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.
Highlights
Antimicrobial peptides (AMPs) are ubiquitous and integral components of innate immunity in virtually every living organism, and are considered promising leads for new antibiotic therapies[1,2]
We have investigated the mechanisms of antimicrobial peptides (AMPs) and ampetoids concurrently, studying their interactions with both model membranes and real bacteria
Based on transmission electron micrographs and soft X-ray tomography of E. coli treated with these antibacterials as well as in vitro work demonstrating peptoid aggregation of bacterial ribosomes, we hypothesize that the non-specific intracellular interactions of AMPs and ampetoids—driven by both electrostatics and the hydrophobic effect—cause flocculation of polyanions such as ribosomes and DNA
Summary
Antimicrobial peptides (AMPs) are ubiquitous and integral components of innate immunity in virtually every living organism, and are considered promising leads for new antibiotic therapies[1,2]. AMPs are highly diverse in both primary and secondary structure, which has confounded efforts to elucidate their mechanisms against bacteria Investigations of their mode of action focused almost entirely on interactions between AMPs and the bacterial cytoplasmic membranes, since many leading researchers in the field hypothesized that membrane permeabilization or disruption could fully explain their activity[3,4,5]. Based on transmission electron micrographs and soft X-ray tomography of E. coli treated with these antibacterials as well as in vitro work demonstrating peptoid aggregation of bacterial ribosomes, we hypothesize that the non-specific intracellular interactions of AMPs and ampetoids—driven by both electrostatics and the hydrophobic effect—cause flocculation of polyanions such as ribosomes and DNA This mechanism causes widespread cytoplasmic disorganization and rapid macromolecular aggregation, leading to a disruption of normal cellular processes and death. Given the rich diversity of AMPs and their apparent mechanisms of actions, there may likewise be diverse mechanisms of actions among their respective peptoid mimics
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