Correlating antimicrobial activity and model membrane leakage induced by nylon-3 polymers and detergents

Abstract

Most antimicrobial peptides act upon target microorganisms by permeabilizing their membranes. The mode of action is often assessed by vesicle leakage experiments that use model membranes, with the assumption that biological activity arises from permeabilization of the lipid bilayer. The current work aims to extend the interpretation of vesicle leakage results and examine the correlation between vesicle leakage and antimicrobial activity. To this end, we used a lifetime-based leakage assay with calcein-loaded vesicles to study the membrane permeabilizing properties of a novel antifungal polymer poly-NM, two of its analogs, and a series of detergents. In conjunction, the biological activities of these compounds against Candida albicans were assessed and correlated with data from vesicle leakage. Poly-NM induces all-or-none leakage in polar yeast lipid vesicles at the polymer’s MIC, 3 μg/mL. At this and higher concentrations, complete leakage after an initial lag time was observed. Concerted activity tests imply that this polymer acts independently of the detergent octyl glucoside (OG) for both vesicle leakage and activity against C. albicans spheroplasts. In addition, Poly-NM was found to have negligible activity against zwitterionic vesicles and red blood cells. Our results provide a consistent, detailed picture of the mode of action of Poly-NM: this polymer induces membrane leakage by electrostatic lipid clustering. In contrast, Poly-MM:CO, a nylon-3 polymer comprised of both cationic and hydrophobic segments, seems to act by a different mechanism that involves membrane asymmetry stress. Vesicle leakage for this polymer is transient (limited to <100%) and graded, non-specific among zwitterionic and polar yeast lipid vesicles, additive with detergent action, and correlates poorly with biological activity. Based on these results, we conclude that comprehensive leakage experiments can provide a detailed description of the mode of action of membrane permeabilizing compounds. Without this thorough approach, it would have been logical to assume that the two nylon-3 polymers we examined act via similar mechanisms; it is surprising that their mechanisms are so distinct. Some, but not all mechanisms of vesicle permeabilization allow for antimicrobial activity.

Alpha-beta chimeric polypeptide molecular brushes display potent activity against superbugs-methicillin resistant Staphylococcus aureus

“…These results imply that the α/β CPMB kill S. aureus quickly by disrupting bacteria cell membrane using its multiple sidechain poly-α-L-lysine that is generally considered as HDP mimics. Similar observations have been reported for HDP and their mimics because these types of antimicrobial agents target cell membrane to have antibacterial activity [72].…”

Biomimetic antimicrobial polymers: recent advances in molecular design

“…149 Gellman et al showed that their HDP-mimetic nylon-3 polymer selectively disrupts lipid membranes depending on the lipid composition; the polymer efficiently disrupts liposomes of DOPG/DOPG (mimicking E. coli membranes), but not those of POPC (mimicking mammalian cell membranes). 147 These data suggest that optimized formulations of HDP-mimetic polymers are inherently more active against membranes that possess anionic charge and negative curvature. Fluorescence lifetime assays can distinguish between all-or-none leakage (a fraction of the vesicles released all of their contents) versus graded leakage (all vesicles released a portion of their contents).…”

“…146 Despite its simplicity, dye leakage provides a useful demonstration of the inherent membrane-disputing ability of a polymer, which may contribute to the mechanism of antibacterial and hemolytic action (at least in part). 147,148 This technique has been employed for polynorbornenes, 56 polymethacrylates, 43 and nylon-3 copolymers. 147 The fundamental basis for cell-type selectivity can be observed by comparing dye leakage from liposomes that mimic anionic bacterial membranes (e.g.…”

“…147,148 This technique has been employed for polynorbornenes, 56 polymethacrylates, 43 and nylon-3 copolymers. 147 The fundamental basis for cell-type selectivity can be observed by comparing dye leakage from liposomes that mimic anionic bacterial membranes (e.g. POPE/POPG) and those that mimic zwitterionic mammalian cell membranes (POPC/chol).…”

“…The type of mechanism depends on polymer hydrophobicity: a nylon-3 derivative consisting solely of cationic units induces all-or-none leakage, whereas a cationic/hydrophobic copolymer exhibits graded leakage. 147 Dye-labeled polymer. The binding affinity of the antimicrobial polymers to lipid bilayers can be estimated based on dye labeling with dansyl, which is sensitive to the polarity of its microenvironment.…”