(Received for publication, November 27, 1995; and in revised form, January 29, 1996)
From the
An amphipathic -helical structure is considered to be a
prerequisite for the lytic activity of most short linear cytolytic
polypeptides that act on both mammalian cells and bacteria. This
structure allows them also to exert diverse pathological and
pharmacological effects, presumably by mimicking protein components
that are involved in membrane-related events. In this study D-amino acid-incorporated analogues (diastereomers) of the
cytolysin pardaxin, which is active against mammalian cells and
bacteria, were synthesized and structurally and functionally
characterized. We demonstrate that the diastereomers do not retain the
-helical structure, which in turn abolishes their cytotoxic
effects on mammalian cells. However, they retain a high antibacterial
activity, which is expressed in a complete lysis of the bacteria, as
revealed by negative staining electron microscopy. The disruption of
the
-helical structure should prevent the diastereomer analogues
from permeating the bacterial wall by forming transmembrane pores but
rather by dissolving the membrane as a detergent. These findings open
the way for a new strategy in developing a novel class of highly potent
antibacterial polypeptides for the treatment of infectious diseases,
due to the increasing resistance of bacteria to the available
antibacterial drugs.
In addition to or complementary to the highly specific
cell-mediated immune response, vertebrates and other organisms have a
defense system made up of distinct groups of broad spectrum
antibacterial peptides(1, 2) . One major group
includes short linear polypeptides (40 amino acids and less),
which have been isolated from diverse species such as insects,
amphibians, and mammals(1, 2) . The largest family
includes those polypeptides that are positively charged and adopt an
amphipathic
-helical structure. Some are cytotoxic to both
bacteria and mammalian cells while others are active against only one
or the other. Although the precise mechanism of the antibacterial
activity of polycationic amphipathic
-helical peptides is not yet
fully understood, accumulating data suggest that they destroy the
energy metabolism of the target organism by increasing the permeability
of energy-transducing membranes(3, 4, 5) .
The novel finding that all D-amino acid polypeptides, which
form a left-handed
-helix, retain the antibacterial activity of
the native peptides suggests that a chiral center is not involved in
the lytic process(6, 7) . Therefore, the target of
these toxins is believed to be the cell membrane. Because of their
amphipathic structure, it has been suggested that these antibacterial
peptides permeate the membrane by forming ion channels/pores via a
``barrel-stave'' mechanism(8, 9) . According
to this model transmembrane amphiphilic
-helices form bundles in
which outwardly directed hydrophobic surfaces interact with the lipid
constituents of the membrane, while inwardly facing hydrophilic
surfaces produce a pore. Alternatively, the peptides bind parallel to
the surface of the membrane, cover the surface of the membrane in a
``carpet''-like manner, and dissolve it like a
detergent(10, 11, 12) .
Pardaxin, a 33-mer polypeptide, is an excitatory neurotoxin that has been purified from the Red Sea Moses sole Pardachirus marmoratus(13, 14) and from the Peacock sole of the western Pacific Pardachirus pavoninus(15) . Pardaxin possesses a variety of biological activities depending upon its concentration (reviewed in (16) ), and recently was found to be endowed with potent antibacterial activity(17) . Its biological roles have been attributed to its interference with the ionic transport of the osmoregulatory system in epithelium and to presynaptic activity by forming ion channels that are voltage-dependent and slightly selective to cations. A ``barrel-stave'' mechanism for insertion of pardaxin into membranes was proposed on the basis of its structure and various biophysical studies (18, 19) (reviewed in (16) ). Pardaxin has a helix-hinge-helix structure; the N-helix includes residues 7-11 and the C-helix includes residues 14-26. The helices are separated by a proline residue situated at position 13(20) . This structural motif is found both in antibacterial peptides that can act specifically on bacteria (e.g. cecropin) and in cytotoxic peptides that can lyse a variety of cells (e.g. melittin).
Herein, functional and structural
studies with D-amino acid-incorporated analogues
(diastereomers) of pardaxin reveal that the -helical structure,
while important for cytotoxicity toward mammalian cells, is not a
prerequisite for antibacterial activity as the diastereomers can lyse
bacteria completely as revealed by negative staining electron
microscopy. The results are discussed in terms of proposed mechanisms
of antibacterial activity as well as the advantages of this novel class
of antibacterial peptides as potential drugs in the treatment of
infectious diseases.
To examine the role of the -helical structure of a
polycationic cytolysin in its cytotoxicity toward mammalian cells and
bacteria, a series of pardaxin-derived peptides (see Table 1)
were synthesized and characterized for their structure, hemolytic
activity on hRBCs, antibacterial activity, and effect on the morphology
of bacteria. The list includes TApar (net charge, +5) in which the
acidic C terminus of pardaxin was converted to a positive one by
transamination with ethylenediamine and three of its diastereomers:
[D]P
, in which the N helix was altered;
[D]L
L
, in which the C helix was
altered; and [D]P
L
L
, in
which both the N and C helices were altered. The D-amino acids
were introduced in the centers of the N and C helices. In addition, the
cytolytic bee venom melittin and the antibacterial peptide dermaseptin (25) were used as controls.
Figure 1:
CD spectra of pardaxin analogues.
