Cholesterol Incorporation in Membranes Attenuates the Disruption Ability of Antimicrobial Peptide Protegrin-1

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 the growing therapeutic void resulting from antibiotic drug resistance. This discriminatory behavior is believed to strongly depend on the chemical and structural properties of the lipids that comprise the cell membrane. For instance, 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 that zwitterionic dimyristoylphosphatidylcholine (DMPC) bilayers display concentration-dependent structural transformations induced by protegrin-1 (PG-1) that progress from fingerlike instabilities at bilayer edges, to the formation of pores, and finally to a network of wormlike micelles. The increasing degree of membrane structural disruption in charge-neutral 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 consideration, specific membrane compositional differences between host and pathogen tunes AMP activity to selectively disrupt microbial membranes rather than those of the host. 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 respectively. In these results we have employed a variety of biophysical techniques to elucidate how increasing cholesterol content in solid-supported DMPC bilayers retards the ability of PG-1 to induce membrane disruption. Atomic force microscopy was used to assess the propensity for pore formation, while neutron reflectivity and oriented circular dichroism studies were advantageous in providing molecular level detail on the location and orientation of PG-1 with respect to the membrane.