apidaecin-ib and 6-carboxyfluorescein

Recent surveillance data on antimicrobial resistance predict the beginning of the post-antibiotic era with pan-resistant bacteria even overcoming polymyxin as the last available treatment option. Thus, new substances using novel modes of antimicrobial action are urgently needed to reduce this health threat. Antimicrobial peptides are part of the innate immune system of most vertebrates and invertebrates and accepted as valid substances for antibiotic drug development efforts. Especially, short proline-rich antimicrobial peptides (PrAMP) of insect origin have been optimized for activity against Gram-negative strains. They inhibit protein expression in bacteria by blocking the 70S ribosome exit tunnel (oncocin-type) or the assembly of the 50S subunit (apidaecin-type binding). Thus, apidaecin analog Api137 and oncocin analog Onc112 supposedly bind to different nearby or possibly partially overlapping binding sites. Here, we synthesized Api137/Onc112-conjugates bridged by ethylene glycol spacers of different length to probe synergistic activities and binding modes. Indeed, the antimicrobial activities against Escherichia coli and Pseudomonas aeruginosa improved for some constructs, although the conjugates did not bind better to the 70S ribosome of E. coli than Api137 and Onc112 using 5(6)-carboxyfluorescein-labelled Api137 and Onc112 in a competitive fluorescence polarization assay. In conclusion, Api137/Onc112-conjugates showed increased antimicrobial activities against P. aeruginosa and PrAMP-susceptible and -resistant E. coli most likely because of improved membrane interactions, whereas the interaction to the 70S ribosome was most likely not improved relying still on the independent apidaecin- and oncocin-type binding modes. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.

Topics: Anti-Bacterial Agents; Antimicrobial Cationic Peptides; Binding Sites; Binding, Competitive; Escherichia coli; Ethylene Glycol; Fluoresceins; Fluorescent Dyes; Kinetics; Microbial Sensitivity Tests; Protein Binding; Pseudomonas aeruginosa; Ribosome Subunits, Large, Bacterial; Ribosome Subunits, Small, Bacterial; Spectrometry, Fluorescence

Contributing factors for the antimicrobial activity enhancement of N-terminally engineered mutants of cell-penetrating apidaecins were analyzed based on their cell-penetration efficiency. The flow cytometric analysis of the engineered apidaecins labeled with carboxyfluorescein (FAM) revealed their enhanced cell-penetrating efficiencies into Escherichia coli that should be one of key factors causing the enhanced antimicrobial activity. It is noteworthy that, for one mutant, the enhancement in antimicrobial activity (18-fold higher than wild type) was greater than that of cell penetration (5.9-fold), suggesting that the N-terminal mutation may reinforce both interaction with unidentified intracellular target(s) and cell-penetration efficiency.

Topics: Amino Acid Sequence; Antimicrobial Cationic Peptides; Escherichia coli; Flow Cytometry; Fluoresceins; Fluorescent Dyes; Molecular Sequence Data; Protein Engineering; Protein Transport