by reversed phase HPLC, that was 50-70% pure peptide by weight. The synthesized peptides were further purified by reversed phase HPLC on a C18 reversed phase Bio-Rad semi-preparative column (250 × 10 mm, 300 Å pore size, 5 µM particle size). The column was eluted in 40 min, using a linear gradient of 5-60% acetonitrile in water, both containing 0.05% trifluoroacetic acid (v/v), at a flow rate of 1.8 ml/min. The purified peptides, which were shown to be homogeneous (>95%) by analytical HPLC, were subjected to amino acid analysis and mass spectroscopy to confirm their composition.
Transamidation of the PeptidesResin-bound peptide (20 mg) was treated for 3 days with a mixture composed of saturated ammonia solution (30%) in methanol and Me2SO (1:1 v/v), which resulted in transamidation of the carboxylate group of the lysine residue located at the C terminus of all analogues. Thus, peptides were obtained in which all the protecting groups remained attached, but whose C-terminal residues were modified by one amide group. The methanol and ammonia were evaporated under a stream of nitrogen, and the Me2SO was evaporated by lyophilization. The resulting protected peptides were then extracted from the resin with fresh Me2SO and precipitated with dry ether. The products were subjected to hydrogen fluoride cleavage and further purified by reversed phase HPLC as described above.
Preparation of LiposomesSmall unilamellar vesicles were prepared by sonication of PC/cholesterol (10:1 w/w) or PE/PG (7:3 w/w) dispersions. Dry lipid mixtures were dissolved in a CHCl3/MeOH mixture (2:1 v/v). The solvents were then evaporated under a stream of nitrogen, and the lipids (at a concentration of 7.2 mg/ml) were subjected to a vacuum for 1 h and then resuspended in the appropriate buffer by vortexing. The resulting lipid dispersions were then sonicated for 5-15 min in a bath type sonicator (Bransonic B3-R sonicator, Branson Ultrasonics Corporation, Danbury, CT) until clear. The lipid concentrations of the resulting preparations were determined by phosphorus analysis (28). Vesicles were visualized using a JEOL JEM 100B electron microscope (Japan Electron Optics Laboratory Co., Tokyo, Japan) as follows: A drop of vesicles was deposited on a carbon-coated grid and negatively stained with uranyl acetate. Examination of the grids demonstrated that the vesicles were unilamellar, with an average diameter of 20-50 nm (29).
Preparation of SerumBlood was collected from five volunteers and allowed to clot at room temperature for 4 h. The blood was then centrifuged for 15 min at 1500 × g, and the serum was removed and pooled. The serum complement was inactivated by heating at 56 °C for 30 min.
CD SpectroscopyThe CD spectra of the peptides were
measured with a Jasco J-500A spectropolarimeter. The spectra were
scanned at 23 °C in a capped, quartz optical cell with a 0.5-mm path
length. Spectra were obtained at wavelengths of 250-190 nm. Eight
scans were taken for each peptide at a scan rate of 20 nm/min. The
peptides were scanned at concentrations of 1.5 × 105-2.0 × 10
5 M in 40%
trifluoroethanol. Fractional helicities (30, 31) were calculated as
follows:
![]() |
(Eq. 1) |
The antibacterial activity of the diastereomers was examined in sterile 96-well plates (Nunc F96 microtiter plates) in a final volume of 100 µl as follows. Aliquots (50 µl) of a suspension containing bacteria at a concentration of 106 colony-forming units/ml LB medium were added to 50 µl of water or 66% pooled normal human serum in PBS, containing the peptide in 2-fold serial dilutions. Growth inhibition was determined by measuring the absorbance at 492 nm with a Microplate autoreader El309 (Bio-tek Instruments), following incubation for 18-20 h at 37 °C. Antibacterial activity is expressed as the minimal inhibitory concentration (MIC), the concentration at which 100% inhibition of growth was observed after 18-20 h of incubation. The bacteria used were: E. coli D21, Acinetobacter calcoaceticus Ac11, Pseudomonas aeruginosa ATCC 27853, Bacillus megaterium Bm11, and Bacillus subtilis ATCC 6051.
Synergistic Effect between Tetracycline and the DiastereomersTo investigate a possible synergistic relationship between the antibiotic drug tetracycline and the diastereomers, tetracycline was tested in 2-fold serial dilutions against P. aeruginosa (ATCC 27853) in the presence of a constant equimolar concentration (1 µM) of [D]-L3,4,8,10-K5L7. Antibacterial activity of the mixtures was determined as described above.
