Antimicrobial action of prototypic amphipathic cationic decapeptides and their branched dimers

Abstract

Toward delineation of antimicrobial action, a prototypic amphipathic, cationic decapeptide Ac-G-X-R-K-X-H-K-X-W-A-NH₂ was designed and peptides for which X was didehydrophenylalanine (ΔFm), α-aminoisobutyric acid (Um), or phenylalanine (Fm) were synthesized. A growth kinetics experiment indicated that the bacteriostatic effects were nil (Um), mild and transient (Fm), and strong and persistent (ΔFm) respectively. Though at par in binding to lipopolysaccharide, ΔFm and Fm, but not Um, caused outer membrane permeabilization. Inner membrane permeabilization was attenuated and membrane architecture rehabilitated with ΔFm but not Fm. Reverse phase high-performance liquid chromatography revealed that ΔFm was translocated into Escherichia coli, while Um and fragments of Fm were detected in the medium. Among these monomers, only ΔFm was modestly antibiotic [minimum inhibitory concentrations (MICs) of 110 μM (E. coli) and 450 μM (Staphylococcus aureus)]. Interestingly, a linear dimer of ΔFm, viz. (ΔFm)₂, turned out to be highly potent against E. coli [MIC of 2 μM and minimum bactericidal concentration (MBC) of 2 μM] and modestly potent against S. aureus (MIC of 20 μM and MBC of 20 μM). In contrast, a lysine-based branched dimer of ΔFm, viz. ΔFd, was found to be a potent antimicrobial against both E. coli (MIC of 2.5 μM) and S. aureus (MIC of 5 μM). Studies with analogous branched dimers of Fm and Um have indicated that dimerization represents a scaffold for potentiation of antimicrobial peptides and that the presence of ΔF confers potent activity against both E. coli and S. aureus. De novo design has identified ΔFd as a potent, noncytotoxic, bacterial cell-permeabilizing and -penetrating antimicrobial peptide, more protease resistant than its monomeric counterpart. We report that in comparison to the subdued and sequential "membrane followed by cell interior" mode of action of the monomeric ΔFm, the strong and simultaneous "membrane along with cell interior" targeting by the dimeric ΔFd potentiates and broadens its antibiotic action across the Gram-negative-Gram-positive divide.