Bactenecin

With the emergence of multidrug-resistant bacteria, antimicrobial peptides (AMPs) are regarded as potential alternatives to traditional antibiotics or chemicals. We designed and synthesized six derivatives of bactenecin (L2C3V10C11, RLCRIVVIRVCR), including R2F3W10L11 (RRFRIVVIRWLR), R2W3W10R11 (RRWRIVVIRWRR), K2W3V10R11 (RKWRIVVIRVRR), W2R3V10R11 (RWRRIVVIRVRR), W2K3K10R11 (RWKRIVVIRKRR), and K2R3R10K11 (RKRRIVVIRRKR), by amino acid substitution to increase the net charge and reduce hydrophobicity gradually. The bioactivity and mechanisms of action of the designed peptides were investigated. The results indicated that the antimicrobial activity of the designed peptides was higher than that of bactenecin. The hemolytic activity and cytotoxicity of the designed peptides were significantly lower than those of bactenecin. The designed peptides exhibited a wide range of antimicrobial activity against food-pathogens, particularly peptides K2W3V10R11 and W2R3V10R11; in addition, the activity was maintained under physiological salt and heat conditions. Mechanism studies indicated that AMPs interacted with negatively charged bacterial cell membranes, resulting in the destruction of cell membrane integrity by increasing membrane permeability and changing transmembrane potential, leading to cell death. The present study suggested that peptides K2W3V10R11 and W2R3V10R11 exhibited potential as alternatives to traditional antibiotics or chemicals for the treatment of food-pathogens. These findings lead to the development of a potential method for the design of novel AMPs.

Cationic antimicrobial peptides (CAMPs) are important antibiotics because they possess a broad spectrum of activity against both Gram-positive and Gram-negative bacteria, including those resistant to traditional antibiotics. The cyclic peptide bactenecin is a 12-amino acid CAMP that contains one intramolecular disulfide bond. To improve the antibacterial activity of bactenecin, we designed and synthesized several bactenecin analogs by applying multiple approaches, including amino acid substitution, use of the d-enantiomeric form, and lipidation. Among the synthetic analogs, d-enantiomeric bactenecin conjugated to capric acid, which we named dBacK-(cap), exhibited a significantly enhanced antibacterial spectrum with MIC values ranging from 1 to 8 μM against both Gram-positive and Gram-negative bacteria, including some drug-resistant bacteria. Upon exposure to dBacK-(cap), S. aureus cells were killed within 1 h at the MIC value, but full inactivation of E. coli required over 2 h. These results indicate that covalent addition of a d-amino acid and a fatty acid to bactenecin is the most effective approach for enhancing its antibacterial activity.

The cathelicidin antimicrobial peptide bactenecin is a beta-hairpin molecule with a single disulfide bond and broad antimicrobial activity. The proform of bactenecin exists as a dimer, however, and it has been proposed that bactenecin is released as a dimer in vivo, although there has been little study of the dimeric form of bactenecin. To investigate the effect of bactenecin dimerization on its biological activity, we characterized the dimer's effect on phospholipid membranes, the kinetics of its bactericidal activity, and its salt sensitivity. We initially synthesized two bactenecin dimers (antiparallel and parallel) and two monomers (beta-hairpin and linear). Under oxidative folding conditions, reduced linear bactenecin preferentially folded into a dimer forming a ladder-like structure via intermolecular disulfide bonding. As compared to the monomer, the dimer had a greater ability to induce lysis of lipid bilayers and was more rapidly bactericidal. Interestingly, the dimer retained antimicrobial activity at physiological salt concentrations (150 mm NaCl), although the monomer was inactivated. This salt resistance was also seen with bactenecin dimer containing one intermolecular disulfide bond, and the bactenecin dimer appears to undergo multimeric oligomerization at high salt concentrations. Overall, dimeric bactenecin shows potent and rapid antimicrobial activity, and resists salt-induced inactivation under physiological conditions through condensation and oligomerization. These characteristics shed light on the features that a peptide would need to serve as an effective therapeutic agent.