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 time-resolved quantification of both membrane-permeabilizing and intracellular effects in Escherichia coli . Applying this pipeline to 12 diverse natural peptides and synthetic peptidomimetics uncovers shared antimicrobial activities, but with different kinetics, forming two classes. With class I peptides, growth arrest is abrupt and predominantly coupled with inner membrane permeabilization and ribosome/DNA reorganization. However, membrane permeabilization leads to rapid peptide absorption by the first exposed bacterial cells, resulting in low efficacy against dense populations. With class II peptides, ribosome/DNA reorganization and growth inhibition occur more gradually, as inner membrane permeabilization is either absent or delayed. This is offset by slower intracellular peptide uptake and greater efficacy against high cell densities. These kinetic differences reveal functional trade-offs between classes that have major immunological and therapeutic implications.