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- EMDB-13247: cryoFIB milling/cryoET of amoeba infected with Legionella pneumophila -

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Basic information

Entry
Database: EMDB / ID: EMD-13247
TitlecryoFIB milling/cryoET of amoeba infected with Legionella pneumophila
Map dataAcanthamoeba castellanii infected with Legionella pneumophila JR32 (2 hpi)
Sample
  • Cell: Acanthamoeba castellanii infected with Legionella pneumophila JR32
Biological speciesLegionella pneumophila (bacteria)
Methodelectron tomography / cryo EM
AuthorsBoeck D / Huesler D
Funding support Switzerland, 2 items
OrganizationGrant numberCountry
Swiss National Science Foundation Switzerland
European Research Council (ERC) Switzerland
CitationJournal: mBio / Year: 2021
Title: The Polar Icm/Dot T4SS Establishes Distinct Contact Sites with the Pathogen Vacuole Membrane.
Authors: Désirée Böck / Dario Hüsler / Bernhard Steiner / João M Medeiros / Amanda Welin / Katarzyna A Radomska / Wolf-Dietrich Hardt / Martin Pilhofer / Hubert Hilbi /
Abstract: Legionella pneumophila, the causative agent of Legionnaires' disease, is a facultative intracellular pathogen that survives inside phagocytic host cells by establishing a protected replication niche, ...Legionella pneumophila, the causative agent of Legionnaires' disease, is a facultative intracellular pathogen that survives inside phagocytic host cells by establishing a protected replication niche, termed the "-containing vacuole" (LCV). To form an LCV and subvert pivotal host pathways, L. pneumophila employs a type IV secretion system (T4SS), which translocates more than 300 different effector proteins into the host cell. The L. pneumophila T4SS complex has been shown to span the bacterial cell envelope at the bacterial poles. However, the interactions between the T4SS and the LCV membrane are not understood. Using cryo-focused ion beam milling, cryo-electron tomography, and confocal laser scanning fluorescence microscopy, we show that up to half of the intravacuolar L. pneumophila bacteria tether their cell pole to the LCV membrane. Tethering coincides with the presence and function of T4SSs and likely promotes the establishment of distinct contact sites between T4SSs and the LCV membrane. Contact sites are characterized by indentations in the limiting LCV membrane and localize juxtaposed to T4SS machineries. The data are in agreement with the notion that effector translocation occurs by close membrane contact rather than by an extended pilus. Our findings provide novel insights into the interactions of the L. pneumophila T4SS with the LCV membrane . Legionnaires' disease is a life-threatening pneumonia, which is characterized by high fever, coughing, shortness of breath, muscle pain, and headache. The disease is caused by the amoeba-resistant bacterium L. pneumophila found in various soil and aquatic environments and is transmitted to humans via the inhalation of small bacteria-containing droplets. An essential virulence factor of L. pneumophila is a so-called "type IV secretion system" (T4SS), which, by injecting a plethora of "effector proteins" into the host cell, determines pathogen-host interactions and the formation of a distinct intracellular compartment, the "-containing vacuole" (LCV). It is unknown how the T4SS makes contact to the LCV membrane to deliver the effectors. In this study, we identify indentations in the host cell membrane in close proximity to functional T4SSs localizing at the bacterial poles. Our work reveals first insights into the architecture of -LCV contact sites.
History
DepositionJul 20, 2021-
Header (metadata) releaseOct 27, 2021-
Map releaseOct 27, 2021-
UpdateNov 17, 2021-
Current statusNov 17, 2021Processing site: PDBe / Status: Released

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Structure visualization

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  • Imaged by UCSF Chimera
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Supplemental images

emd_13247.png

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Map

FileDownload / File: emd_13247.map.gz / Format: CCP4 / Size: 249.8 MB / Type: IMAGE STORED AS SIGNED BYTE
AnnotationAcanthamoeba castellanii infected with Legionella pneumophila JR32 (2 hpi)
Voxel sizeX=Y=Z: 17.14 Å
Density
Minimum - Maximum-128.0 - 127.0
Average (Standard dev.)22.680704 (±38.310806)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-1617147
Dimensions928960294
Spacing960928294
CellA: 16454.398 Å / B: 15905.92 Å / C: 5039.1597 Å
α=β=γ: 90.0 °

CCP4 map header:

modeenvelope stored as signed bytes (from -128 lowest to 127 highest)
Å/pix. X/Y/Z17.13999791666717.1417.14
M x/y/z960928294
origin x/y/z0.0000.0000.000
length x/y/z16454.39815905.9205039.160
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS17-16147
NC/NR/NS960928294
D min/max/mean-128.000127.00022.681

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Supplemental data

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Sample components

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Entire : Acanthamoeba castellanii infected with Legionella pneumophila JR32

EntireName: Acanthamoeba castellanii infected with Legionella pneumophila JR32
Components
  • Cell: Acanthamoeba castellanii infected with Legionella pneumophila JR32

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Supramolecule #1: Acanthamoeba castellanii infected with Legionella pneumophila JR32

SupramoleculeName: Acanthamoeba castellanii infected with Legionella pneumophila JR32
type: cell / ID: 1 / Parent: 0
Details: Legionella-containing vacuole (LCV) at 2 hpi in the amoeba host A. castellanii
Source (natural)Organism: Legionella pneumophila (bacteria) / Strain: JR32

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Experimental details

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Structure determination

Methodcryo EM
Processingelectron tomography
Aggregation statecell

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Sample preparation

BufferpH: 7
GridModel: Quantifoil R2/2 / Material: GOLD / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE-PROPANE / Chamber humidity: 90 % / Chamber temperature: 280 K / Instrument: FEI VITROBOT MARK IV
DetailscryoFIB milling of plunge-frozen infected amoeba and subsequent cryoET of the resulting lamellae
SectioningFocused ion beam - Instrument: OTHER / Focused ion beam - Ion: OTHER / Focused ion beam - Voltage: 30 kV / Focused ion beam - Current: 0.025 nA / Focused ion beam - Duration: 3600 sec. / Focused ion beam - Temperature: 120 K / Focused ion beam - Initial thickness: 1000 nm / Focused ion beam - Final thickness: 200 nm
Focused ion beam - Details: The value given for _emd_sectioning_focused_ion_beam.instrument is FEI Helios NanoLab600i. This is not in a list of allowed values {'OTHER', 'DB235'} so OTHER is written into the XML file.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Average electron dose: 1.25 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Final reconstructionNumber images used: 61

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