Attack on Single Escherichia Coli Spheroplasts by Antimicrobial Peptides

Abstract

Studies of the molecular mechanisms of AMPs are mostly performed with lipid bilayers.Thus there is a persistent question as to whether the action of AMPs on bacterial membranes can be reproduced on lipid bilayers. Recently Weisshaar and coworkers have studied the actions of AMPs on E. coli and Bacillus subtilis by time-lapse fluorescence microscopy. The direct observation of the action of AMPs on bacteria revealed two key steps. The first is growth halting due to direct interference of AMP with cell wall synthesis and is recoverable. The second is permeabilization of the cytoplasmic membrane which is not recoverable. Here we study the direct action of AMPs on the cytoplasmic membranes by using E. coli spheroplasts, the cell form from which the outer membranes have been removed. The purpose is to compare the action on bacterial membranes with that on lipid bilayers. The key question is how to reveal the mechanisms of AMPs on bacterial membranes. First we observe the action of AMPs on giant unilamellar vesicles (GUVs) made of E. coli total lipid extract.We used the aspiration method to hold the GUV in a solution containing a soluble dye molecule and a fluorescence labeled melittin to obtain the lipid bilayer response to an AMP. The same method was applied to spheroplasts to observe the action of AMPs on bacterial membranes. In both cases, we found first the binding of peptides expanded the membrane area. As the membrane area increased to ∼2-3%, the dye molecules began to leak in. After a time, we found the spheroplasts lost their phase contrast indicating the lose of the cell content. The same experiments were repeated with LL37 and magainin. We found that sytox green is not a good indicator of membrane permeability.