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

Welin, Amanda; Weber, Samuël; Hilbi, Hubert

doi:10.1128/aem.00158-18

Cited by 15 publications

(16 citation statements)

References 76 publications

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“…Imaging flow cytometry (IFC) is a well‐established technique for the study of infection biology, especially infection induced by intracellular pathogens as well as parasitic protozoa such as the phagocytic responses in Entamoeba sp. . Here we describe a protocol and the first report using IFC to analyze the opportunistic bacterial pathogen P. aeruginosa and its interactions with the free‐living amoeba A. polyphaga .…”

Section: Discussionmentioning

confidence: 99%

Interactions of Pseudomonas aeruginosa with Acanthamoeba polyphaga Observed by Imaging Flow Cytometry

et al. 2019

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Pseudomonas aeruginosa is a Gram‐negative bacterium that is abundant in the environment and water systems, with strains that cause serious infections, especially in patients with compromised immune systems. In times of stress or as part of its natural life cycle, P. aeruginosa can adopt a viable but not culturable (VBNC) state, which renders it undetectable by current conventional food and water testing methods and makes it highly resistant to antibiotic treatment. Specific conditions can resuscitate these coccoid VBNC P. aeruginosa cells, which returns them to their active, virulent rod‐shaped form. Underreporting the VBNC cells of P. aeruginosa by standard culture‐based methods in water distribution systems may therefore pose serious risks to public health. As such, being able to accurately detect and quantify the presence of VBNC P. aeruginosa, especially in a hospital setting, is of critical importance. Herein, we describe a method to analyze VBNC P. aeruginosa using imaging flow cytometry. With this technique, we can accurately distinguish between active and VBNC forms. We also show here that association of VBNC P. aeruginosa with Acanthamoeba polyphaga results in resuscitation of P. aeruginosa to an active form within 2 h. Our approach could provide an alternative, reliable detection method of VBNC P. aeruginosa when coupled with species‐specific staining. Most importantly, our experiments demonstrate that the coculture with amoebae can lead to a resuscitation of P. aeruginosa of culturable morphology after only 2 h, indicating that VBNC P. aeruginosa could potentially resuscitate in piped water (healthcare) environments colonized with amoebae. © 2019 International Society for Advancement of Cytometry

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Section: Discussionmentioning

confidence: 99%

Interactions of Pseudomonas aeruginosa with Acanthamoeba polyphaga Observed by Imaging Flow Cytometry

et al. 2019

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“…Finally, we quantified the accumulation of PtdIns3 P using a recently established imaging flow cytometry method employing dually labelled D . discoideum (Welin, Weber, & Hilbi, ). Using this unbiased, quantitative, and high‐throughput approach, we found that significantly more GFP‐2×FYVE accumulated on MCVs harbouring Δtpd or the RD1 mutant strain compared with wild‐type M .…”

Section: Resultsmentioning

confidence: 99%

“…In focus, single cells that were positive for GFP and had phagocytosed a single M . marinum bacterium were gated as described in Welin et al (). Subsequently, a mask that identified the MCV was created, defined as [Dilate (Spot(M04, Mm, Bright, 8.5, 1), 5) And Not Spot(M04, Mm, Bright, 8.5, 1)].…”

Section: Methodsmentioning

confidence: 99%

DistinctMycobacterium marinumphosphatases determine pathogen vacuole phosphoinositide pattern, phagosome maturation, and escape to the cytosol

Koliwer‐Brandl

Knobloch

Barisch

et al. 2019

Cellular Microbiology

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The causative agent of tuberculosis, Mycobacterium tuberculosis, and its close relative Mycobacterium marinum manipulate phagocytic host cells, thereby creating a replication-permissive compartment termed the Mycobacterium-containing vacuole (MCV). The phosphoinositide (PI) lipid pattern is a crucial determinant of MCV formation and is targeted by mycobacterial PI phosphatases. In this study, we establish an efficient phage transduction protocol to construct defined M. marinum deletion mutants lacking one or three phosphatases, PtpA, PtpB, and/or SapM. These strains were defective for intracellular replication in macrophages and amoebae, and the growth defect was complemented by the corresponding plasmid-borne genes. Fluorescence microscopy of M. marinum-infected Dictyostelium discoideum revealed that MCVs harbouring mycobacteria lacking PtpA, SapM, or all three phosphatases accumulate significantly more phosphatidylinositol-3-phosphate (PtdIns3P) compared with MCVs containing the parental strain. Moreover, PtpA reduced MCV acidification by blocking the recruitment of the V-ATPase, and all three phosphatases promoted bacterial escape from the pathogen vacuole to the cytoplasm. In summary, the secreted M. marinum phosphatases PtpA, PtpB, and SapM determine the MCV PI pattern, compartment acidification, and phagosomal escape.

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“…L. pneumophila grows more efficiently in D. discoideum lacking Dd5P4, and thus, the pleiotropic PI 5-phosphatase restricts intracellular bacterial growth. Mechanistic details of this process are not known, but Dd5P4 modulates the recruitment of calnexin, Rab1 and retromer components to LCVs, which might account for growth restriction (156,164).…”

Section: Subversion Of Host Phosphoinositide Kinases and Phosphatasesmentioning

confidence: 99%

Phosphoinositides and the Fate of Legionella in Phagocytes

Swart

Hilbi

2020

Front. Immunol.

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Legionella pneumophila is the causative agent of a severe pneumonia called Legionnaires' disease. The environmental bacterium replicates in free-living amoebae as well as in lung macrophages in a distinct compartment, the Legionella-containing vacuole (LCV). The LCV communicates with a number of cellular vesicle trafficking pathways and is formed by a plethora of secreted bacterial effector proteins, which target host cell proteins and lipids. Phosphoinositide (PI) lipids are pivotal determinants of organelle identity, membrane dynamics and vesicle trafficking. Accordingly, eukaryotic cells tightly regulate the production, turnover, interconversion, and localization of PI lipids. L. pneumophila modulates the PI pattern in infected cells for its own benefit by (i) recruiting PI-decorated vesicles, (ii) producing effectors acting as PI interactors, phosphatases, kinases or phospholipases, and (iii) subverting host PI metabolizing enzymes. The PI conversion from PtdIns(3)P to PtdIns(4)P represents a decisive step during LCV maturation. In this review, we summarize recent progress on elucidating the strategies, by which L. pneumophila subverts host PI lipids to promote LCV formation and intracellular replication.

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

Cited by 15 publications

References 76 publications

Interactions of Pseudomonas aeruginosa with Acanthamoeba polyphaga Observed by Imaging Flow Cytometry

Interactions of Pseudomonas aeruginosa with Acanthamoeba polyphaga Observed by Imaging Flow Cytometry

DistinctMycobacterium marinumphosphatases determine pathogen vacuole phosphoinositide pattern, phagosome maturation, and escape to the cytosol

Phosphoinositides and the Fate of Legionella in Phagocytes

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