title: The Legionella pneumophila phosphatidylinositol-4 phosphate-binding type IV substrate SidC recruits endoplasmic reticulum vesicles to a replication-permissive vacuole.
abstract: Legionella pneumophila, the causative agent of Legionnaires' disease, uses the intracellular multiplication/defective organelle trafficking (Icm/Dot) type IV secretion system to establish within amoebae and macrophages an endoplasmic reticulum (ER)-derived replication-permissive compartment, the Legionella-containing vacuole (LCV). The Icm/Dot substrate SidC and its paralogue SdcA anchor to LCVs via phosphatidylinositol-4 phosphate [PtdIns(4)P]. Here we identify the unique 20 kDa PtdIns(4)P-binding domain of SidC, which upon heterologous expression in Dictyostelium binds to LCVs and thus is useful as a PtdIns(4)P-specific probe. LCVs harbouring L. pneumophilaDeltasidC-sdcA mutant bacteria recruit ER and ER-derived vesicles less efficiently and carry endosomal but not lysosomal markers. The phenotypes are complemented by supplying sidC on a plasmid. L. pneumophilaDeltasidC-sdcA grows at wild-type rate in calnexin-negative LCVs, suggesting that communication with the ER is dispensable for establishing a replicative compartment. The amount of SidC and calnexin is directly proportional on isolated LCVs, and in a cell-free system, the recruitment of calnexin-positive vesicles to LCVs harbouring DeltasidC-sdcA mutant bacteria is impaired. Beads coated with purified SidC or its 70 kDa N-terminal fragment recruit ER vesicles in Dictyostelium and macrophage lysates. Our results establish SidC as an L. pneumophila effector protein, which anchors to PtdIns(4)P on LCVs and recruits ER vesicles to a replication-permissive vacuole.
authors.affiliation: ETH Zürich, Institute of Microbiology, Wolfgang-Pauli-Strasse 10, 8093 Zürich, Switzerland.
authors.forename: Curdin
authors.initials: C
authors.lastname: Ragaz
authors.forename: Holger
authors.initials: H
authors.lastname: Pietsch
authors.forename: Simon
authors.initials: S
authors.lastname: Urwyler
authors.forename: André
authors.initials: A
authors.lastname: Tiaden
authors.forename: Stefan S
authors.initials: SS
authors.lastname: Weber
authors.forename: Hubert
authors.initials: H
authors.lastname: Hilbi
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D037281:Calnexin / Q000032:analysis; D022162:Cytoplasmic Vesicles / Q000737:chemistry / Q000378:metabolism* / Q000382:microbiology* / Q000648:ultrastructure; D017353:Gene Deletion; D005816:Genetic Complementation Test; D006801:Humans; D016952:Legionella pneumophila / Q000472:pathogenicity*; D018613:Microscopy, Confocal; D046529:Microscopy, Electron, Transmission; D037521:Virulence Factors / Q000235:genetics / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; C534216:SidC protein, Legionella pneumophila; D037521:Virulence Factors; D037281:Calnexin