Diastereoisomers of cytolysins, a novel class of potent antibacterial peptides.

An amphipathic alpha-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 alpha-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 alpha-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 cellmediated 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 (ϳ40 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 mem-branes (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 -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 Ref. 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 voltagedependent 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 Ref. 16). Pardaxin has a helixhinge-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 acidincorporated 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.

MATERIALS AND METHODS
Peptide Synthesis and Purification-The peptides were synthesized by a solid phase method on (phenylacetamido)methyl-amino acid resin (0.5 mEq) (21). The resin-bound peptides were then transaminated with 30% ethylenediamine in dimethylformamide for 3 days, followed by filtration of the resin, precipitation of the protected peptides with ether, and removal of the protecting groups with HF. The synthetic peptides were purified (Ͼ95% homogenicity) by reverse-phase high performance liquid chromatography on a C 18 column using a linear gradient of 25-80% acetonitrile in 0.1% trifluoroacetic acid for 40 min and then subjected to amino acid analysis to confirm their composition.
Antibacterial Activity Assay-The antibacterial activity assay was performed in sterilized 96-well plates (Nunc F96 microtiter plates) in a final volume of 100 l as follows. Fifty microliters of a suspension containing bacteria at 1 ϫ 10 6 colony-forming units/ml in culture medium (LB medium) was added to 50 l of water containing the peptide in serial 2-fold dilutions in water. Inhibition of growth was determined by measuring the absorbance at 492 nm, using a Microplate autoreader * This research was supported by the Israel Academy of Sciences and Humanities and by the Pasteur-Weizmann Research Foundation. 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.
Hemolytic Assay-The hemolytic activity of the peptides was determined using human red blood cells (hRBCs). 1 Fresh hRBCs with EDTA were rinsed 3 times with PBS (35 mM phosphate buffer, 0.15 M NaCl, pH 7.0), centrifuged for 15 min at 900 ϫ g, and resuspended in PBS. Peptides dissolved in PBS were then added to 50 l of a 10% solution of the stock hRBC in PBS to a final volume of 100 l. The suspension was agitated during incubation for 30 min at 37°C. The samples were then centrifuged at 1000 ϫ g for 5 min. Release of hemoglobin was monitored by measuring the absorbance of the supernatant at 540 nm. Zero hemolysis (blank) and 100% hemolysis controls were determined using hRBC suspended in PBS and 1% Triton, respectively.
CD Spectroscopy-CD spectra were obtained using a Jasco J-500A spectropolarimeter. The spectra were scanned with a quartz optical cell with 0.5-mm pathlength at 23°C. Each spectrum was the average of four scans at wavelengths of 250 -195 nm. Fractional helicities (22) were calculated as described elsewhere (23).
Preparation of Lipid Vesicles-Small unilamellar vesicles were prepared by sonicating PC/PS/cholesterol (10:10:1, w/w) and PC/cholesterol (10:1), as has been described in detail elsewhere (10). The lipid concentrations of the liposome suspensions were determined by phosphorus analysis.
Membrane Permeability Studies-Membrane destabilization, in the form of diffusion potential collapse, was detected fluorimetrically as described previously (23,24).
Visualization of Bacterial Lysis by Electron Microscopy-Samples containing Escherichia coli (1 ϫ 10 6 colony-forming units/ml) in LB medium were incubated with the different peptides at their minimal inhibitory concentration (MIC) and at one dilution less than MIC, for 16 h, and then centrifuged for 10 min at 3000 ϫ g. Pellets were resuspended, and a drop containing the bacteria was deposited onto a carboncoated grid and then negatively stained with 2% phosphotungstic acid, pH 6.8. The grids were examined using a JEOL JEM 100B electron microscope (Japan Electron Optics Laboratory Co., Tokyo, Japan).

