The outermost layer of Gram negative bacteria is composed of a lipopolysaccharide (LPS) network that forms a dense protective hydrophilic barrier against entry of hydrophobic drugs. At low μM concentrations, a large family of cationic polypeptides known as antimicrobial peptides (AMPs) are able to penetrate the LPS layer and permeabilize the outer membrane (OM) and the cytoplasmic membrane (CM), causing cell death. Cecropin A is a well-studied cationic AMP from moth. Here a battery of time-resolved, single-cell microscopy experiments explores how deletion of sugar layers and/or phosphoryl negative charges from the core oligosaccharide layer (core OS) of K12 E. coli alters the timing of OM and CM permeabilization induced by Cecropin A. Deletion of sugar layers, or phosphoryl charges, or both from the core OS shortens the time to the onset of OM permeabilization to periplasmic GFP and also the lag time between OM permeabilization and CM permeabilization. Meanwhile, the 12-h minimum inhibitory concentration (MIC) changes only twofold with core OS alterations. The results suggest a two-step model in which the core oligosaccharide layers act as a kinetic barrier to penetration of Cecropin A to the lipid A outer leaflet of the OM. Once a threshold concentration has built up at the lipid A leaflet, nucleation occurs and the OM is locally permeabilized to GFP and, by inference, to Cecropin A. Whenever Cecropin A permeabilizes the OM, CM permeabilization always follows, and cell growth subsequently halts and never recovers on the 45 min observation timescale.

Antimicrobial peptides (AMPs) cause bacterial membrane permeabilization and ultimately cell death at low μM concentrations. The membrane permeabilization action of a moth derived AMP Cecropin A on E. coli cells in exponential growth (mid-log phase) is well studied. At 1×MIC concentration, Cecropin A penetrates the lipopolysaccharide (LPS) barrier and causes outer membrane (OM) and cytoplasmic membrane (CM) permeabilization. For non-septating cells, permeabilization of both membranes begins at one pole. For septating cells, OM permeabilization begins at the septal region and CM permeabilization begins at one pole. However, in nature bacteria are frequently found in nutrient-starved conditions. Here we extend our single-cell microscopy assays to the attack of Cecropin A on E. coli cells in early stationary phase. Stationary phase E. coli is much more resistant to membrane permeabilization by Cecropin A than mid-log phase E. coli. A tenfold higher concentration of Cecropin A is required to observe CM permeabilization in the majority of stationary phase cells, and even then permeabilization proceeds more slowly. In addition, the spatial pattern of initial CM permeabilization changes from localized at one pole to global. Studies of lipid mutant strains suggest that a sufficient localized concentration of the anionic phospholipid phosphatidylglycerol (PG) guides the position of initial attack of the cationic AMP Cecropin A on the CM.

Synthetic, sequence-random polymers that feature a wide range of backbone and side chain structures have been reported to function as mimics of natural host-defense peptides, inhibiting bacterial growth while exerting little or no toxicity toward eukaryotic cells. The common themes among these materials are net positive charge, which is thought to confer preferential action toward prokaryotic vs eukaryotic cells, and the presence of hydrophobic components, which are thought to mediate membrane disruption. This study is based on a set of new binary cationichydrophobic nylon-3 copolymers that was designed to ask whether factors beyond net charge and net hydrophobicity influence the biological activity profile. In previous work, we found that nonpolar subunits preorganized by a ring led to copolymers with a diminished tendency to disrupt human cell membranes (as measured via lysis of red blood cells) relative to copolymers containing more flexible nonpolar subunits. An alternative mode of conformational restriction, involving geminal substitution, also minimized hemolysis. Here, we asked whether combining a cyclic constraint and geminal substitution would be synergistic; the combination was achieved by introducing backbone methyl groups to previously described cyclopentyl and cyclohexyl subunits. The new cyclic subunits containing two quaternary backbone carbons (i.e, two sites of geminal substitution) were comparable or slightly superior in terms of antibacterial potency but markedly superior in terms of low hemolytic activity, relative to cyclic subunits lacking the quaternary carbons. However, new cyclic units containing only one quaternary carbon were very hemolytic, which was unanticipated. Variations in net hydrophobicity cannot explain the trend in hemolysis, in contrast to the standard perspective in this field. The impact of each new polymer on live E. coli cells was evaluated via fluorescence microscopy. All new polymers moved rapidly across the outer membrane without large-scale disruption of barrier function. Increasing the number of quaternary carbons in the nonpolar subunit correlated with an increased propensity to permeabilize the cytoplasmic membrane of E. coli cells. Collectively, these findings show that relationships between nonpolar subunit identity and biological activity are influenced by factors in addition to hydrophobicity and charge. We propose that the variation of subunit conformational properties may be one such factor.

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angle between Arg and His in the helices, and significantly increased when two hydrophilic residues are located at the same side of the helix. The peptides containing other high hydrophilic residues at the same side of the helices also had flip-promotion abilities, whereas the substitution of either Arg or His with a lower hydrophilic or hydrophobic residue inhibited the activities. Therefore, the scramblase activities of the peptides were attributed to the hydrophilicity of the residues at the same side. We also found that the peptides with hydrophilic residues at the deeper position in the membrane showed higher flip-promotion ability. These results provided the possibility that the proteins whose transmembrane regions have similar physicochemical properties as those of the peptides used in this study might be involved in the flipflop promotion in the ER. 495-Pos Board B265Cyclodextrin-Mediated Lipid Exchange Monitored with FRET