{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,8,25]],"date-time":"2026-08-25T16:57:49Z","timestamp":1787677069405,"version":"build-2784847793"},"reference-count":64,"publisher":"American Society for Microbiology","issue":"9","license":[{"start":{"date-parts":[[2025,9,10]],"date-time":"2025-09-10T00:00:00Z","timestamp":1757462400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"},{"start":{"date-parts":[[2025,9,10]],"date-time":"2025-09-10T00:00:00Z","timestamp":1757462400000},"content-version":"tdm","delay-in-days":0,"URL":"https:\/\/journals.asm.org\/non-commercial-tdm-license"}],"funder":[{"DOI":"10.13039\/100000060","name":"National Institute of Allergy and Infectious Diseases","doi-asserted-by":"publisher","award":["R01AI157205"],"award-info":[{"award-number":["R01AI157205"]}],"id":[{"id":"10.13039\/100000060","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["journals.asm.org"],"crossmark-restriction":true},"short-container-title":["mBio"],"published-print":{"date-parts":[[2025,9,10]]},"abstract":"<jats:title>ABSTRACT<\/jats:title>\n                  <jats:sec>\n                    <jats:title\/>\n                    <jats:p>\n                      Cell-to-cell spread is a major mechanism used by the bacterial pathogen\n                      <jats:italic toggle=\"yes\">Listeria monocytogenes<\/jats:italic>\n                      to disseminate within its host. In this mechanism, bacteria are directly transferred from the cytosol of an infected donor cell to a recipient cell via formation of an intercellular protrusion. The intercellular protrusion resolves into a vacuole that is disrupted by\n                      <jats:italic toggle=\"yes\">L. monocytogenes<\/jats:italic>\n                      to reach the cytosol of the recipient cell, where it divides and starts new cell-to-cell spread cycles. Here, we studied the spatiotemporal dynamics of the donor and recipient host cell plasma membranes during intercellular protrusion formation, resolution, and disruption, as well as the role of the bacterial pore-forming toxin listeriolysin O (LLO) and two phospholipases C in these dynamics. Leveraging three-dimensional live-cell microscopy, we co-cultured and imaged infected epithelial cells expressing different fluorescent plasma membrane markers to distinguish donor from recipient cell membranes. We characterized, for the first time, the dynamics of the canonical cell-to-cell spread pathway in which protrusions resolve into double-membrane vacuoles, followed by the successive disruption of donor and recipient membranes. We found that LLO controls protrusion resolution into a double-membrane vacuole and disruption of donor and recipient membranes, whereas the phospholipases C (PLCs) control donor membrane disruption and prevent bacterial entrapment into autophagosomes. Furthermore, we identified a second, more efficient \u201cnon-canonical\u201d cell-to-cell spread pathway strictly requiring LLO\/PLCs cooperation, which resolves into a single-membrane vacuole that is rapidly disrupted. Altogether, this study provides the first detailed analysis of membrane dynamics within intercellular protrusions and vacuoles, establishing an advanced model for\n                      <jats:italic toggle=\"yes\">L. monocytogenes<\/jats:italic>\n                      cell-to-cell spread.\n                    <\/jats:p>\n                    <jats:sec>\n                      <jats:title>IMPORTANCE<\/jats:title>\n                      <jats:p>\n                        Intracellular bacterial pathogens, including\n                        <jats:italic toggle=\"yes\">L. monocytogenes<\/jats:italic>\n                        , spread from cell to cell to effectively disseminate within host tissues and cause systemic infections. We developed a fluorescence microscopy-based method to visualize and distinguish in real time the dynamics of donor and recipient host cell plasma membranes during the different stages of the cell-to-cell spread process: (i) formation of the intercellular protrusion-containing\n                        <jats:italic toggle=\"yes\">L. monocytogenes<\/jats:italic>\n                        , (ii) protrusion resolution into a vacuole, and (iii) disruption of the vacuolar membranes until the bacterium is released into the cytosol of the recipient cell. We measured the kinetics of the two identified\u2014canonical and non-canonical\u2014cell-to-cell spread pathways of\n                        <jats:italic toggle=\"yes\">L. monocytogenes<\/jats:italic>\n                        in epithelial cells. This work also revealed that the virulence factors listeriolysin O and phospholipases C differentially control donor and recipient membrane remodeling and bacterial capture into autophagosomes, providing an advanced model for\n                        <jats:italic toggle=\"yes\">L. monocytogenes<\/jats:italic>\n                        cell-to-cell spread.\n                      <\/jats:p>\n                    <\/jats:sec>\n                  <\/jats:sec>","DOI":"10.1128\/mbio.01183-25","type":"journal-article","created":{"date-parts":[[2025,8,18]],"date-time":"2025-08-18T13:00:43Z","timestamp":1755522043000},"update-policy":"https:\/\/doi.org\/10.1128\/asmj-crossmark-policy-page","source":"Crossref","is-referenced-by-count":6,"title":["Role of listeriolysin O and phospholipases C in\n                    <i>L. monocytogenes<\/i>\n                    intercellular protrusion dynamics, resolution, and autophagy avoidance"],"prefix":"10.1128","volume":"16","author":[{"ORCID":"https:\/\/orcid.org\/0009-0003-9855-7973","authenticated-orcid":false,"given":"Madalyn","family":"Fields","sequence":"first","affiliation":[{"id":[{"id":"https:\/\/ror.org\/00rs6vg23","id-type":"ROR","asserted-by":"publisher"}],"name":"Department of Microbial Infection and Immunity, The Ohio State University Medical College","place":["Columbus, USA"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Abigail","family":"Spencer","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/00rs6vg23","id-type":"ROR","asserted-by":"publisher"}],"name":"Department of Microbial Infection and Immunity, The Ohio State University Medical College","place":["Columbus, USA"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sara J.","family":"Mason","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/00rs6vg23","id-type":"ROR","asserted-by":"publisher"}],"name":"Department of 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