{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,26]],"date-time":"2026-09-26T13:28:10Z","timestamp":1790429290160,"version":"4.1.0"},"reference-count":77,"publisher":"American Society for Microbiology","issue":"11","license":[{"start":{"date-parts":[[2018,6,1]],"date-time":"2018-06-01T00:00:00Z","timestamp":1527811200000},"content-version":"tdm","delay-in-days":0,"URL":"https:\/\/journals.asm.org\/non-commercial-tdm-license"}],"funder":[{"DOI":"10.13039\/501100001711","name":"Swiss National Science Foundation","doi-asserted-by":"crossref","award":["31003A_153200"],"award-info":[{"award-number":["31003A_153200"]}],"id":[{"id":"10.13039\/501100001711","id-type":"DOI","asserted-by":"crossref"}]},{"name":"OPO Foundation"},{"DOI":"10.13039\/501100004359","name":"Swedish Research Council","doi-asserted-by":"crossref","award":["2014-396"],"award-info":[{"award-number":["2014-396"]}],"id":[{"id":"10.13039\/501100004359","id-type":"DOI","asserted-by":"crossref"}]},{"DOI":"10.13039\/501100002347","name":"Bundesministerium f\u00fcr Bildung und Forschung","doi-asserted-by":"publisher","award":["031A410A"],"award-info":[{"award-number":["031A410A"]}],"id":[{"id":"10.13039\/501100002347","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100004784","name":"Novartis Foundation for Medical-Biological Research","doi-asserted-by":"publisher","id":[{"id":"10.13039\/501100004784","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["journals.asm.org"],"crossmark-restriction":true},"short-container-title":["Appl Environ Microbiol"],"published-print":{"date-parts":[[2018,6]]},"abstract":"<jats:title>ABSTRACT<\/jats:title>\n          <jats:p>\n            The ubiquitous environmental bacterium\n            <jats:named-content content-type=\"genus-species\">Legionella pneumophila<\/jats:named-content>\n            survives and replicates within amoebae and human macrophages by forming a\n            <jats:named-content content-type=\"genus-species\">Legionella<\/jats:named-content>\n            -containing vacuole (LCV). In an intricate process governed by the bacterial Icm\/Dot type IV secretion system and a plethora of effector proteins, the nascent LCV interferes with a number of intracellular trafficking pathways, including retrograde transport from endosomes to the Golgi apparatus. Conserved retrograde trafficking components, such as the retromer coat complex or the phosphoinositide (PI) 5-phosphatase\n            <jats:named-content content-type=\"genus-species\">D. discoideum<\/jats:named-content>\n            5-phosphatase 4 (Dd5P4)\/oculocerebrorenal syndrome of Lowe (OCRL), restrict intracellular replication of\n            <jats:named-content content-type=\"genus-species\">L. pneumophila<\/jats:named-content>\n            by an unknown mechanism. Here, we established an imaging flow cytometry (IFC) approach to assess in a rapid, unbiased, and large-scale quantitative manner the role of retrograde-linked PI metabolism and actin dynamics in the LCV composition. Exploiting\n            <jats:named-content content-type=\"genus-species\">Dictyostelium discoideum<\/jats:named-content>\n            genetics, we found that Dd5P4 modulates the acquisition of fluorescently labeled LCV markers, such as calnexin, the small GTPase Rab1 (but not Rab7 and Rab8), and retrograde trafficking components (Vps5, Vps26, Vps35). The actin-nucleating protein and retromer interactor WASH (Wiskott-Aldrich syndrome protein [WASP] and suppressor of cAMP receptor [SCAR] homologue) promotes the accumulation of Rab1 and Rab8 on LCVs. Collectively, our findings validate IFC for the quantitative and unbiased analysis of the pathogen vacuole composition and reveal the impact of retrograde-linked PI metabolism and actin dynamics on the LCV composition. The IFC approach employed here can be adapted for a molecular analysis of the pathogen vacuole composition of other amoeba-resistant pathogens.\n          <\/jats:p>\n          <jats:p>\n            <jats:bold>IMPORTANCE<\/jats:bold>\n            <jats:named-content content-type=\"genus-species\">Legionella pneumophila<\/jats:named-content>\n            is an amoeba-resistant environmental bacterium which can cause a life-threatening pneumonia termed Legionnaires' disease. In order to replicate intracellularly, the opportunistic pathogen forms a protective compartment, the\n            <jats:named-content content-type=\"genus-species\">Legionella<\/jats:named-content>\n            -containing vacuole (LCV). An in-depth analysis of the LCV composition and the complex process of pathogen vacuole formation is crucial for understanding the virulence of\n            <jats:named-content content-type=\"genus-species\">L. pneumophila<\/jats:named-content>\n            . Here, we established an imaging flow cytometry (IFC) approach to assess in a rapid, unbiased, and quantitative manner the accumulation of fluorescently labeled markers and probes on LCVs. Using IFC and\n            <jats:named-content content-type=\"genus-species\">L. pneumophila<\/jats:named-content>\n            -infected\n            <jats:named-content content-type=\"genus-species\">Dictyostelium discoideum<\/jats:named-content>\n            or defined mutant amoebae, a role for phosphoinositide (PI) metabolism, retrograde trafficking, and the actin cytoskeleton in the LCV composition was revealed. In principle, the powerful IFC approach can be used to analyze the molecular composition of any cellular compartment harboring bacterial pathogens.\n          <\/jats:p>","DOI":"10.1128\/aem.00158-18","type":"journal-article","created":{"date-parts":[[2018,3,29]],"date-time":"2018-03-29T13:27:21Z","timestamp":1522330041000},"update-policy":"https:\/\/doi.org\/10.1128\/asmj-crossmark-policy-page","source":"Crossref","is-referenced-by-count":18,"title":["Quantitative Imaging Flow Cytometry of Legionella-Infected Dictyostelium Amoebae Reveals the Impact of Retrograde Trafficking on Pathogen Vacuole Composition"],"prefix":"10.1128","volume":"84","author":[{"given":"Amanda","family":"Welin","sequence":"first","affiliation":[{"name":"Institute of Medical Microbiology, University of Z\u00fcrich, Z\u00fcrich, Switzerland"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Stephen","family":"Weber","sequence":"additional","affiliation":[{"name":"Institute of Medical Microbiology, University of Z\u00fcrich, Z\u00fcrich, Switzerland"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Hubert","family":"Hilbi","sequence":"additional","affiliation":[{"name":"Institute of Medical Microbiology, University of Z\u00fcrich, Z\u00fcrich, Switzerland"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"235","reference":[{"key":"e_1_3_3_2_2","first-page":"1","article-title":"Antigenic and genetic characterization of Legionella proteins: contributions to taxonomy, diagnosis and pathogenesis","volume":"70","author":"Bangsborg JM","year":"1997","unstructured":"Bangsborg JM. 1997. 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