Previous studies identified lysine- and tryptophan-rich sequences within various cationic antimicrobial peptides. In the present study, we synthesized a series of peptides composed of lysine (K)-tryptophan (W) repeats (KW)n (where n equals 2, 3, 4 or 5) with amidation of the C-terminal to increase cationicity. We found that increases in chain length up to (KW)4 enhanced the peptides’ antibacterial activity; (KW)5 exhibited somewhat less bactericidal activity than (KW)4. Cytotoxicity, measured as lysis of human red blood cells, also increased with increasing chain length. With (KW)5, reduced antibacterial activity and increased cytotoxicity correlated with greater hydrophobicity and self-aggregation in the aqueous environment. The peptides acted by inducing rapid collapse of the bacterial transmembrane potential and induction of membrane permeability. The mode of interaction of the peptides and the phosphate groups of lipopolysaccharide was dependent upon the peptides’ ability to permeate the membrane. Longer peptides [(KW)4 and (KW)5] but not shorter peptides [(KW)2 and (KW)3] strongly bound and partially inserted into negatively charged, anionic lipid bilayers. These longer peptides also induced membrane permeabilization and aggregation of lipid vesicles. The peptides had a disordered structure in aqueous solution, and only (KW)4 and (KW)5 displayed a folded conformation on lipid membranes. Moreover, (KW)4 destroyed and agglutinated bacterial cells, demonstrating its potential as an antimicrobial agent. Collectively, the results show (KW)4 to be the most efficacious peptide in the (KW)n series, exhibiting strong antibacterial activity with little cytotoxicity.Electronic supplementary materialThe online version of this article (doi:10.1007/s00726-012-1388-6) contains supplementary material, which is available to authorized users.

Circular dichroism (CD) is a useful technique for monitoring changes in the conformation of antimicrobial peptides or gelatin. In this study, interactions between cationic peptides and gelatin were observed without affecting the triple helical content of the gelatin, which was more strongly affected by anionic surfactant. The peptides did not adopt a secondary structure in the presence of aqueous solution or Tween 80, but a peptide secondary structure formed upon the addition of sodium dodecyl sulfate (SDS). The peptides bound to the phosphate group of lipopolysaccharide (LPS) and displayed an alpha-helical conformation while (KW) 4 adopted a folded conformation. Further, the peptides did not specifically interact with the fungal cell wall components of mannan or laminarin. Tryptophan blue shift assay indicated that these peptides interacted with SDS, LPS, and gelatin but not with Tween 80, mannan, or laminarin. The peptides also displayed antibacterial activity against P. aeruginosa without cytotoxicity against HaCaT cells at MIC, except for HPA3NT3-analog peptide. In this study, we used a CD spectroscopic method to demonstrate the feasibility of peptide characterization in numerous environments. The CD method can thus be used as a screening method of gelatin-peptide interactions for use in wound healing applications. OPEN ACCESSInt. J. Mol. Sci. 2012, 13 3230

fThe increasing prevalence of drug-resistant pathogens highlights the need to identify novel antibiotics. Here we investigated the efficacies of four new antimicrobial peptides (AMPs) for potential drug development. The antibacterial activities, synergistic effects, and antibiofilm properties of the four chimeric AMPs were tested against Acinetobacter baumannii, an emerging Gramnegative, nosocomial, drug-resistant pathogen. Nineteen A. baumannii strains resistant to ampicillin, cefotaxime, ciprofloxacin, tobramycin, and erythromycin were isolated at a hospital from patients with cholelithiasis. All four peptides exhibited significant antibacterial effects (MIC ‫؍‬ 3.12 to 12.5 M) against all 19 strains, whereas five commercial antibiotics showed little or no activity against the same pathogens. An exception was polymyxin, which was effective against all of the strains tested. Each of the peptides showed synergy against one or more strains when administered in combination with cefotaxime, ciprofloxacin, or erythromycin. The peptides also exhibited an ability to prevent biofilm formation, which was not seen with cefotaxime, ciprofloxacin, or erythromycin, though polymyxin also inhibited biofilm formation. Indeed, when administered in combination with ciprofloxacin, the AMP HPMA exerted a potent synergistic effect against A. baumannii biofilm formation. Collectively, our findings indicate that the AMPs tested have no cytotoxicity but possess potent antibacterial and antibiofilm activities and may act synergistically with commercial antibiotics.

In recent years, the increasing number of antibiotic-resistant bacteria has become a serious health concern. Antimicrobial peptides (AMPs) are an important component of the innate immune system of most organisms. A better understanding of their structures and mechanisms of action would lead to the design of more potent and safer AMPs as alternatives for current antibiotics. For detailed investigations, effective recombinant production which allows the facile modification of the amino acid sequence, the introduction of unnatural amino acids, and labeling with stable isotopes for nuclear magnetic resonance (NMR) studies is desired. Several expression strategies have been introduced in previous reports; however, their effectiveness has been limited to a select few AMPs. Here, we have studied calmodulin (CaM) as a more universal carrier protein to express many types of AMPs in E. coli. We have discovered that the unique architecture of CaM, consisting of two independent target binding domains with malleable methionine-rich interaction surfaces, can accommodate numerous amino acid sequences containing basic and hydrophobic residues. This effectively masks the toxic antimicrobial activities of many amphipathic AMPs and protects them from degradation during expression and purification. Here, we demonstrate the expression of various AMPs using a CaM-fusion expression system, including melittin, fowlicidin-1, tritrpticin, indolicidin, puroindoline A peptide, magainin II F5W, lactoferrampin B, MIP3α51-70, and human β-defensin 3 (HBD-3), the latter requiring three disulfide bonds for proper folding. In addition, our approach was extended to the transmembrane domain of the cell adhesion protein l-selectin. We propose the use of the CaM-fusion system as a universal approach to express many cationic amphipathic peptides that are normally toxic and would kill the bacterial host cells.

Cationic antimicrobial peptides (AMPs) are essential components of the innate immune system, offering protection against invading pathogenic bacteria. In nature, AMPs serve as antibiotics with broad-spectrum antimicrobial and anti-biofilm properties. However, low effective stability in high-salt environments and physiological instability in biological membranes limit the applicability of naturally occurring AMPs as novel therapeutics. We therefore designed short synthetic cationic peptides by substituting key residues in myxinidin, an AMP derived from the epidermal mucus of hagfish, with lysine (Lys, K), arginine (Arg, R), and tryptophan (Trp, W). The resultant myxinidin analogs exhibited strong antimicrobial activity against both Gram-positive and Gram-negative bacteria, including multidrug-resistant strains, even under high-salt conditions. Moreover, these peptides showed high binding affinity for both lipopolysaccharides and lipoteichoic acids and inhibited biofilm formation by most bacteria, but did not cause significant lysis of human red blood cells and were not cytotoxic to normal human keratinocytes. Circular dichroism analysis revealed that myxinidin and its analogs assumed α-helical or β-sheet structures within artificial liposomes and bacterial membranes. In addition, bacterial killing and membrane permeation experiments demonstrated that the myxinidin analogs permeated through bacterial membranes, leading to cytoplasmic disruption and cell death. Taken together, these findings suggest myxinidin analogs may be promising candidate antibiotic agents for therapeutic application against antibiotic-resistant bacteria.