Melittin-Induced Permeabilization, Re-sealing, and Re-permeabilization of E. coli Membranes

• Published in: Biophysical journal 2018-01, Vol.114 (2), p.368-379
• Main Authors: Yang, Zhilin, Choi, Heejun, Weisshaar, James C.
• Format: Article
• Language: English
• Subjects: Antimicrobial peptides; Bubbles; Cationic peptides; Cations; Cell walls; Crosslinking; Curvature; Cytoplasm; E coli; Fluorescence; Fluorescence microscopy; Green fluorescent protein; Lipid bilayers; Lipopolysaccharides; Mechanical properties; Membranes; Microscopy; Minimum inhibitory concentration; Modulus of elasticity; Peptides; Periplasm; Permeability; Sealing; Shrinkage; Turgor
• ISSN: 0006-3495; 1542-0086
• DOI: 10.1016/j.bpj.2017.10.046

The permeabilization of model lipid bilayers by cationic peptides has been studied extensively over decades, with the bee-sting toxin melittin perhaps serving as the canonical example. However, the relevance of these studies to the permeabilization of real bacterial membranes by antimicrobial peptides remains uncertain. Here, we employ single-cell fluorescence microscopy in a detailed study of the interactions of melittin with the outer membrane (OM) and the cytoplasmic membrane (CM) of live Escherichia coli. Using periplasmic green fluorescent protein (GFP) as a probe, we find that melittin at twice the minimum inhibitory concentration first induces abrupt cell shrinkage and permeabilization of the OM to GFP. Within ∼4 s of OM permeabilization, the CM invaginates to form inward facing “periplasmic bubbles.” Seconds later the bubbles begin to leak periplasmic GFP into the cytoplasm. Permeabilization is localized, consistent with possible formation of toroidal pores. Within ∼20 s, first the OM and then the CM re-seals to GFP. Some 2–20 min later, both CM and OM are re-permeabilized to GFP. We invoke a mechanism based on curvature stress concepts derived from model bilayer studies. The permeabilization and re-sealing events involve sequential, time-dependent build-up of melittin density within the outer and inner leaflets of each bilayer. We also propose a mechanical explanation for the early cell shrinkage event induced by melittin and a variety of other cationic peptides. As peptides gain access to the periplasm, they bind to the anionic peptido-crosslinks of the lipopolysaccharide layer, increasing its longitudinal elastic modulus. The cell wall shrinks because it can withstand the same turgor pressure with smaller overall extension. Shrinkage in turn induces invagination of the CM, preserving its surface area. We conclude by comparing the behavior of different peptides.