Disruption of Escherichia coli outer membranes by EM 49. A new membrane active peptide antibiotic

Abstract

A new peptide antibiotic, EM 49, is shown to disrupt the structure of Escherichia coli outer membranes and release outer membrane fragments into the surrounding media. Evidence supporting this conclusion indludes EM 49 stimulated release of outer membrane phospholipids, lipopolysaccharide, and membrane fragments having a phospholipid and polypeptide composition similar to outer membranes. The density of the membrane fragments released by EM 49 was 1.22 g/cm3, which was identical to isolated outer membranes. Approximately 10 to 15% of the E. coli lipopolysaccharide was released upon treatment with EM 49. Both scanning and transmission electron microscopy revealed that the antibiotic caused the formation of numerous protrusions or blebs on the surface of E. coli with apparent release of membrane vesicles from the cells. Direct interaction between EM 49 and outer membranes was demonstrated using outer membranes labeled with the fluorescent dye diphenylhexatriene. Treatment of the fluorescent-labeled outer membranes with EM 49 increased fluorescence intensity and decreased polarization, indicating that the peptide perturbed outer-membrane structure. In addition, strong interactions between EM 49 and purified E. coli phospholipids were detected using the Hummel and Dreyer technique. Association constants between the peptide and phospholipids were approximately 10(5) M-1. A model for the disruptive effect of EM 49 on outer-membrane structure is proposed in which the fatty acid chain of the antibiotic is inserted into the hydrophobic core of the membrane. This orientation would allow the polycationic, peptide portion of the antibiotic to disrupt the antibiotic to disrupt the normal electrostatic interactions between divalent cations and components of the outer membrane. Evidence supporting this conclusion includes specific protection of E. coli from EM 49 by Mg2+ and Ca2+ and inhibition of EM 49 stimulated phospholipid release by these cations. Disruption of the antibiotic to penetrate to the inner membrane, which is probably the primary killing site of EM 49.

Isolation of a Paenibacillus sp. Strain and Structural Elucidation of Its Broad-Spectrum Lipopeptide Antibiotic

“…This structure may help paenibacterin to disrupt the cytoplasmic membrane of target cells. Disruption of the cytoplasmic membrane and leakage of cell components are considered the mechanism of action of many cationic antimicrobial peptides, such as polymyxin, octapeptin, and magainin (9,11,35,42). Polymyxin, which is produced by Paenibacillus polymyxa strains, contains six diaminobutyric acids and has five net positive charges (12).…”

Anionic lipid domains: correlation with functional topography in a mammalian cell membrane.

“…This duration of incubation, however, proved to be unnecessary because the distribution of fluorescence did not change after the first 10 min in the medium. Addition of 0.4 ml of 10 (13,14,19,35).…”

Mechanism of Action of EM 49, Membrane-Active Peptide Antibiotic

“…The permeability of phos-MECHANISM OF ACTION OF EM 49 671 pholipid liposomes for polar molecules has been shown to be increased by polymyxin B (7,8). Inhibition of bacterial growth, binding of the antibiotics to membranes and lipids, and perturbation of membrane structure are all specifically inhibited by Ca2+ and Mg2+ ions (6,11,22,25). These observations suggest that polymyxin and EM 49 may competitively displace magnesium or calcium from negatively charged phosphate groups on membrane lipids.…”

“…These antibiotics bind to and disrupt the structure of phospholipid and lipopolysaccharide aggregates (4,15,22). The permeability of phos-MECHANISM OF ACTION OF EM 49 671 pholipid liposomes for polar molecules has been shown to be increased by polymyxin B (7,8).…”

“…Recently, a new peptide antibiotic, EM 49 (recently renamed octapeptin), has been isolated (17) that disrupts membrane structure (19,22). EM 49 resembles the polymyxins structurally; however, the antimicrobial spectra of these peptides are significantly different.…”