Intracellular biomass flocculation as a key mechanism of rapid bacterial killing by cationic, amphipathic antimicrobial peptides and peptoids

Abstract

Many organisms rely on antimicrobial peptides (AMPs) as a first line of defense against pathogens. In general, most AMPs are thought to kill bacteria by binding to and disrupting cell membranes. However, certain AMPs instead appear to inhibit biomacromolecule synthesis, while causing less membrane damage. Despite an unclear understanding of mechanism(s), there is considerable interest in mimicking AMPs with stable, synthetic molecules. Antimicrobial N-substituted glycine (peptoid) oligomers (“ampetoids”) are structural, functional and mechanistic analogs of helical, cationic AMPs, which offer broad-spectrum antibacterial activity and better therapeutic potential than peptides. Here, we show through quantitative studies of membrane permeabilization, electron microscopy, and soft X-ray tomography that both AMPs and ampetoids trigger extensive and rapid non-specific aggregation of intracellular biomacromolecules that correlates with microbial death. We present data demonstrating that ampetoids are “fast killers”, which rapidly aggregate bacterial ribosomes in vitro and in vivo. We suggest intracellular biomass flocculation is a key mechanism of killing for cationic, amphipathic AMPs, which may explain why most AMPs require micromolar concentrations for activity, show significant selectivity for killing bacteria over mammalian cells, and finally, why development of resistance to AMPs is less prevalent than developed resistance to conventional antibiotics.

Rigidification of the Escherichia coli cytoplasm by the human antimicrobial peptide LL-37 revealed by superresolution fluorescence microscopy

“…Even after rinsing the permeabilized cells for 80 min with fresh, aerated medium lacking LL-37 the high intracellular density of LL-37 persists and growth does not resume. This live-cell study corroborates and extends a recent fixed-cell study from the Barron laboratory that used TEM and X-ray tomography to demonstrate agglomeration of ribosomes after treatment with LL-37 and a variety of cationic peptoids (9).…”

“…For example, bulk methods can distinguish disruption of the outer membrane (OM) from disruption of the cytoplasmic membrane (CM) using fluorogenic dyes, measure real-time release of K + from the cytoplasm, monitor dissipation of the proton motive force, and detect many additional effects (6, 7). Recent work has employed imaging methodologies such as transmission electron microscopy (TEM) (8), immunofluorescence, and soft X-ray tomography (9) to directly observe the effects of AMPs on single cells. Those studies necessarily involve fixation and permeabilization of the cells, and they are typically carried out at a single time point after addition of the AMP.…”

Halogenation as a tool to tune antimicrobial activity of peptoids

“…The aim of the study is to investigate the link between the nature of the halogen, the amount of halogen substitution, their ability to self-assemble into nanostructures and their antimicrobial activity. Next, we proceeded to incorporate halogen atoms into the scaffold of the well-known Barron lab compound, Peptoid 1 34 , 35 , in an attempt to increase its antimicrobial activity, or to modulate its cytotoxicity.…”

Peptoid drug discovery and optimization via surface X‐ray scattering

“…There are several mechanisms of membrane disruption in bacteria described for AMPs including barrel‐stave, carpet‐like, and formation of toroidal pores, yet none of them have been confirmed for peptoid substituents. Membrane damaging effect is suggested to be the primary mode of action by peptoids, while the potential intracellular targeting of ATP efflux, ribosome aggregation, enzymatic and mitochondrial activity, as well as nucleic acid synthesis, might be also involved. Peptoid amphipathic topology allows for electrostatic binding toward the lipid phosphate groups, implicated in membrane depolarization .…”