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Here, we present a time-resolved single-cell pipeline for quantifying these effects in\n                  <jats:italic>Escherichia coli<\/jats:italic>\n                  . Applying this pipeline to 18 diverse natural peptides and synthetic peptidomimetics reveals shared core activities, but with different kinetics, defining two classes with opposite trade-offs. Class I peptides cause abrupt growth arrest, predominantly coupled with inner membrane permeabilization and ribosome\/DNA reorganization, conferring fast, multipronged action. However, rapid intracellular absorption by the first permeabilized cells depletes the extracellular pool, rendering them ineffective against dense populations, including biofilms. Class II peptides act more gradually, with delayed or absent inner membrane permeabilization, limiting their speed of action. However, this results in slower intracellular absorption and greater efficacy at high cell densities and against biofilms. These opposing functional trade-offs point to important immunological and therapeutic implications.\n                <\/jats:p>","DOI":"10.1101\/2025.04.10.648262","type":"posted-content","created":{"date-parts":[[2025,4,11]],"date-time":"2025-04-11T00:55:11Z","timestamp":1744332911000},"source":"Crossref","is-referenced-by-count":2,"title":["Time-resolved phenotyping at subcellular resolution reveals shared principles and key trade-offs across antimicrobial peptide activities"],"prefix":"10.64898","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-8366-2998","authenticated-orcid":false,"given":"Alessio","family":"Fragasso","sequence":"first","affiliation":[{"name":"Sarafan ChEM-H Institute, Stanford University, Stanford, CA, USA"},{"name":"Department of Biology, Stanford University, Stanford, CA, USA"},{"name":"Howard Hughes Medical Institute, Stanford University, Stanford, CA, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-8430-3954","authenticated-orcid":false,"given":"Tatjana","family":"Schlechtweg","sequence":"additional","affiliation":[{"name":"Sarafan ChEM-H Institute, Stanford University, Stanford, CA, USA"},{"name":"Howard Hughes Medical Institute, Stanford University, Stanford, CA, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-8177-5892","authenticated-orcid":false,"given":"Wei-Hsiang","family":"Lin","sequence":"additional","affiliation":[{"name":"Sarafan ChEM-H Institute, Stanford University, Stanford, CA, USA"},{"name":"Howard Hughes Medical Institute, Stanford University, Stanford, CA, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0735-6873","authenticated-orcid":false,"given":"Annelise E.","family":"Barron","sequence":"additional","affiliation":[{"name":"Department of Bioengineering, Stanford University, Schools of Medicine and of Engineering, Stanford, CA, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0980-5334","authenticated-orcid":false,"given":"Christine","family":"Jacobs-Wagner","sequence":"additional","affiliation":[{"name":"Sarafan ChEM-H Institute, Stanford University, Stanford, CA, USA"},{"name":"Department of Biology, Stanford University, Stanford, CA, USA"},{"name":"Howard Hughes Medical Institute, Stanford University, Stanford, CA, USA"},{"name":"Department of Bioengineering, Stanford University, Schools of Medicine and of Engineering, Stanford, CA, USA"},{"name":"Department of Microbiology and Immunology, Stanford University, School of Medicine, Stanford, CA, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"54368","reference":[{"key":"2026091719150814000_2025.04.10.648262v4.1","doi-asserted-by":"publisher","DOI":"10.1111\/j.1365-2958.2012.08081.x"},{"key":"2026091719150814000_2025.04.10.648262v4.2","doi-asserted-by":"publisher","DOI":"10.1038\/nmeth.4509"},{"key":"2026091719150814000_2025.04.10.648262v4.3","doi-asserted-by":"publisher","DOI":"10.1039\/C9AN01185D"},{"key":"2026091719150814000_2025.04.10.648262v4.4","doi-asserted-by":"publisher","DOI":"10.1038\/nbt0102-83"},{"key":"2026091719150814000_2025.04.10.648262v4.5","doi-asserted-by":"publisher","DOI":"10.1101\/2023.03.09.531820"},{"key":"2026091719150814000_2025.04.10.648262v4.6","doi-asserted-by":"publisher","DOI":"10.1128\/IAI.61.7.2978-2984.1993"},{"key":"2026091719150814000_2025.04.10.648262v4.7","unstructured":"Ca\u00f1as-Duarte, S. 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