Journal: 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.
Cryogen name: ETHANE-PROPANE / Chamber humidity: 90 % / Chamber temperature: 280 K / Instrument: FEI VITROBOT MARK IV
Details
cryoFIB milling of plunge-frozen infected amoeba and subsequent cryoET of the resulting lamellae
Sectioning
Focused 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
Microscope
FEI TITAN KRIOS
Electron beam
Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
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Legionella pneumophila (bacteria)
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Switzerland, 2 items 
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emd_13246.png
http://ftp.pdbj.org/pub/emdb/structures/EMD-13246
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