Abstract

Cecropin A is a naturally occurring, linear, cationic, 37-residue antimicrobial peptide. The precise mechanism by which it kills bacteria is not known, but its site of action is believed to be the cell membrane. To investigate the nature of its membrane activity, we examined the ability of cecropin A to alter membrane permeability in synthetic lipid vesicles and in Gram-negative bacteria. Cecropin A exerted distinctly different types of membrane activity depending on its concentration. In synthetic lipid vesicles, cecropin A dissipated transmembrane electrochemical ion gradients at relatively low concentrations, but much higher concentrations were required to release an encapsulated fluorescent probe. Cecropin A dissipated ion gradients whether or not the vesicle membranes contained anionic lipid, although the presence of anionic lipid dramatically increased peptide binding, and modestly increased the release of an encapsulated probe. Cholesterol did not prevent the dissipation of ion gradients by low concentrations of peptide, but it did inhibit release of the encapsulated probe by high concentrations of peptide. At the highest concentrations examined, cecropin A remained monomeric in solution, and did not aggregate, lyse, or otherwise alter vesicle size. In Gram-negative bacteria, cecropin A was potently bactericidal at concentrations which dissipated ion gradients in lipid vesicles, but much higher concentrations were required to cause the release of cytoplasmic contents. These findings point to the conclusion that cecropin A kills bacteria by dissipating transmembrane electrochemical ion gradients. They weigh against theories comparing the antimicrobial activity of cecropin A to the release of encapsulated probes from lipid vesicles, and against roles for cholesterol or anionic lipid headgroups in the selectivity of peptide action against bacteria.

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