A Pore Model or the Carpet Model? The Mode of Action of AMPs on E. Coli Spheroplasts

One fundamental issue in the field of AMPs has been whether the action of AMPs produces pores in the membranes or disintegrates the membranes by the saturated surface binding of AMPs (the carpet model). In the absence of clear evidence, either mechanism seems to explain the phenomena of bacteria lysis. Even if the issue were resolved in the model membrane studies, it is not clear if the result of model membranes extends to bacterial membranes. Here we investigated the action of AMPs on E. coli spheroplasts. The absence of the outer membrane made it possible to observe the action of AMPs on the cytoplasmic membranes. Previously we found that the properties of the bacterial cell membranes are dominated by a membrane reservoir, significantly different from that of a GUV. Furthermore, these unique properties of bacterial membranes are metabolically maintained. We would like to know how the actions of AMPs on spheroplast membranes compare with the actions on GUVs. We compared the actions of human AMP LL-37 and melittin on spheroplasts and GUVs. We developed a fluorescence recovery after photobleaching (FRAP) technique to examine the dye leakage through the bacterial membranes. AMP binding did not increase the apparent membrane area of a spheroplast, contrary to the responses of GUVs. The permeability through the bacterial membrane increased in a sigmoidal fashion as the AMP binding increased in time, exhibiting a cooperative behavior of AMPs. The analysis of FRAP showed that the fluxes of dye molecules in and out of the cell were consistent with diffusion of molecules through a number of pores that increased with binding of AMPs and then saturated to a steady level. The effects of LL37 and melittin are qualitatively the same.