EcDBS1R6: A novel cationic antimicrobial peptide derived from a signal peptide sequence.

BACKGROUND: Bacterial infections represent a major worldwide health problem the antimicrobial peptides (AMPs) have been considered as potential alternative agents for treating these infections. Here we demonstrated the antimicrobial activity of EcDBS1R6, a peptide derived from a signal peptide sequence of Escherichia coli that we previously turned into an AMP by making changes through the Joker algorithm. METHODS: Antimicrobial activity was measured by broth microdilution method. Membrane integrity was measured using fluorescent probes and through scanning electron microscopy imaging. A sliding window of truncated peptides was used to determine the EcDBS1R6 active core. Molecular dynamics in TFE/water environment was used to assess the EcDBS1R6 structure. RESULTS: Signal peptides are known to naturally interact with membranes; however, the modifications introduced by Joker transformed this peptide into a membrane-active agent capable of killing bacteria. The C-terminus was unable to fold into an α-helix whereas its fragments showed poor or no antimicrobial activity, suggesting that the EcDBS1R6 antibacterial core was located at the helical N-terminus, corresponding to the signal peptide portion of the parent peptide. CONCLUSION: The strategy of transforming signal peptides into AMPs appears to be promising and could be used to produce novel antimicrobial agents. GENERAL SIGNIFICANCE: The process of transforming an inactive signal peptide into an antimicrobial peptide could open a new venue for creating new AMPs derived from signal peptides.

Fast and potent bactericidal membrane lytic activity of PaDBS1R1, a novel cationic antimicrobial peptide.

Antimicrobial peptides (AMPs) are promising candidates for the development of future antibiotics. In an attempt to increase the efficacy of therapeutic AMPs, computer-based design methods appear as a reliable strategy. In this study, we evaluated the antimicrobial efficiency and mechanism of action of a novel designed AMP named PaDBS1R1, previously designed by means of the Joker algorithm, using a fragment of the ribosomal protein L39E from the archaeon Pyrobaculum aerophilum as a template. PaDBS1R1 displayed low micromolar broad-spectrum antimicrobial activity against Gram-negative (MIC of 1.5 µM) and Gram-positive (MIC of 3 µM) bacteria, including carbapenem-resistant Klebsiella pneumoniae (MIC of 6.25 µM) and methicillin-resistant Staphylococcus aureus (MIC of 12.5 µM), without cytotoxicity towards HEK-293 cells. In addition, membrane permeabilization and depolarization assays, combined with time-kill studies and FEG-SEM imaging, indicated a fast membrane permeation and further leakage of intracellular content. Biophysical studies with lipid vesicles show a preference of PaDBS1R1 for Gram-negative bacteria-like membranes. We investigated the three-dimensional structure of PaDBS1R1 by CD and NMR analyses. Our results suggest that PaDBS1R1 adopts an amphipathic α-helix upon interacting with hydrophobic environments, after an initial electrostatic interaction with negative charges, suggesting a membrane lytic effect. This study reveals that PaDBS1R1 has potential application in antibiotic therapy.

Selective amino acid substitution reduces cytotoxicity of the antimicrobial peptide mastoparan.

The emergence of antibiotic-resistant clinical isolates and the decreased rate of development of new antibiotics are a constant threat to human health. In this context, the therapeutic value of mastoparan (MP), a toxin from wasp venom, has been extensively studied. However, since MP shows significant cytotoxic activities, further optimization is needed. Here we evaluated the antimicrobial and cytolytic activities of an MP analog created by Ala-substitution in positions 5 and 8, named [I5, R8] mastoparan ([I5, R8] MP). We found that [I5, R8] MP displayed a broad-spectrum antimicrobial activity against bacteria and fungi (MIC in the range 3-25µM), without being hemolytic or cytotoxic toward HEK-293 cells. In addition, [I5, R8] MP-amide was highly potent (MIC=3µM) against antibiotic-resistant bacteria. The interaction with microbial membranes was investigated revealing that [I5, R8] MP is able to form an active amphipathic α-helix conformation and to disturb membranes causing lysis and cell death. Based on our findings, we hypothesize that [I5, R8] MP follows a mechanism of action similar to that proposed for MP, where the pore-forming activity leads to cell death. Our results indicate that hydrophobic moment modified by amino acid substitution may enhance MP selectivity.