Spectra were taken at peptide concentrations of 0.8-2.0
10
M in 40% TFE/water. Solid line,
TApar; dotted line, [D]P
; dashed
line, [D]L
L
; dash-dot
line,
[D]P
L
L
.
Figure 2:
Dose-response curve of the hemolytic
activity of the peptides toward hRBCs. The assay was performed as
described under ``Materials and Methods.'' The inset shows the assay results at low concentration. Designations are as
follows: filled squares, melittin; filled triangles,
TApar; filled circles, [D]P; empty
circles, [D]L
L
; empty
squares, [D]P
L
L
; empty triangles, dermaseptin.
Figure 3:
Maximal dissipation of the diffusion
potential in vesicles induced by the peptides. The peptides were added
to isotonic K free buffer containing small unilamellar
vesicles composed of PC/PS (A) or PC (B),
pre-equilibrated with the fluorescent dye diS-C
-5 and
valinomycin. Fluorescence recovery was measured 10-20 min after
the peptides were mixed with the vesicles. Designations are as follows: filled triangles, TApar; filled circles,
[D]P
; empty circles,
[D]L
L
; empty squares,
[D]P
L
L
.
Figure 4:
Electron micrographs of negatively
stained E. coli untreated and treated with
[D]PL
L
. A,
control; B, after treatment of the bacteria with the peptide
at a concentration lower than the MIC; C, after treatment of
the bacteria with the peptide at the MIC
concentration.
Numerous studies have led to the conclusion that a net
positive charge and an amphipathic -helical structure are
prerequisites for the activity of most of the linear antibacterial
peptides studied so far. We therefore used TApar (net charge, +5),
which has various cytotoxic and histopathological effects(16) ,
as a case study.
Herein we demonstrate that TApar has an
-helical structure and is endowed with high antibacterial activity
on Gram-negative and Gram-positive bacteria and with hemolytic activity
on human erythrocytes. However, D-amino acids incorporated
into TApar dramatically reduced its
-helical structure (Fig. 1). This in turn reduced the hemolytic activity of the
diastereomeric analogues, which indicates the importance of this
structure in the cytotoxicity of the peptide to mammalian cells.
However, the amphipathic
-helical structure seems not to be
crucial for antibacterial activity, since with most of the bacteria
tested there was no significant decrease in the antibacterial activity
of the peptides when there was a reduction of the
-helical
structure. As an extreme case
[D]P
L
L
, which lost
almost all
-helical structure, is practically non-hemolytic but
yet is endowed with high antibacterial activity. The lack of a
significant
-helical structure should prevent this analogue from
inserting and forming a transmembrane pore, and hence a
``barrel-stave'' mechanism (9, 27) is
unlikely as its mode of action. Its final effect, as revealed by the
electron microscope photographs (Fig. 4), was total lysis of the
bacterial wall, as was also the case with all the other analogues
including the wild type (data are not shown). Therefore, the peptide
probably acts as a detergent, in what has been described as a
``carpet''-like
mechanism(10, 11, 12) .
Besides the
contribution of this study to the understanding of structural elements
that determine cytotoxicity toward mammalian cells and bacteria, the
strategy of local amino acid substitution opens a new avenue for the
design of antibacterial peptides that should have some advantageous
properties as discussed below. (i) Several cytolytic amphipathic
-helical peptides have been shown to exert diverse pathological
and pharmacological effects, presumably by mimicking protein components
that are involved in membrane-related events. For example, Staphylococcus
-toxin, the antibacterial peptide
alamethicin, cobra direct lytic factor, and pardaxin have several
histopathological effects on various cells as a result of pore
formation and activation of the arachidonic acid cascade. Furthermore,
cytolysins have been shown to increase intracellular calcium and induce
eicosanoid release in pheochromocytoma PC12 cell cultures(28) .
None of these effects has been observed with the D-amino
acid-incorporated analogues investigated herein. (
)It should
be noted that insect (cecropins A) and pig (cecropin P1) antibacterial
peptides and related amphipathic peptides can mimic mitochondrial
presequences (which adopt amphipathic
-helical structures) in
their ability to release respiratory control, inhibit protein import,
and at higher concentrations to inhibit respiration(29) . It
has also been found that many amphipathic
-helical peptides bind
to calmodulin to elicit several cell responses. Furthermore, even all D-amino acid
-helices (e.g. melittin) are
endowed with similar activity(30) . The disruption of the
-helical structure of a particular cytolysin can therefore abolish
many side effects. (ii) Local D-amino acid substitution should
enable controlled clearance of the antibacterial peptides by
proteolytic enzymes rather than the total protection acquired by
complete D-amino substitution(6) . Total resistance of
a lytic peptide to degradation might be a disadvantage in therapeutic
use. Furthermore, short fragments containing D and L amino acids have a dramatically altered antigenicity as compared
with their entire L- or D-amino acid parent
molecules(31) . (iii) It is evident from the electron
micrographs that total inhibition of bacterial growth is associated
with total lysis of the bacterial wall. Therefore, it might be more
difficult for the bacteria to develop resistance to such a destructive
mechanism, as compared with the more specific mechanisms of the
commonly used drugs.