Biochemical property and membrane-peptide interactions of de novo antimicrobial peptides designed by helix-forming units.

Typical peptides composed of Phe, Ile, and Arg residues have not been reported, and the effect of the helix-forming unit (HFU) composed of the tripeptide core on biological activity remains unclear. In this study, multimers of the 3-residue HFU were designed to investigate the structure-function relationships. The in vitro biological activities of the peptides were determined. We used synthetic lipid vesicles and intact bacteria to assess the interactions of the peptides with cell membranes. The well-studied peptide melittin was chosen as a control peptide. The results showed that the antimicrobial and hemolytic activities of the peptides increased with the number of HFUs. HFU3 had optimal cell selectivity as determined by the therapeutic index. HFU3 and HFU4 exhibited strong resistance to salts, pH, and heat. CD spectra revealed that the peptides except HFU2 displayed α-helix-rich secondary structures in the presence of SDS or trifluoroethanol (TFE). The peptides interacted weakly with zwitterionic phospholipids (mimicking mammalian membranes) but strongly with negatively charged phospholipids (mimicking bacterial membranes), which corresponds well with the data for the biological activities. There was a correlation between the cell selectivity of the peptides and their high binding affinity with negatively charged phospholipids. Cell membrane permeability experiments suggest that the peptides targeted the cell membrane, and HFU3 showed higher permeabilization of the inner membrane but lower permeabilization of the outer membrane than melittin. These findings provide the new insights to design antimicrobial peptides with antimicrobial potency by trimers.

Cell selectivity and interaction with model membranes of Val/Arg-rich peptides.

Antimicrobial peptides are major components of the innate self-defence system and a large number of peptides have been designed to study the mechanism of action. In the present study, a small combinatorial library was designed to study whether the biological activity of Val/Arg-rich peptides is associated with targeted cell membranes. The peptides were produced by segregating hydrophilic residues on the polar side and hydrophobic residues on the opposite side. The peptides displayed strong antimicrobial activity against Gram-negative and Gram-positive bacteria, but weak haemolysis even at a concentration of 256 µM. CD spectra showed that the peptides formed α-helical-rich structure in the presence of negatively charged membranes. The tryptophan fluorescence and quenching experiments indicated that the peptides bound preferentially to negatively charged phospholipids over zwitterionic phospholipids, which corresponds well with the biological activity data. In the in vivo experiment, the peptide G6 decreased the bacterial counts in the mouse peritoneum and increased survival after 7 days. Overall, a high binding affinity with negatively charged phospholipids correlated closely with the cell selectivity of the peptides and some peptides in this study may be likely candidates for the development of antibacterial agents.

The influence of isoleucine and arginine on biological activity and peptide-membrane interactions of antimicrobial peptides from the bactericidal domain of AvBD4.

In this study, the influence of isoleucine and arginine on the biological activity and peptide-membrane interactions of linear avian ß-defensin-4 (RL38) analogs was investigated. Results of biological activities showed that the antimicrobial activities of AvBD-4 analogs were closely related to hydrophobicity and amphipathicity. The peptide GLI19 with high hydrophobicity value and amphipathicity displayed broad spectrum antimicrobial activity against both gram-negative and gram-positive, whereas GLR19 with increasing multiple charges only exhibited activity against gram-negative. The interaction between peptides and the liposome membrane demonstrated that the peptides preferentially bound to negatively charged phospholipids over zwitterionic phospholipids, which supported the antimicrobial activity data. The outer membranes assay further demonstrated that GLI19 had a greater capacity than the other tested peptides to penetrate the cell membrane at a low concentration. Collectively, the peptides derived from the bactericidal domain of linear ß- defensins by truncation and hydrophobic amino acid substitution may be effective high-potential antibacterial agents.

Influence of truncation of avian ß-defensin-4 on biological activity and peptide-membrane interaction.