Hemolysis of Human Red Blood CellsHemolytic activity of the peptides were tested against human red blood cells (hRBC). Fresh hRBC with EDTA were rinsed three times with PBS (35 mM phosphate buffer/0.15 M NaCl, pH 7.3) by centrifugation for 10 min at 800 × g and resuspended in PBS. Peptides dissolved in PBS were then added to 50 µl of a solution of the stock hRBC in PBS to reach a final volume of 100 µl (final erythrocyte concentration, 5% v/v). The resulting suspension was incubated under agitation for 30 min at 37 °C. The samples were then centrifuged at 800 × g for 10 min. Release of hemoglobin was monitored by measuring the absorbance of the supernatant at 540 nm. Controls for zero hemolysis (blank) and 100% hemolysis consisted of hRBC suspended in PBS and Triton 1%, respectively.
Peptides Induced Membrane PermeationMembrane permeation
was assessed utilizing the diffusion potential assay (32, 33) as
described previously (34, 35). In a typical experiment, in a glass
tube, 4 µl of a liposome suspension (final phospholipid
concentration, 32 µM) in a K+ containing
buffer (50 mM K2SO4, 25 mM HEPES-SO42, pH 6.8),
was diluted in 1 ml of an isotonic K+ free buffer (50 mM Na2SO4, 25 mM
HEPES-SO4
2, pH 6.8), and the
fluorescent, potential-sensitive dye diS-C2-5 was then
added. Valinomycin (1 µl, 10
7 M) was added
to the suspension to slowly create a negative diffusion potential
inside the vesicles, which led to a quenching of the dye's
fluorescence. Once the fluorescence had stabilized, which took 3-10
min, peptides were added. The subsequent dissipation of the diffusion
potential, as reflected by an increase in fluorescence, was monitored
on a Perkin-Elmer LS-50B spectrofluorometer, with the excitation set at
620 nm, the emission set at 670 nm, and the gain adjusted to 100%. The
percentage of fluorescence recovery, Ft, was
defined as:
![]() |
(Eq. 2) |
Samples containing E. coli (106 colony-forming units/ml) in LB medium were incubated with the various peptides at their MIC and 80% of the MIC for 1 h and then centrifuged for 10 min at 3000 × g. The pellets were resuspended, and a drop containing the bacteria was deposited onto a carbon-coated grid, which was then negatively stained with 2% phosphotungstic acid, pH 6.8. The grids were examined using a JEOL JEM 100B electron microscope.
Four diastereomers of linear and short
(12 amino acids long) model peptides composed of varying ratios of
lysine-to-leucine were synthesized in order 1) to examine whether a
balance between hydrophobicity and a net positive charge may be a
sufficient criteria necessary for selective bacterial lysis and 2) to
gain insight into the mechanism underlying this effect. The location of
D-amino acids remained constant in all peptides, because it
was constructed for maximum disruption of -helical structure.
D-Amino acids were distributed along the peptide, leaving
only very short stretches of 1-3 consecutive L-amino
acids. The peptides were then characterized with regard to their
structure, biological function, and interaction with bacteria and model
membranes composed of either zwitterionic or negatively charged
phospholipids. The following peptides were synthesized;
[D]-L3,4,8,10-K3L9,
[D]-L3,4,8,10-K4L8,
[D]-L3,4,8,10-K5L7, and
[D]-L3,4,8,10-K7L5 (Table
I). The hydrophobicities (36) and net positive charge of
the peptides are listed in Table I.
|
The extent of the -helical structure of
the diastereomers was determined from their CD spectra in 40%
trifluoroethanol, a solvent that strongly promotes
-helical
structure. As expected, after incorporation of D-amino
acids, no signal was observed for all the diastereomers, demonstrating
the lack of any specific secondary structure (data not shown). In a
recent study, a peptide with a sequence identical to that of
[D]-L3,4,8,10-K4L8 but composed
of only L-amino acids was found to have ~40%
-helical
structure in methanol and in
L-
-dimyristoylphosphatidylcholine vesicles (22).
The
hemolytic activity of the peptides against the highly cytolytically
susceptible human erythrocytes was tested. A dose-response curve for
the hemolytic activity of the peptides is shown in Fig. 1. The hemolytic activity of bee venom melittin served
as a control. A direct correlation was found between the hydrophobicity
(Table I) and the hemolytic activity of the diastereomers.
