title: The machinery at endoplasmic reticulum-plasma membrane contact sites contributes to spatial regulation of multiple Legionella effector proteins.
abstract: The Dot/Icm system of the intracellular pathogen Legionella pneumophila has the capacity to deliver over 270 effector proteins into host cells during infection. Important questions remain as to spatial and temporal mechanisms used to regulate such a large array of virulence determinants after they have been delivered into host cells. Here we investigated several L. pneumophila effector proteins that contain a conserved phosphatidylinositol-4-phosphate (PI4P)-binding domain first described in the effector DrrA (SidM). This PI4P binding domain was essential for the localization of effectors to the early L. pneumophila-containing vacuole (LCV), and DrrA-mediated recruitment of Rab1 to the LCV required PI4P-binding activity. It was found that the host cell machinery that regulates sites of contact between the plasma membrane (PM) and the endoplasmic reticulum (ER) modulates PI4P dynamics on the LCV to control localization of these effectors. Specifically, phosphatidylinositol-4-kinase IIIα (PI4KIIIα) was important for generating a PI4P signature that enabled L. pneumophila effectors to localize to the PM-derived vacuole, and the ER-associated phosphatase Sac1 was involved in metabolizing the PI4P on the vacuole to promote the dissociation of effectors. A defect in L. pneumophila replication in macrophages deficient in PI4KIIIα was observed, highlighting that a PM-derived PI4P signature is critical for biogenesis of a vacuole that supports intracellular multiplication of L. pneumophila. These data indicate that PI4P metabolism by enzymes controlling PM-ER contact sites regulate the association of L. pneumophila effectors to coordinate early stages of vacuole biogenesis.
authors.affiliation: Section of Microbial Pathogenesis, Yale University School of Medicine, New Haven, Connecticut, United States of America; Research Institute for Microbial Diseases, Osaka University, 3-1 Yamadaoka, Suita, Osaka, Japan.
authors.forename: Andree
authors.initials: A
authors.lastname: Hubber
authors.affiliation: Section of Microbial Pathogenesis, Yale University School of Medicine, New Haven, Connecticut, United States of America; School of Life Sciences, Tokyo University of Pharmacy and Life Sciences, Horinouchi, Hachioji, Tokyo, Japan.
authors.forename: Kohei
authors.initials: K
authors.lastname: Arasaki
authors.affiliation: Department of Cell Biology, Yale University School of Medicine, New Haven, Connecticut, United States of America.
authors.forename: Fubito
authors.initials: F
authors.lastname: Nakatsu
authors.affiliation: Section of Microbial Pathogenesis, Yale University School of Medicine, New Haven, Connecticut, United States of America.
authors.forename: Camille
authors.initials: C
authors.lastname: Hardiman
authors.affiliation: Program in Molecular Medicine and Department of Biochemistry & Molecular Pharmacology, University of Massachusetts Medical School, Worcester, Massachusetts, United States of America.
authors.forename: David
authors.initials: D
authors.lastname: Lambright
authors.affiliation: Research Institute for Microbial Diseases, Osaka University, 3-1 Yamadaoka, Suita, Osaka, Japan.
authors.forename: Pietro
authors.initials: P
authors.lastname: De Camilli
authors.affiliation: Research Institute for Microbial Diseases, Osaka University, 3-1 Yamadaoka, Suita, Osaka, Japan.
authors.forename: Hiroki
authors.initials: H
authors.lastname: Nagai
authors.affiliation: Section of Microbial Pathogenesis, Yale University School of Medicine, New Haven, Connecticut, United States of America.
authors.forename: Craig R
authors.initials: CR
authors.lastname: Roy
mesh_terms: D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics / Q000276:immunology*; D002462:Cell Membrane / Q000235:genetics / Q000276:immunology* / Q000473:pathology; D004721:Endoplasmic Reticulum / Q000235:genetics / Q000276:immunology* / Q000382:microbiology / Q000473:pathology; D020662:Guanine Nucleotide Exchange Factors / Q000235:genetics / Q000276:immunology*; D057809:HEK293 Cells; D006367:HeLa Cells; D006801:Humans; D016952:Legionella pneumophila / Q000235:genetics / Q000276:immunology / Q000472:pathogenicity; D007877:Legionnaires' Disease / Q000235:genetics / Q000276:immunology* / Q000473:pathology; D051379:Mice; D037521:Virulence Factors / Q000235:genetics / Q000276:immunology*; D020761:rab1 GTP-Binding Proteins / Q000235:genetics / Q000276:immunology
chemical_list: D001426:Bacterial Proteins; D020662:Guanine Nucleotide Exchange Factors; C539733:SidM protein, Legionella pneumophila; D037521:Virulence Factors; D020761:rab1 GTP-Binding Proteins