Studying the Mechanisms of Hybrid Peptides Containing Permeabalizing and Cell Penetrating Domains

Abstract

The ability of antimicrobial peptides (AMPs) to target and lyse the harmful microbial membrane over that of a host's is a unique characteristic, making these innate immune effectors promising candidates to fill a growing therapeutic void resulting from antibiotic drug resistance. This selectivity is believed to depend on the chemical and structural properties of the lipids that comprise the cell membrane. The selectivity of AMPs can be based on the electrostatic attraction of these predominately cationic peptides for the bacterial membrane surface heavily populated with negatively charged lipid components. We have previously shown with atomic force microscopy that zwitterionic dimyristoylphosphatidylcholine (DMPC) bilayers display concentration-dependent structural transformations induced by protegrin-1 (PG-1) that progress from finger-like instabilities at bilayer edges, to the formation of pores, and finally to a network of worm-like micelles. The increasing degree of membrane disruption in chargeneutral membranes demonstrates that a more complex interaction than that suggested by a simple electrostatic argument is needed to explain AMP selectivity. We propose that in addition to an electrostatic element, specific membrane compositional differences between host and pathogen tunes AMP activity to selectively disrupt microbial membranes. We have tailored our investigations to utilize membrane components which eukaryotes and prokaryotes contain in drastically different proportions, specifically the presence and absence of cholesterol. In these results we have employed a variety of biophysical techniques to elucidate how increasing cholesterol content in both phospholipid monolayers and bilayers attenuates the ability of PG-1 to induce membrane disruption. Atomic force microscopy and isothermal titration calorimetry were used to assess the propensity for peptide insertion and pore formation. X-ray and neutron reflectivity measurements were advantageous in providing molecular level detail on the location and orientation of PG-1 with respect to the membrane. 2793-Pos Board B223What Vesicle Leakage Reveals about Antimicrobial Activity (and What It Doesn't) The mode of action of antimicrobial peptides and their mimics is often assessed by vesicle leakage experiments, and most of these compounds are believed to act by membrane permeabilization. This work aims at improving the interpretation of vesicle leakage data in general, and at understanding and optimizing the fungicidal activity of nylon-3 polymers. We have studied the membrane permeabilizing properties of the cationic homopolymer poly-NM (Liu, R et al. JACS, 2013, 135, 5270), which displays significant antifungal activity, and two related cationic/hydrophobic binary copolymers using the lifetime-based leakage assay of calcein-loaded vesicles. We compared the results with biological activities against Candida albicans. Poly-NM induces all-or-none leakage of vesicles that are made from yeast polar lipid extract (YPLE), at the polymer's MIC against C. albicans (3 m...