Cryptonin

Dermaseptin B2 (Drs B2) is a 33-residue-long cationic, alpha-helical antimicrobial peptide endowed with membrane-damaging activity against a broad spectrum of microorganisms, including bacteria, yeasts, fungi, and protozoa, but its precise mechanism of action remained ill-defined. A detailed characterization of peptide-membrane interactions of Drs B2 was undertaken in comparison with a C-terminal truncated analogue, [1-23]-Drs B2, that was virtually inactive on bacteria despite retaining the cationic charge of the full-length peptide. Both peptides were tested on living cells using membrane permeabilization assays and on large unilamellar and multilamellar phospholipid vesicles composed of binary lipid mixtures by dye leakage assay, fluorescence spectroscopy, circular dichroism, and differential scanning calorimetry and also on SDS micelles using NMR spectroscopy. The results indicate that Drs B2 induces a strong perturbation of anionic lipid bilayers, resides at the hydrocarbon core-water interface, parallel to the plane of the membrane, and interacts preferentially with the polar head groups and glycerol backbone region of the anionic phospholipids, as well as the region of the lipid acyl chain near the bilayer surface. The interfacial location of Drs B2 induces a positive curvature of the bilayer and clustering of anionic lipids, consistent with a carpet mechanism, that may lead to the formation of mixed peptide-phospholipid toroidal, transient pores and membrane permeation/disruption once a threshold peptide accumulation is reached. In constrast, the truncated [1-23]-Drs B2 analogue interacts at the head group level without penetrating and perturbing the hydrophobic core of the bilayer. NMR study in SDS micelles showed that [1-23]-Drs B2 adopts a well-defined helix encompassing residues 2-20, whereas Drs B2 was previously found to adopt helical structures interrupted around the Val(9)-Gly(10) segment. Thus the antibacterial activity of Drs B2 depends markedly on a threshold number of hydrophobic residues to be present on both extremities of the helix. In a membrane environment with a strong positive curvature strain, Drs B2 can adopt a flexible helix-hinge-helix structure that facilitates the concomitant insertion of the strongly hydrophobic N- and C-termini of the peptide into the acyl core of the membrane.

Toward delineation of antimicrobial action, a prototypic amphipathic, cationic decapeptide Ac-G-X-R-K-X-H-K-X-W-A-NH(2) was designed and peptides for which X was didehydrophenylalanine (DeltaFm), alpha-aminoisobutyric acid (Um), or phenylalanine (Fm) were synthesized. A growth kinetics experiment indicated that the bacteriostatic effects were nil (Um), mild and transient (Fm), and strong and persistent (DeltaFm) respectively. Though at par in binding to lipopolysaccharide, DeltaFm and Fm, but not Um, caused outer membrane permeabilization. Inner membrane permeabilization was attenuated and membrane architecture rehabilitated with DeltaFm but not Fm. Reverse phase high-performance liquid chromatography revealed that DeltaFm was translocated into Escherichia coli, while Um and fragments of Fm were detected in the medium. Among these monomers, only DeltaFm was modestly antibiotic [minimum inhibitory concentrations (MICs) of 110 microM (E. coli) and 450 microM (Staphylococcus aureus)]. Interestingly, a linear dimer of DeltaFm, viz. (DeltaFm)(2), turned out to be highly potent against E. coli [MIC of 2 microM and minimum bactericidal concentration (MBC) of 2 microM] and modestly potent against S. aureus (MIC of 20 microM and MBC of 20 microM). In contrast, a lysine-based branched dimer of DeltaFm, viz. DeltaFd, was found to be a potent antimicrobial against both E. coli (MIC of 2.5 microM) and S. aureus (MIC of 5 microM). Studies with analogous branched dimers of Fm and Um have indicated that dimerization represents a scaffold for potentiation of antimicrobial peptides and that the presence of DeltaF confers potent activity against both E. coli and S. aureus. De novo design has identified DeltaFd as a potent, noncytotoxic, bacterial cell-permeabilizing and -penetrating antimicrobial peptide, more protease resistant than its monomeric counterpart. We report that in comparison to the subdued and sequential "membrane followed by cell interior" mode of action of the monomeric DeltaFm, the strong and simultaneous "membrane along with cell interior" targeting by the dimeric DeltaFd potentiates and broadens its antibiotic action across the Gram-negative-Gram-positive divide.

The structure-activity relations and mechanism of action of parasin I, a 19-amino acid histone H2A-derived antimicrobial peptide, were investigated. Parasin I formed an amphipathic alpha-helical structure (residues 9-17) flanked by two random coil regions (residues 1-8 and 18-19) in helix-promoting environments. Deletion of the lysine residue at the N-terminal [Pa(2-19)] resulted in loss of antimicrobial activity, but did not affect the alpha-helical content of the peptide. The antimicrobial activity was recovered when the lysine residue was substituted with another basic residue, arginine ([R(1)]Pa), but not with polar, neutral, or acidic residues. Progressive deletions from the C-terminal [Pa(1-17), Pa(1-15)] slightly increased the antimicrobial activity (1-4 microg/ml) without affecting the alpha-helical content of the peptide. However, further deletion [Pa(1-14)] resulted in nearly complete loss of antimicrobial activity and alpha-helical structure. Confocal microscopic analysis and membrane permeabilization assays showed that parasin I and its analogs with comparable antimicrobial activities localized to the cell membrane and subsequently permeabilized the outer and cytoplasmic membranes. Pa(1-14) also localized to the cell membrane, but lost membrane-permeabilizing activity, whereas Pa(2-19) showed poor membrane-binding and -permeabilizing activities. The results indicate that the basic residue at the N-terminal is essential for the membrane-binding activity of parasin I, and among the membrane-binding parasin I analogs, the alpha-helical structure is necessary for the membrane-permeabilizing activity.