Single-cell, real-time detection of oxidative stress induced in Escherichia coli by the antimicrobial peptide CM15

Abstract

Antibiotics target specific biochemical mechanisms in bacteria. In response to new drugs, pathogenic bacteria rapidly develop resistance. In contrast, antimicrobial peptides (AMPs) have retained broad spectrum antibacterial potency over millions of years. We present single-cell fluorescence assays that detect reactive oxygen species (ROS) in the Escherichia coli cytoplasm in real time. Within 30 s of permeabilization of the cytoplasmic membrane by the cationic AMP CM15 [combining residues 1-7 of cecropin A (from moth) with residues 2-9 of melittin (bee venom)], three fluorescence signals report oxidative stress in the cytoplasm, apparently involving O 2 − , H 2 O 2 , and •OH. Mechanistic studies indicate that active respiration is a prerequisite to the CM15-induced oxidative damage. In anaerobic conditions, signals from ROS are greatly diminished and the minimum inhibitory concentration increases 20-fold. Evidently the natural human AMP LL-37 also induces a burst of ROS. Oxidative stress may prove a significant bacteriostatic mechanism for a variety of cationic AMPs. If so, host organisms may use the local oxygen level to modulate AMP potency.antimicrobial peptide | oxidative stress | CM15 | real-time ROS assay | E. coli

HaloTag Assay Suggests Common Mechanism of E. coli Membrane Permeabilization Induced by Cationic Peptides

“…11 Flow of 10 μM CM15 (2X MIC) plus 100 nM JF 646 ligand across zy3720 cells began at t = 0. In an attempt to estimate the kinetic response time of the new assay, the time resolution was increased to 3 sec/cycle.…”

“…5, 6 Our lab has been developing single-cell, time resolved, fluorescence-based assays that monitor a variety of AMP-induced “symptoms”. 7–15 We have focused primarily on the Gram negative species E. coli under attack by the polycationic AMPs LL-37, 7, 14 Cecropin A, 8 CM15, 11 and Melittin, 15 as well as by the synthetic cationic random β-peptide copolymer MM 63 :CHx 37 (Table 1). 13 Thus far the methods enable direct determination of the timing of events such as outer membrane permeabilization (OMP) to GFP, cytoplasmic membrane permeabilization (CMP) to GFP and a Sytox dye, cell shrinkage, the halting of growth, and the onset of oxidative stress.…”

Controlling macromolecular structures towards effective antimicrobial polymers

“…This could be attainable by using labeled antimicrobial nanoparticles. In addition, it would be interesting to find out if synthetic antimicrobial polymers can induce oxidative stress on microbes as some antimicrobial peptides do [186].…”

Lights, Camera, Action! Antimicrobial Peptide Mechanisms Imaged in Space and Time

“…The time resolution can be sub-minute or sub-second, as the phenomena require. We have used this approach to study the interactions of several AMPs with E. coli (Gram-negative) [26–29] and B. subtilis (Gram-positive) [30]. Each cell exhibits a series of ‘symptoms’ on its own peculiar timescale (Figure 5, Key Figure).…”

“…For observations under aerobic conditions, fresh, aerated medium flows over the plated cells for the entire observation period, typically 60 min after initiation of flow of the AMP. Observations under anaerobic conditions are also feasible [26]. Use of a small microfluidics chamber enables us to flow growth medium plus AMP without undue expense.…”