Direct Visualization of Antibiotic-induced Pores in Phospholipid Vesicles by Cryo Electron Microscope

Abstract

Cytolytic peptides, such as Magainin, Melittin, and Alamethicin are ubiquitously present within the animal kingdom as a part of the host-defense system. Magainin-2 lyses a wide range of both gram-negative and gram-positive bacteria and a range of cancer cells. Unlike most commercial antibiotics, which interact with specific protein targets, Magainin 2 and other peptides in this class have been shown to interact directly with the lipid bilayer; therefore, it is believed that bacteria will be largely unable to develop resistance to this class of antibiotics. It is believed to initially interact with acidic lipids in the bacterial membranes through electrostatic interactions, forming an amphiphillic helix, followed by hydrophobic interactions inducing pore formation, but the issue remains controversial. A number of methods have been used to study the structure of possible pores; however, none of those methods could directly observe the pores themselves. We present a new method for studying peptide/lipid interactions, which employs cryo-EM to directly image Magainin-induced pores in phospholipid vesicles. Images of DMPC/DMPG lipid vesicles with Magainin showed both perturbed and unperturbed vesicles, while vesicles without Magainin were unperturbed: perturbed vesicles exhibited power spectra similar to neutron scattering experiments in the presence of Magainin. To estimate pore size, we completed a set of simulations with randomly distributed pores on spherical vesicles. The mean pore size obtained by simulation was ∼83A, which is compatible with prior neutron scattering data. In addition, since the vesicle images are projections, we performed cryo-electron tomography experiments to reconstruct the 3-D structure of the pores. For the first time, we were able to visualize antibiotic peptide-induced pores on phospholipids vesicles, and the pore size is consistent with the simulation result.