Time-resolved phenotyping at subcellular resolution reveals shared principles and key trade-offs across antimicrobial peptide activities

Cationic antimicrobial peptides are a large family of host defense molecules with diverse sequences and structures. Here, we present a computational and experimental pipeline for quantifying the membrane-permeabilizing effects, as well as the intracellular impacts on ribosome and DNA organization, of cationic peptides in growing Escherichia coli cells at high temporal and subcellular resolution. Applying this pipeline to 11 diverse natural peptides and synthetic peptidomimetics uncovered shared antibacterial activities, but with different kinetics that categorized them into two classes: class I, where inner membrane permeabilization predominantly correlates with growth inhibition, and class II, where it does not. With the class I peptides, intracellular ribosome and DNA reorganization, along with growth inhibition, occurred abruptly and was coupled with inner membrane permeabilization. However, this coupling led to rapid peptide absorption by the first exposed cells and poor antibacterial activity against dense cell populations. With the class II peptides, ribosome/DNA reorganization and growth inhibition occurred more gradually, as inner membrane permeabilization was either absent or delayed. This was offset by slower intracellular uptake and greater efficacy against high cell densities. These kinetic differences reveal functional trade-offs between classes that have immunological and therapeutic implications.