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The rapid emergence of antibiotic resistance has been of great concern in recent years (7). Thus, there is great interest in the development of new classes of antimicrobial agents (2). Among the possible candidates, a group of antimicrobial cationic peptides has attracted increasing research and clinical interest due to their unique properties (3, 5). Cationic peptides have been found in a variety of sources, from prokaryotes to eukaryotes (4). In recent years, it has become clear that these endogenous peptide antibiotics constitute part of the first line of host defense; for more primitive life forms, like insects and plants, they constitute a host's primary defense system (1).

Bactenecin (also called bovine dodecapeptide) from bovine neutrophils (8) is the smallest natural cationic antimicrobial peptide, being only 12 amino acids long, including 4 arginine residues, 2 cysteine residues, and 6 other hydrophobic residues. The two cysteine residues form a disulfide bond to make bactenecin a loop molecule.

Bactenecin was previously shown to form a -turn structure regardless of its environment (9). It tended to be weakly active only against gram-negative bacteria. Studies of its mechanism of action suggested that it was taken up across the outer membrane by the process of self-promoted uptake and that it failed to cause substantial depolarization of the cytoplasmic membrane, in contrast to most other peptides (9, 10). In contrast, when bactenecin was linearized either by reduction of the disulfide bridge or by alteration of the cysteines to serine residues, the peptide lost its activity against gram-negative bacteria, except for mutants that were generally supersusceptible to antibiotics due to an altered outer membrane barrier. The linearized peptides were dramatically altered in their interaction with cells. They interacted poorly with the outer membrane but were quite effective in permeabilizing (depolarizing) the cytoplasmic membrane. In addition, they adopted a different structure, being unstructured in free solution and adopting a -turn structure upon interacting with membranes. Thus, its small size, unique mechanistic properties, and single disulfide bond make bactenecin an interesting candidate for research and drug development.

It is known that hydrophobicity, positive charge, disulfide bridging, and amphipathicity are important factors in the antimicrobial activities of cationic peptides (4). Analogues were designed to investigate the effects of modification of these factors on antimicrobial activities (Table 1). Peptides were designed by computer modeling with the program Insight II on a Silicon Graphics Indy computer. They were synthesized by N-9-fluorenylmethoxycarbonyl chemistry with an Applied Biosystems, Inc. (Foster City, Calif.), model 431 peptide synthesizer. The purchased bactenecin and its derivatives were in their fully reduced form. The disulfide bond was formed by air oxidation in 0.01 M Tris buffer, pH 7.6, at 23°C for 24 h, and then the oxidized form was purified as described previously (9). Concentrations of bactenecin and its derivatives were determined by amino acid analysis. Control experiments demonstrated that the disulfide bonds of linearized (reduced) bactenecin did not re-form under the experimental conditions employed for MIC measurements.

All bacterial strains used in these studies are listed in Table 2, footnote a. MICs were examined by the broth dilution microtiter method, modified for use with cationic peptides (9). Bacterial strains for antimicrobial activity testing were grown in Luria broth (10 g of Bacto-tryptone per liter and 5 g of Bacto-yeast extract per liter [both from Difco Laboratories]), except for the Streptococcus strains, which were grown in Todd-Hewitt broth (500 g of beef heart infusion per liter, 20 g of Bacto-neopeptone per liter, 2 g of Bacto-dextrose per liter, 2 g of sodium chloride per liter, 0.4 g of disodium phosphate per liter, and 2.5 g of sodium carbonate per liter).

