{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,6,2]],"date-time":"2026-06-02T13:16:13Z","timestamp":1780406173952,"version":"3.54.1"},"update-to":[{"DOI":"10.1371\/journal.ppat.1012707","type":"new_version","label":"New version","source":"publisher","updated":{"date-parts":[[2025,6,13]],"date-time":"2025-06-13T00:00:00Z","timestamp":1749772800000}}],"reference-count":52,"publisher":"Public Library of Science (PLoS)","issue":"5","license":[{"start":{"date-parts":[[2025,5,16]],"date-time":"2025-05-16T00:00:00Z","timestamp":1747353600000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/100006492","name":"Division of Intramural Research, National Institute of Allergy and Infectious Diseases","doi-asserted-by":"publisher","award":["R01 AI153455"],"award-info":[{"award-number":["R01 AI153455"]}],"id":[{"id":"10.13039\/100006492","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100006492","name":"Division of Intramural Research, National Institute of Allergy and Infectious Diseases","doi-asserted-by":"publisher","award":["R01AI073904"],"award-info":[{"award-number":["R01AI073904"]}],"id":[{"id":"10.13039\/100006492","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["www.plospathogens.org"],"crossmark-restriction":false},"short-container-title":["PLoS Pathog"],"abstract":"<jats:p><jats:italic>Shigella flexneri<\/jats:italic> is a human intracellular pathogen responsible for bacillary dysentery (bloody diarrhea). <jats:italic>S. flexneri<\/jats:italic> invades colonic epithelial cells and spreads from cell to cell, leading to massive epithelial cell fenestration, a critical determinant of pathogenesis. Cell-to-cell spread relies on actin-based motility, which leads to formation of membrane protrusions, as bacteria project into adjacent cells. Membrane protrusions resolve into intermediate structures termed vacuole-like protrusions (VLPs), which remain attached to the primary infected cell by a membranous tether. The resolution of the membranous tether leads to formation of double-membrane vacuoles (DMVs), from which <jats:italic>S. flexneri<\/jats:italic> escapes to gain access to the cytosol of adjacent cells. Here, we identify the class III PI3K family member PIK3C3 as a critical determinant of <jats:italic>S. flexneri<\/jats:italic> cell-to-cell spread. Inhibition of PIK3C3 decreased the size of infection foci formed by <jats:italic>S. flexneri<\/jats:italic> in HT-29 cells. Tracking experiments using live-fluorescence confocal microscopy showed that PIK3C3 is required for efficient resolution of VLPs into DMVs. PIK3C3-dependent accumulation of PtdIns(3)P at the VLP membrane in adjacent cells correlated with the transient recruitment of the membrane scission machinery component Dynamin 2 at the neck of VLPs at the time of DMV formation. By contrast, <jats:italic>Listeria monocytogenes<\/jats:italic> did not form VLPs and protrusions resolved directly into DMVs. However, PIK3C3 was also required for <jats:italic>L. monocytogenes<\/jats:italic> dissemination, but at the stage of vacuole escape. Finally, we showed that PIK3C3 inhibition decreased <jats:italic>S. flexneri<\/jats:italic> dissemination in the infant rabbit model of shigellosis. We propose a model of <jats:italic>Shigella<\/jats:italic> dissemination in which vacuole formation relies on the PIK3C3-dependent accumulation of PtdIns(3)P at the VLP stage of cell-to-cell spread, thereby supporting the resolution of VLPs into DMVs through recruitment of the membrane scission machinery component, DNM2.<\/jats:p>","DOI":"10.1371\/journal.ppat.1012707","type":"journal-article","created":{"date-parts":[[2025,5,16]],"date-time":"2025-05-16T17:31:47Z","timestamp":1747416707000},"page":"e1012707","update-policy":"https:\/\/doi.org\/10.1371\/journal.ppat.corrections_policy","source":"Crossref","is-referenced-by-count":1,"title":["Host PIK3C3 promotes Shigella flexneri spread from cell to cell through vacuole formation"],"prefix":"10.1371","volume":"21","author":[{"given":"Steven J.","family":"Rolland","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Zachary 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F.","family":"Agaisse","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"340","published-online":{"date-parts":[[2025,5,16]]},"reference":[{"issue":"11","key":"ppat.1012707.ref001","doi-asserted-by":"crossref","first-page":"1229","DOI":"10.1016\/S1473-3099(18)30475-4","article-title":"Morbidity and mortality due to shigella and enterotoxigenic Escherichia coli diarrhoea: the Global Burden of Disease Study 1990-2016","volume":"18","author":"IA Khalil","year":"2018","journal-title":"Lancet Infect Dis"},{"issue":"10122","key":"ppat.1012707.ref002","doi-asserted-by":"crossref","first-page":"801","DOI":"10.1016\/S0140-6736(17)33296-8","article-title":"Shigellosis","volume":"391","author":"KL Kotloff","year":"2018","journal-title":"Lancet"},{"key":"ppat.1012707.ref003","article-title":"Morphology of rectal mucosa of patients with shigellosis","author":"MM Mathan","year":"1991","journal-title":"Rev Infect 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