Effects of alterations of the E. coli lipopolysaccharide layer on membrane permeabilization events induced by Cecropin A

Abstract

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.

Resistance of early stationary phase E. coli to membrane permeabilization by the antimicrobial peptide Cecropin A

“…Next we performed live-cell imaging experiments to measure the time course of Cecropin A action on single E. coli cells growing exponentially or in stationary phase. These experiments were performed using the imaging assays described in our previous work [14]. Briefly, for E. coli cells in exponential growth phase, a solution of Cecropin A and 5 nM Sytox Orange in fresh EZRDM flowed through the microfluidic chamber containing plated mid-log cells (Methods).…”

Studies on the interactions of neutral Galleria mellonella cecropin D with living bacterial cells

“…There are studies indicating that the LPS layer plays a crucial role in the attraction and control of AMPs binding to the surface of Gram-negative bacteria (Ding et al 2003;Papo and Shai 2005;Agrawal and Weisshaar 2018). Ebbensgaard et al (2015) demonstrated protective effect of LPS against antimicrobial activity of many AMPs (including cecropin B and cecropin P1) manifested by a higher susceptibility of the E. coli LPS defective mutants in comparison with their parental strains: O6 serotype E. coli ATCC 25,922 and rough E. coli BW25113K-12.…”

“…Furthermore, Agrawal and Weisshaar (2018) using single-cell fluorescence microscopy showed that deletion of sugar layers or/and phosphate groups from the core oligosaccharide in E. coli K-12 LPS accelerated permeabilization of the outer membrane and cytoplasmic membrane by cecropin A. They postulated that the core oligosaccharide part of LPS functions as a kinetic barrier slowing cecropin A passage to the lipid A leaflet (Agrawal and Weisshaar 2018).…”

Electrophysiological Analysis of Antimicrobial Peptides in Diverse Species

“…We mixed the bacterial solution with the Mueller-Hinton broth, and the potential bacterial increase was checked by examining the turbidity after 24 h of incubation at 35 °C. The value of cecropin A, 46 pardaxin P5, 47 and SL-37 29 have previously reported and used the value in this study.…”