RESULTS
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 I) 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, ϩ5) 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 7 , in which the N helix was altered; [D]L 18 L 19 , in which the C helix was altered; and [D]P 7 L 18 L 19 , 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.
Secondary Structure of the Peptides-The secondary structures of the peptides were evaluated from their CD spectra in 40% TFE, a solvent that strongly promotes an ␣-helical structure, and in PBS (35 mM phosphate buffer, 0.15 M NaCl, pH 7.0). As expected, a dramatic decrease in the ␣-helix content of the peptides was observed as more D-amino acids were incorporated, as reflected by the minima at 208 and 222 nm in 40% TFE (Fig. 1). There was a more than 90% decrease in the ␣-helix content between TApar (50% ␣-helix) and [D]P 7 L 18 L 19 (4%). The ␣-helix contents of [D]P 7 and [D]L 18 L 19 were 25 and 15%, respectively. It should be noted that proline at position 7 does not introduce a kink in the structure but rather participates in the formation of the N helix as revealed by NMR spectroscopy (20). In PBS, pardaxin gave a low value of ϳ12% ␣-helix content while all the D-amino acid incorporated analogues gave very low signals that could not be attributed to specific structures (data not shown).
Hemolytic and Antibacterial Activity of the Peptides-The peptides were then examined for their hemolytic activity toward the highly susceptible human erythrocytes and for their potential to inhibit the growth of different species of bacteria. In addition, the cytotoxic bee venom melittin, the antibacterial peptide dermaseptin, and the antibiotic tetracycline were used as controls. Fig. 2 shows the dose-response curves of the hemolytic activity of the peptides. Table II gives the MIC of the peptides for a representative set of test bacteria, which includes two Gram-negative species, E. coli and Acinetobacter calcoaceticus, and two Gram-positive species, Bacillus megaterium and Bacillus subtilis. The data reveal that (i) D-amino acids introduced into TApar dramatically reduced its hemolytic activity, which correlates with the loss of ␣-helix content in the corresponding analogues. TApar with the highest ␣-helix content is the most potent, while [D]P 7 L 18 L 19 with the lowest ␣-helix content is practically devoid of hemolytic activity up to the maximum concentration tested (50 M). (ii) Despite the dramatic decrease in the ␣-helix content and hemolytic activity of the diastereomeric analogues, they all retained most of the potent antibacterial activity of the parent peptide, which is comparable with that of known native non-hemolytic antibacterial peptides (2) (Fig. 2).
Membrane Destabilization by the Peptides-A common property of all of the ␣-helical, positively charged, naturally occurring antibacterial peptides studied so far is their ability to interact and permeate negatively charged phospholipids better than zwitterionic phospholipids. The relevance of these findings to their biological target membranes has been attributed to the fact that the surface of bacteria contains lipopolysaccharides (in Gram-negative bacteria) and polysaccharides (teichoic acids, in Gram-positive bacteria), both of which are acidic, while normal mammalian cells (e.g. erythrocytes) express the predominantly zwitterionic phospholipid PC on their outer leaflet. Herein we demonstrated that [D]P 7 L 18 L 19 , the only diastereomer that is devoid of hemolytic activity but retains antibacterial activity, permeates negatively charged phospholipids significantly better than zwitterionic phospholipids (

G F F A L I P K I I S S P L F K T L L S A V G S A L S S S G G Q E-(NH 2 ) 2
[D]P 7

G F F A L I P K I I S S P L F K T L L S A V G S A L S S S G G Q E-(NH 2 ) 2
[D]L 18 L 19

G F F A L I P K I I S S P L F K T L L S A V G S A L S S S G G Q E-(NH 2 ) 2
[D]P 7 L 18 L 19

G F F A L I P K I I S S P L F K T L L S A V G S A L S S S G G Q E-(NH 2 ) 2
Melittin b G I G A V L K V L T T G L P A L I S W I K R K R Q Q-(NH 2 ) 2 a Underlined and bold amino acids were substituted with their D-enantiomers. E-(NH 2 ) 2 stands for glutamic acid in which the two -COOH groups were modified to two -CO-NH-CH 2 CH 2 -NH 2 groups by transamination with ethylenediamine.
b Underlined sequences designate the N and C helices, respectively.
Peptides Derived from Diastereomers of Cytolysins 7306 bic N helix or the amphipathic C helix intact, which is sufficient to promote strong binding to both types of vesicles via hydrophobic interactions.
Visualization of Bacterial Lysis Using Electron Microscopy-The effect of the peptides on the morphology of intact and treated bacteria was visualized using negative staining electron microscopy. Fig. 4 shows the photographs obtained with the non-hemolytic analogue [D]P 7 L 18 L 19 as an example. It was found that at the MIC the peptide lysed the bacteria completely, and only small fragments could be observed (Fig. 4C).
However, at concentrations lower than the MIC, patches were observed (Fig. 4B), which might indicate the initial step involved in the lytic process. DISCUSSION 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, ϩ5), 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 Gramnegative 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   Peptides Derived from Diastereomers of Cytolysins 7307 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 7 L 18 L 19 , 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 -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. 2 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.