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Quantitative Imaging Flow Cytometry of Legionella-Infected Dictyostelium Amoebae Reveals the Impact of Retrograde Trafficking on Pathogen Vacuole Composition


Welin, Amanda; Weber, Stephen; Hilbi, Hubert (2018). Quantitative Imaging Flow Cytometry of Legionella-Infected Dictyostelium Amoebae Reveals the Impact of Retrograde Trafficking on Pathogen Vacuole Composition. Applied and Environmental Microbiology, 84(11):e00158.

Abstract

The ubiquitous environmental bacterium survives and replicates within amoebae and human macrophages by forming a -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 5-phosphatase 4 (Dd5P4)/oculocerebrorenal syndrome of Lowe (OCRL), restrict intracellular replication of 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 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. 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 -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 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 -infected 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.

Abstract

The ubiquitous environmental bacterium survives and replicates within amoebae and human macrophages by forming a -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 5-phosphatase 4 (Dd5P4)/oculocerebrorenal syndrome of Lowe (OCRL), restrict intracellular replication of 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 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. 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 -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 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 -infected 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.

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Item Type:Journal Article, refereed, original work
Communities & Collections:04 Faculty of Medicine > Institute of Medical Microbiology
Dewey Decimal Classification:570 Life sciences; biology
610 Medicine & health
Language:English
Date:1 June 2018
Deposited On:26 Oct 2018 10:48
Last Modified:24 Sep 2019 23:49
Publisher:American Society for Microbiology
ISSN:0099-2240
OA Status:Green
Free access at:PubMed ID. An embargo period may apply.
Publisher DOI:https://doi.org/10.1128/AEM.00158-18
PubMed ID:29602783

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