[D]-L3,4,8,10-K3L9, which has the
highest hydrophobicity, was the most hemolytic peptide. However, its
hemolytic activity is very low in comparison with that of melittin
(>60-fold less activity). All the other peptides showed no significant
hemolytic activity up to the maximum concentration tested (100 µm) (Fig. 1). It should be noted that although
[D]-L3,4,8,10-K4L8 is not
hemolytic at concentrations >100-fold of those required for
significant hemolysis by melittin, its entirely L-amino
acid form, [D]-L3,4,8,10-K4L8,
has hemolytic activity close to that of melittin (~5-fold less)
(22).
The peptides were also tested for their antibacterial activity against a representative set of bacteria, including three Gram-negative species (E. coli, A. calcoaceticus, and P. aeruginosa) and two Gram-positive species (B. megaterium and B. subtilis). The antibiotic tetracycline, the native antibacterial peptide dermaseptin S, and the cytolytic peptide melittin served as controls. The resultant MICs are shown in Table II. The data show that the antibacterial activity of the diastereomers was modulated by the balance between hydrophobicity and positively charged amino acids. Both the most hydrophobic peptide, [D]-L3,4,8,10-K3L9, and the most hydrophilic peptide, [D]-L3,4,8,10-K7L5, displayed the lowest range in antibacterial activity (Table II). However, [D]-L3,4,8,10-K4L8 and [D]-L3,4,8,10-K5L7 displayed high antibacterial activity against most of the bacteria tested with the former being slightly more potent. Furthermore, each peptide had a unique spectrum of antibacterial activity, and each was active more against Gram-positive as compared with Gram-negative bacteria.
|
We observed a synergistic effect between the antibiotic drug tetracycline and the diastereomer [D]-L3,4,8,10-K5L7. Tetracycline shows little activity against P. aeruginosa. However, when mixed with 1 µM solution of [D]-L3,4,8,10-K5L7, a concentration that is 10-fold lower than that required for lytic activity against P. aeruginosa, an 8-fold increase in the activity of tetracycline was observed (Table III). A possible explanation for the synergistic effect is that the peptide slightly disrupts the bacterial wall, which improves partitioning of tetracycline into the bacteria. This is supported by electron microscopy studies showing that below its MIC [D]-L3,4,8,10-K5L7 causes morphological changes in the bacterial wall (see Fig. 3). In addition, the effect of pooled human serum on the antibacterial activity of [D]-L3,4,8,10-K5L7 and the native antibacterial peptide dermaseptin against P. aeruginosa and E. coli was found to differ considerably (Table III). Although dermaseptin was 8-10-fold less active in the presence of serum, [D]-L3,4,8,10-K5L7 retained its antibacterial activity.
|
Peptide-induced Membrane Permeation
Various concentrations of
peptides were mixed with vesicles that had been pretreated with the
fluorescent dye, diS-C2-5, and valinomycin. The kinetics of
the fluorescence recovery was monitored, and the maximum fluorescence
level was determined as a function of peptide concentration (Fig.
2). PC/cholesterol vesicles (10:1) served as a model of
the phospholipid composition of the outer erythrocyte leaflet (37), and
PE/PG vesicles (7:3) were used to mimic the phospholipid composition of
E. coli (38). We found a direct correlation between the
potential of the peptides to permeate model phospholipid membranes and
their lytic activity against erythrocytes and E. coli. Only
the hemolytic peptide
[D]-L3,4,8,10-K3L9 permeated the
zwitterionic phospholipid vesicles. Furthermore, the ability of the
peptides to permeate PE/PG vesicles correlates with the antibacterial
activity of the peptides against E. coli (Table II).
[D]-L3,4,8,10-K7L5, which has the
lowest antibacterial activity, also had significantly decreased ability
to permeate PE/PG vesicles compared with the other three peptides.
Electron Microscopy Study of Bacterial Lysis
The effect of the diastereomers on the morphology of treated E. coli was visualized using transmission electron microscopy. All the peptides caused total lysis of the bacteria at the MIC (data not shown). However, when the peptides were utilized at concentrations corresponding to 80% of their MIC, some differences in the morphology of the treated bacteria were observed, depending upon the peptide used. The most hydrophobic peptide, [D]-L3,4,8,10-K3L9, caused the most damage to the cell wall and membranes, whereas the least hydrophobic peptide, [D]-L3,4,8,10-K7L5, only caused local perturbations (Fig. 3).