Linear peptides. In a previous study (9), two linear derivatives of bactenecin, Bac2S (here called Lin Bac2S, to be consistent with the other linear derivatives) and reduced bactenecin (Lin Bac), were described. They were found to have high selectivity for gram-positive bacteria and little activity against the wild-type gram-negative bacteria Escherichia coli, Pseudomonas aeruginosa, and Salmonella typhimurium. Amidation of the C terminus partially restored activity against gram-negative organisms to Lin Bac2S-NH₂ (9). To further confirm this observation, two more linear derivatives of bactenecin were made (Table 1), Lin Bac2A-NH₂ (with two Cys-to-Ala replacements) and Lin BacS-NH₂ (with a single Cys-to-Ser replacement at position 3). The hydroxyl groups in serine residues and the sulfhydryl groups in cysteine residues are capable of hydrogen bonding to water and are thus hydrophilic, which would tend to make linear bactenecin more hydrophilic than cyclic bactenecin, since the SH groups in native bactenecin form a disulfide bridge. To ensure that the hydrophobicity of linear bactenecin was as similar as possible to that of native bactenecin, Lin Bac2A-NH₂ had alanine substitutions at both cysteine positions, since alanines are hydrophobic residues. Lin Bac2A-NH₂ was similar to Lin Bac2S-NH₂ in that both were more active against both gram-negative and gram-positive bacteria than linear (reduced) bactenecin (Table 2) and were almost as active as cyclic bactenecin against the gram-negative bacteria E. coli, P. aeruginosa, and S. typhimurium. Overall, these peptides demonstrated good activity against gram-positive bacteria. In contrast, BacS-NH₂, with only a single alteration from Cys-3 to Ser-3, was two- to fourfold less active than Bac2S-NH₂.

Cyclic peptides. Native bactenecin has a type I -turn structure, with two arginine residues at positions 4 and 9 adjacent to the disulfide bond (8). Previous studies indicated that native cyclic bactenecin was selective entirely for gram-negative organisms and had little activity against the gram-positive bacteria Staphylococcus aureus, Staphylococcus epidermidis, and Enterococcus faecalis (9). When a more extensive group of gram-positive bacteria were examined (Table 2), it was found that bactenecin had reasonable MICs (1 to 2 µg/ml) for Corynebacterium xerosis and Streptococcus mitis and measurable MICs for Streptococcus pyogenes and Listeria monocytogenes. Increasing the positive charge from +3 to +5 in BacR (9) led to improved activity against most gram-negative and gram-positive bacteria, with the exception of S. aureus, C. xerosis, and S. pneumoniae (Table 2). In this study, a series of peptide variants were made to test the importance of ring size (numbers of amino acids between the cysteine residues), charge, and amphipathicity (Table 1). Peptides with the same charge as BacR, BacP3R, and BacP3R-V had similar activities, with BacR having about twofold-lower MICs. Interestingly, the peptide BacP3R-V had only six residues between the two cysteines but had better antimicrobial activity than bactenecin. It was the position of the positive charges rather than the number that was important, since Bac2I-NH₂ and BacP2R-NH₂ (with charges of +4 and +5, respectively) appeared to have no advantages over bactenecin (the latter also had three charged residues in the ring, destroying the hydrophobicity of this portion of the peptide).

Agglutination activities of bactenecin and its derivatives. Hemolysis and hemagglutination by these peptides were tested in a multiwell dilution assay for 8 h with fresh human erythrocytes with the buffy coat removed by centrifugation and suspended in 0.85% saline, as previously described (6). Bactenecin and its derivatives did not lyse human erythrocytes. However, some of the cyclic molecules did cause agglutination of these cells (Table 2) at 32 to 64 µg/ml. In general, the reduced forms of the cyclic bactenecin derivatives showed two- to eightfold-higher agglutination activities than their oxidized equivalents. For example, linear (reduced) bactenecin caused agglutination of erythrocytes at a concentration of 16 µg/ml, four times lower than the hemagglutination concentration of native bactenecin (64 µg/ml). Reduced Lin BacW2R and Lin BacW caused agglutination at lower concentrations than did their disulfide-bridged equivalents, at 8 µg/ml (fourfold) and 4 µg/ml (eightfold), respectively. It seemed that the formation of the disulfide bond inhibited the agglutination of human erythrocytes by bactenecin peptides. On the other hand, the linear derivatives Lin Bac2A-NH₂, Lin BacS-NH₂, and Lin Bac2S-NH₂ did not agglutinate erythrocytes. The low agglutinating activities of many bactenecin derivatives, especially those that had a broad spectrum of antimicrobial activity, make these peptides interesting and valuable candidates for drug development.