Defensins are important components in host defense systems. The therapeutic use of ß-defensins is limited by their innate toxicity and high cost due to the size and complex disulfide pairing. In this study, we used linear avian ß- defensin-4 (RL38) without disulfide bonds as model peptide to derive two peptides by the truncation. GL23 is the C-terminal truncated sequence of RL38, and GLI23 is the derivative of GL23 by the replacement of cysteines with isoleucines. Results showed that these peptides exhibited strong antibacterial activity against gram-negative and gram-positive bacteria. An exception was that GL23 showed weak antimicrobial activity against gallinaceous pathogenic bacteria Salmonella Pullorum C79-13. Two truncated peptides GL23 and GLI23 displayed no or weak hemolysis, which was in accordance with little blue shifts of the peptides in the presence of synthetic eukaryotic membranes. CD spectroscopy demonstrated that these peptides appeared to be unfolded in aqueous solution but acquire structure in the presence of membrane- mimicking phospholipids. GLI23 kept the antibacterial activity at high concentrations of NaCl or low concentration of divalent cations (Mg2+ and Ca2+). The peptides preferentially bound to negatively charged phospholipids over zwitterionic phospholipids, which led to greater cell selectivity. The outer and inner membranes assay displayed that GLI23 killed bacteria by targeting the cell membrane. These results suggest the peptides derived by truncation of linear ß-defensins may be a promising candidate for future antibacterial agent.

Biochemical property and in vivo efficacies of novel Val/Arg-rich antimicrobial peptide.

A novel α-helical antimicrobial peptide G6 rich in Val/Arg residues has been screened previously. In this study, we further evaluated the biochemical stability, interaction with whole bacteria, and in vivo therapeutic or prophylactic role of the peptide in Salmonella typhimurium-infected mice. The results showed that G6 exhibited strong resistance to pH, heat, and salts. But G6 lost the antimicrobial activity when treated with proteolytic enzymes. G6 had no toxicity on mammalian cell. An intraperitoneal model of sepsis caused by Salmonella typhimurium was established in mice. G6 was administered intraperitoneally 1 h before or after mice were infected with Salmonella typhimurium. For the mice given peptide post-bacterial infection, the mortality of the mice and the peritoneal bacterial counts were significantly lower in the groups that were administered 2.5 mg/kg BW and 5.0 mg/kg BW of G6 (P < 0.05) compared to the PBS-treated group. Similar trend was obtained when G6 was given 1 h prior to Salmonella typhimurium infection. Peptide-membrane experiments showed that G6 was effective in permeabilizing the outer and inner membrane in a dose dependent manner, indicating that the peptide targets the cell membrane. Taken together, the results revealed that the peptide G6 may provide a useful alternative to antibiotic agents to treat or prevent bacterial infections.

Novel design of short antimicrobial peptides derived from the bactericidal domain of avian ß-defensin-4.

Short antimicrobial peptides were designed and synthesized by C-terminal truncation and residue substitution of avian ß-defensin-4. The biological activity of these peptides was examined to elucidate the quantitative structure-activity relationships and find a lead peptide for the development of a novel antimicrobial peptide. The results showed that the truncation of the avian ß-defensin-4 eliminated the hemolysis of the peptide. The GLI13 derivative, developed by substituting the Cys of the truncated peptide with Ile, led to increased antimicrobial activity. These results suggest that the peptides with antimicrobial activity can be derived by truncating the avian ß-defensin-4. We further developed the GLI13 derivative with an increased net charge by residue substitution. The results showed that the GLI13-5 with 5 net charges had the highest cell selectivty. An increase in the net charge from 6 to 8 did not result in the improvement of antimicrobial potency. Membrane-simulating experiments showed that the peptides preferentially bound to negatively charged phospholipids over zwitterionic phospholipids, which led to greater cell selectivity. A membrane depolarization assay showed that GLI13-5 killed bacteria by targeting the cytoplasmic membrane. These results suggest that the short peptide developed by truncation of linear ß-defensin may be a promising candidate for future antibacterial agents.

The effects of Leu or Val residues on cell selectivity of α-helical peptides.

In this study, the peptides were designed to compare the effect of multiple Leu or Val residues as the hydrophobic side of an α-helical model on their structure, function, and interaction with model membranes. The Leu-rich peptides displayed 4- to 16-fold stronger antimicrobial activity than Val-rich peptides, while Val-containing peptides showed no haemolysis and weak cytotoxicity. The peptides LR and VR showed an α-helical-rich structure under a membranemimicking environment. Different cell selectivity for Leu- or Val-containing peptides correlated with the targeted cell membranes. The Leu-rich peptide LR(W) and Val-rich peptide VR(W) interacted preferentially with negatively charged phospholipids over zwitterionic phospholipids. VR(W) displayed no interaction with zwitterionic phospholipids, which was consistent with its lack of haemolytic activity. The ability of LR to depolarize bacterial cells was much greater than that of VR. Val- and Leu-rich peptides appeared to kill bacteria in a membrane-targeted fashion, with different modes of action. Leu-rich peptides appeared to be active via a membrane-disrupting mode, while Val-rich peptides were active via the formation of small channels.