title: The Legionella longbeachae Icm/Dot substrate SidC selectively binds phosphatidylinositol 4-phosphate with nanomolar affinity and promotes pathogen vacuole-endoplasmic reticulum interactions.
abstract: Legionella spp. cause the severe pneumonia Legionnaires' disease. The environmental bacteria replicate intracellularly in free-living amoebae and human alveolar macrophages within a distinct, endoplasmic reticulum (ER)-derived compartment termed the Legionella-containing vacuole (LCV). LCV formation requires the bacterial Icm/Dot type IV secretion system (T4SS) that translocates into host cells a plethora of different "effector" proteins, some of which anchor to the pathogen vacuole by binding to phosphoinositide (PI) lipids. Here, we identified by unbiased pulldown assays in Legionella longbeachae lysates a 111-kDa SidC homologue as the major phosphatidylinositol 4-phosphate [PtdIns(4)P]-binding protein. The PI-binding domain was mapped to a 20-kDa P4C [PtdIns(4)P binding of SidC] fragment. Isothermal titration calorimetry revealed that SidC of L. longbeachae (SidC(Llo)) binds PtdIns(4)P with a K(d) (dissociation constant) of 71 nM, which is 3 to 4 times lower than that of the SidC orthologue of Legionella pneumophila (SidC(Lpn)). Upon infection of RAW 264.7 macrophages with L. longbeachae, endogenous SidC(Llo) or ectopically produced SidC(Lpn) localized in an Icm/Dot-dependent manner to the PtdIns(4)P-positive LCVs. An L. longbeachae ΔsidC deletion mutant was impaired for calnexin recruitment to LCVs in Dictyostelium discoideum amoebae and outcompeted by wild-type bacteria in Acanthamoeba castellanii. Calnexin recruitment was restored by SidC(Llo) or its orthologues SidC(Lpn) and SdcA(Lpn). Conversely, calnexin recruitment was restored by SidC(Llo) in L. pneumophila lacking sidC and sdcA. Together, biochemical, genetic, and cell biological data indicate that SidC(Llo) is an L. longbeachae effector that binds through a P4C domain with high affinity to PtdIns(4)P on LCVs, promotes ER recruitment to the LCV, and thus plays a role in pathogen-host interactions.
authors.affiliation: Max von Pettenkofer Institute, Department of Medicine, Ludwig-Maximilians University Munich, Munich, Germany.
authors.forename: Stephanie
authors.initials: S
authors.lastname: Dolinsky
authors.affiliation: Max von Pettenkofer Institute, Department of Medicine, Ludwig-Maximilians University Munich, Munich, Germany Institute of Medical Microbiology, Department of Medicine, University of Zürich, Zürich, Switzerland.
authors.forename: Ina
authors.initials: I
authors.lastname: Haneburger
authors.affiliation: Center for Integrated Protein Science Munich, Department of Chemistry, Technical University Munich, Garching, Germany.
authors.forename: Adam
authors.initials: A
authors.lastname: Cichy
authors.affiliation: Center for Integrated Protein Science Munich, Department of Chemistry, Technical University Munich, Garching, Germany.
authors.forename: Mandy
authors.initials: M
authors.lastname: Hannemann
authors.affiliation: Center for Integrated Protein Science Munich, Department of Chemistry, Technical University Munich, Garching, Germany.
authors.forename: Aymelt
authors.initials: A
authors.lastname: Itzen
authors.affiliation: Max von Pettenkofer Institute, Department of Medicine, Ludwig-Maximilians University Munich, Munich, Germany Institute of Medical Microbiology, Department of Medicine, University of Zürich, Zürich, Switzerland hilbi@imm.uzh.ch.
authors.forename: Hubert
authors.initials: H
authors.lastname: Hilbi
mesh_terms: D048248:Acanthamoeba castellanii / Q000382:microbiology; D000818:Animals; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D037281:Calnexin / Q000378:metabolism; D002151:Calorimetry; D002460:Cell Line; D002874:Chromosome Mapping; D004023:Dictyostelium / Q000382:microbiology; D004721:Endoplasmic Reticulum / Q000382:microbiology*; D017353:Gene Deletion; D054884:Host-Pathogen Interactions; D007700:Kinetics; D044128:Legionella longbeachae / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D008264:Macrophages / Q000382:microbiology; D051379:Mice; D008970:Molecular Weight; D018129:Phosphatidylinositol Phosphates / Q000378:metabolism*; D011485:Protein Binding; D014617:Vacuoles / Q000382:microbiology*
chemical_list: D001426:Bacterial Proteins; D018129:Phosphatidylinositol Phosphates; C037178:phosphatidylinositol 4-phosphate; D037281:Calnexin