Previous studies with model peptides used to elucidate the
structure-function study of antibacterial peptides focused on three parameters: helical structure, hydrophobicity, and charge (20-24). Each change in one of these parameters simultaneously resulted in
changes in the other two, making it difficult to clarify the unique
contribution of each parameter to the overall antibacterial activity.
In this study, the effect of the helical structure was eliminated,
which therefore permitted the study of only two parameters, namely,
hydrophobicity and net positive charge, by varying the ratio of leucine
and lysine. For this purpose, we chose to investigate diastereomers of
short model peptides (12 amino acids long) containing stretches of only
1-3 consecutive L-amino acids, which are too short to form
an -helical structure.
CD spectroscopy revealed that the diastereomers studied here are indeed
totally devoid of -helical structure (data not shown), unlike the
diastereomers of melittin and pardaxin, which retain low
-helical
structure (25, 26). Nevertheless, the diastereomers exhibit potent
antibacterial activity similar to or greater than that of native
antibacterial peptides such as dermaseptin S or the antibiotic drug
tetracycline (Table II). Moreover, the most potent peptides,
[D]-L3,4,8,10-K4L8 and
[D]-L3,4,8,10-K5L7, were devoid
of hemolytic activity against the highly cytolytically susceptible
human erythrocytes. It should be noted that
[D]-L3,4,8,10-K3L9, which is
devoid of
-helical structure, has considerable hemolytic activity
that approaches that of the native cytolytic peptide, pardaxin (13).
This could indicate that the balance between hydrophobicity and
positive charge compensates for the amphipathic
-helical structure.
However, increasing the positive charge drastically reduced the
hemolytic activity while antibacterial activity was preserved,
demonstrating that
-helical structure is not required for
antibacterial activity.
The interaction of the diastereomers with both negatively charged and zwitterionic phospholipid membranes was examined to elucidate the basis of their selective cytotoxicity against bacteria. Negatively charged PE/PG vesicles were used to mimic the lipid composition of E. coli (38), and the zwitterionic PC vesicles to mimic the outer leaflet of human erythrocytes (37). The biological activity of the peptides on erythrocytes (Fig. 1) and E. coli (Table II) correlates well with their ability to permeate model membranes. The only peptide that permeated PC vesicles was the only peptide with significant hemolytic activity. These results suggest that the phospholipid composition of the bacterial membrane plays a role in permeation by this family of antibacterial peptides. The ability of antibacterial and nonhemolytic peptides to bind and permeate negatively charged but not zwitterionic phospholipid vesicles is characteristic of of native antibacterial peptides (15-17, 39) and has been attributed to the fact that the bacterial surface contains lipopolysaccharides (in Gram-negative bacteria) and polysaccharides (teichoic acids, in Gram-positive bacteria), and their inner membranes contain PG, all of which are negatively charged, whereas normal eukaryotic cells such as erythrocytes predominantly express the zwitterionic phospholipid PC on their outer leaflet.
The antibacterial peptide, magainin, is a nonhemolytic peptide, whereas
melittin, pardaxin, and a model peptide with a sequence similar to that
of [D]-L3,4,8,10-K4L8 but
composed of entirely L-amino acids are hemolytic, mainly due to their high hydrophobicity. When the -helical structure of
magainin was disrupted by the introduction of three D-amino acids, the resulting diastereomer had no antibacterial activity (19),
even though its net positive charge is similar to that of native
magainin. Thus, an optimal balance that already exists between the
-helical structure, hydrophobicity, and net positive charge of
native magainin allows selective antibacterial activity, and any change
in one of these properties could cause a loss in magainin's
antibacterial activity. Contrastingly, hydrophobicity appears to play a
major role in compensating for the loss of
-helical structure in
melittin, pardaxin, and the diastereomers studied in this paper.
