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LCV formation is governed by the bacterial Icm\/Dot type IV secretion system that translocates ~300 different \u201ceffector\u201d proteins into host cells. Some of the translocated effectors anchor to the LCV membrane via phosphoinositide (PI) lipids. Here, we use the soil amoeba\n            <jats:named-content content-type=\"genus-species\">Dictyostelium discoideum<\/jats:named-content>\n            , producing fluorescent PI probes, to analyze the LCV PI dynamics by live-cell imaging. Upon uptake of wild-type or Icm\/Dot-deficient\n            <jats:named-content content-type=\"genus-species\">L. pneumophila<\/jats:named-content>\n            , PtdIns(3,4,5)\n            <jats:italic>P<\/jats:italic>\n            <jats:sub>3<\/jats:sub>\n            transiently accumulated for an average of 40\u00a0s on early phagosomes, which acquired PtdIns(3)\n            <jats:italic>P<\/jats:italic>\n            within 1\u00a0min after uptake. Whereas phagosomes containing \u0394\n            <jats:italic>icmT<\/jats:italic>\n            mutant bacteria remained decorated with PtdIns(3)\n            <jats:italic>P<\/jats:italic>\n            , more than 80% of wild-type LCVs gradually lost this PI within 2\u00a0h. The process was accompanied by a major rearrangement of PtdIns(3)\n            <jats:italic>P<\/jats:italic>\n            -positive membranes condensing to the cell center. PtdIns(4)\n            <jats:italic>P<\/jats:italic>\n            transiently localized to early phagosomes harboring wild-type or \u0394\n            <jats:italic>icmT L.\u00a0pneumophila<\/jats:italic>\n            and was cleared within minutes after uptake. During the following 2\u00a0h, PtdIns(4)\n            <jats:italic>P<\/jats:italic>\n            steadily accumulated only on wild-type LCVs, which maintained a discrete PtdIns(4)\n            <jats:italic>P<\/jats:italic>\n            identity spatially separated from calnexin-positive endoplasmic reticulum (ER) for at least 8\u00a0h. The separation of PtdIns(4)\n            <jats:italic>P<\/jats:italic>\n            -positive and ER membranes was even more pronounced for LCVs harboring \u0394\n            <jats:italic>sidC<\/jats:italic>\n            -\n            <jats:italic>sdcA<\/jats:italic>\n            mutant bacteria defective for ER recruitment, without affecting initial bacterial replication in the pathogen vacuole. These findings elucidate the temporal and spatial dynamics of PI lipids implicated in LCV formation and provide insight into host cell membrane and effector protein interactions.\n          <\/jats:p>\n          <jats:p>\n            <jats:bold>IMPORTANCE<\/jats:bold>\n            The environmental bacterium\n            <jats:named-content content-type=\"genus-species\">Legionella pneumophila<\/jats:named-content>\n            is the causative agent of Legionnaires\u2019 pneumonia. The bacteria form in free-living amoebae and mammalian immune cells a replication-permissive compartment, the\n            <jats:italic>Legionella<\/jats:italic>\n            -containing vacuole (LCV). To subvert host cell processes, the bacteria secrete the amazing number of ~300 different proteins into host cells. Some of these proteins bind phosphoinositide (PI) lipids to decorate the LCV. PI lipids are crucial factors involved in host cell membrane dynamics and LCV formation. Using\n            <jats:italic>Dictyostelium<\/jats:italic>\n            amoebae producing one or two distinct fluorescent probes, we elucidated the dynamic LCV PI pattern in high temporal and spatial resolution. Notably, the endocytic PI lipid PtdIns(3)\n            <jats:italic>P<\/jats:italic>\n            was slowly cleared from LCVs, thus incapacitating the host cell\u2019s digestive machinery, while PtdIns(4)\n            <jats:italic>P<\/jats:italic>\n            gradually accumulated on the LCV, enabling critical interactions with host organelles. The LCV PI pattern underlies the spatiotemporal configuration of bacterial effector proteins and therefore represents a crucial aspect of LCV formation.\n          <\/jats:p>","DOI":"10.1128\/mbio.00839-13","type":"journal-article","created":{"date-parts":[[2014,1,29]],"date-time":"2014-01-29T03:34:59Z","timestamp":1390966499000},"update-policy":"https:\/\/doi.org\/10.1128\/asmj-crossmark-policy-page","source":"Crossref","is-referenced-by-count":115,"title":["Live-Cell Imaging of Phosphoinositide Dynamics and Membrane Architecture during\n            <i>Legionella<\/i>\n            Infection"],"prefix":"10.1128","volume":"5","author":[{"given":"Stephen","family":"Weber","sequence":"first","affiliation":[{"name":"Max von Pettenkofer Institute, Department of Medicine, Ludwig-Maximilians University Munich, Munich, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Maria","family":"Wagner","sequence":"additional","affiliation":[{"name":"Max von Pettenkofer Institute, 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