Kiyota et al. recently examined how the
hydrophobic-hydrophilic balance of apolar and polar surfaces of
amphipathic positively charged helices affect membrane lytic activity
(40). The results demonstrate that model peptides with a hydrophobic
face larger than their hydrophilic face immerse their hydrophobic
regions in lipid bilayers, whereas peptides with a hydrophilic
positively charged face larger than hydrophobic face interact with the
anionic lipid head groups on liposome surfaces. Thus, there appear to be two stages in the lytic process: binding and immersion into the
hydrophobic core of the lipid bilayer, which then leads to membrane
lysis. The concept of peptide hydrophobic-hydrophilic balance may also
be applicable in the diastereomeric model peptides, even though they
lack -helicity. Electrostatic interactions between the positively
charged diastereomers and the negatively charged phospholipid membranes
seem to have an important role in initial interactions and selectivity,
but biological activity appears to be driven by the hydrophobic
interactions between the nonpolar amino acids and the hydrophobic core
of the lipid bilayer. High hydrophobicity may force the immersion of
the peptide into the hydrophobic core of the lipid bilayer, regardless
of the phospholipid head group, thereby permeabilizing membranes of
eukaryotic and prokaryotic cells, as found in the case of
[D]-L3,4,8,10-K3L9. On the other
hand high positive charge may prevent efficient immersion step, so that
the peptide interacts predominantly with the negatively charged lipid
head group as found in the case of [D]-L3,4,8,10-K7L5 (Table II).
Within the constraints of high hydrophobicity and high positive charge,
the peptide may acquire selective activity as a result of electrostatic
interactions with lipid head groups and high antibacterial activity
driven by hydrophobicity that immerses the peptide into the hydrophobic
core of the lipid bilayer, resulting in lysis.
The results presented here, together with those obtained with diastereomers of pardaxin (25) and melittin (26), suggest a new strategy for the design of a repertoire of short, simple, and easily manipulated antibacterial peptides. Each of the diastereomeric model peptides has a unique spectrum of activity (Table II). The existence of a repertoire of diastereomeric antibacterial peptides will enable one to choose the most efficacious peptide with regard to the target cell. Furthermore, simultaneous administration of multiple forms of diastereomers peptides, acting separately or in concert, also has a selective survival value and provides a better shielding against a wider range of infectious microbes. All the peptides displayed increased antibacterial activity against Gram-positive in comparison to Gram-negative bacteria. These results are important considering the increasing resistance of Gram-positive bacteria such as Staphylococcus aureus, enterococci, and pneumococci to conventional antibiotics (1). In addition, unlike the native antibacterial peptide dermaseptin S, [D]-L3,4,8,10-K5L7 retained its antibacterial activity in the presence of pooled human serum.
Diastereomeric peptides should have several advantages over known
antibacterial peptides: 1) The peptides should lack the diverse
pathological and pharmacological effects induced by -helical lytic
cytolysins. For example, staphylococcus
-toxin, the antibacterial peptide alamethicin, cobra direct lytic factor, and pardaxin exert several histopathological effects on various cells due to pore formation and activation of the arachidonic acid cascade. However, pardaxin diastereomers do not exert these activities (25). In addition,
many amphipathic
-helical peptides bind to calmodulin and elicit
several cell responses, and even all D-amino acid
-helices, including melittin have similar activity (41).
Diastereomers with disrupted
-helical structure are not expected to
bind to calmodulin. 2) Local D-amino acid substitution
would result in controlled clearance of the antibacterial peptides by
proteolytic enzymes, as opposed to the total protection acquired by
complete D-amino acids substitution (42). Total resistance
of a lytic peptide to degradation is disadvantageous for therapeutic
use. Furthermore, the antigenicity of short fragments containing
D,L-amino acids is dramatically altered as
compared with their wholly L- or D-amino acid
parent molecules (43). 3) Total inhibition of bacterial growth induced
by the diastereomers is associated with total lysis of the bacterial
wall, as shown by electron microscopy (Fig. 3). Therefore, bacteria
might not easily develop resistance to drugs that trigger such a
destructive mechanism. 4)
[D]-L3,4,8,10-K5L7 has the
ability to perturb the cell wall of bacteria at concentrations lower
than their MIC, as seen by electron microscopy (Fig. 3). The
simultaneous administration of clinically used antibiotics, which have
no activity due to their inability to penetrate the bacterial cell
wall, together with
[D]-L3,4,8,10-K5L7 may present a
solution to this resistance mechanism of bacteria.
In summary, the results obtained with pardaxin, melittin, and the model peptide diastereomers indicate that neither a specific sequence, length, or position of D-amino acids are required for a polypeptide to have antibacterial activity. However, these factors seem to be more crucial for cytotoxicity toward mammalian cells. Our results indicate that only modulating the hydrophobicity and net positive charge of linear cytotoxic polypeptides is sufficient in the design of a repertoire of potent antibacterial diastereomeric polypeptides for the treatment of infectious diseases.