The Machinery at Endoplasmic Reticulum-Plasma Membrane Contact Sites Contributes to Spatial Regulation of Multiple Legionella Effector Proteins

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.

[1]  T. Balla,et al.  A novel probe for phosphatidylinositol 4-phosphate reveals multiple pools beyond the Golgi , 2014, The Journal of cell biology.

[2]  X. Darzacq,et al.  The SNARE Sec22b has a non-fusogenic function in plasma membrane expansion , 2014, Nature Cell Biology.

[3]  P. Janmey,et al.  Structural basis for PI(4)P-specific membrane recruitment of the Legionella pneumophila effector DrrA/SidM. , 2014, Structure.

[4]  J. Prestegard,et al.  Interaction of Fapp1 with Arf1 and PI4P at a membrane surface: an example of coincidence detection. , 2014, Structure.

[5]  S. Colombo,et al.  PI(4,5)P2-Dependent and Ca2+-Regulated ER-PM Interactions Mediated by the Extended Synaptotagmins , 2013, Cell.

[6]  T. Mazel,et al.  RabGEFs are a major determinant for specific Rab membrane targeting , 2013, The Journal of cell biology.

[7]  Sang Yoon Lee,et al.  PtdIns4P synthesis by PI4KIIIα at the plasma membrane and its impact on plasma membrane identity , 2012, The Journal of cell biology.

[8]  T. Balla,et al.  PI4P and PI(4,5)P2 Are Essential But Independent Lipid Determinants of Membrane Identity , 2012, Science.

[9]  Y. Mao,et al.  Structural basis for substrate recognition by a unique Legionella phosphoinositide phosphatase , 2012, Proceedings of the National Academy of Sciences.

[10]  R. Goody,et al.  Reversible phosphocholination of Rab proteins by Legionella pneumophila effector proteins , 2012, The EMBO journal.

[11]  R. Goody,et al.  Posttranslational modifications of Rab proteins cause effective displacement of GDP dissociation inhibitor , 2012, Proceedings of the National Academy of Sciences.

[12]  D. Toomre,et al.  The Legionella pneumophila effector DrrA is sufficient to stimulate SNARE-dependent membrane fusion. , 2012, Cell host & microbe.

[13]  Zhao‐Qing Luo,et al.  Legionella pneumophila regulates the small GTPase Rab1 activity by reversible phosphorylcholination , 2011, Proceedings of the National Academy of Sciences.

[14]  D. Rigden.  Identification and modelling of a PPM protein phosphatase fold in the Legionella pneumophila deAMPylase SidD , 2011, FEBS letters.

[15]  J. Galán,et al.  Modulation of Rab GTPase function by a protein phosphocholine transferase , 2011, Nature.

[16]  A. Yergey,et al.  De-AMPylation of the Small GTPase Rab1 by the Pathogen Legionella pneumophila , 2011, Science.

[17]  Zhao‐Qing Luo,et al.  Legionella pneumophila SidD is a deAMPylase that modifies Rab1 , 2011, Nature.

[18]  R. Goody,et al.  A structural basis for Lowe syndrome caused by mutations in the Rab‐binding domain of OCRL1 , 2011, The EMBO journal.

[19]  S. Emr,et al.  Osh Proteins Regulate Phosphoinositide Metabolism at ER-Plasma Membrane Contact Sites , 2011, Cell.

[20]  D. Zamboni,et al.  A Method for Generation of Bone Marrow-Derived Macrophages from Cryopreserved Mouse Bone Marrow Cells , 2010, PloS one.

[21]  R. Goody,et al.  The Legionella Effector Protein DrrA AMPylates the Membrane Traffic Regulator Rab1b , 2010, Science.

[22]  P. De Camilli,et al.  The inositol 5-phosphatase SHIP2 regulates endocytic clathrin-coated pit dynamics , 2010, The Journal of cell biology.

[23]  R. Goody,et al.  High‐affinity binding of phosphatidylinositol 4‐phosphate by Legionella pneumophila DrrA , 2010, EMBO reports.

[24]  S. Emr,et al.  Crystal structure of the yeast Sac1: implications for its phosphoinositide phosphatase function , 2010, The EMBO journal.

[25]  C. Roy,et al.  Legionella pneumophila Promotes Functional Interactions between Plasma Membrane Syntaxins and Sec22b , 2010, Traffic.

[26]  Yan Zhou,et al.  Structural mechanism of host Rab1 activation by the bifunctional Legionella type IV effector SidM/DrrA , 2010, Proceedings of the National Academy of Sciences.

[27]  P. De Camilli,et al.  Two closely related endocytic proteins that share a common OCRL-binding motif with APPL1 , 2010, Proceedings of the National Academy of Sciences.

[28]  R. Goody,et al.  RabGDI displacement by DrrA from Legionella is a consequence of its guanine nucleotide exchange activity. , 2009, Molecular cell.

[29]  H. Stenmark.  Rab GTPases as coordinators of vesicle traffic , 2009, Nature Reviews Molecular Cell Biology.

[30]  M. Lowe,et al.  Differential Clathrin Binding and Subcellular Localization of OCRL1 Splice Isoforms* , 2009, Journal of Biological Chemistry.

[31]  H. Hilbi,et al.  Pathogen trafficking pathways and host phosphoinositide metabolism , 2009, Molecular microbiology.

[32]  H. Hilbi,et al.  The inositol polyphosphate 5‐phosphatase OCRL1 restricts intracellular growth of Legionella, localizes to the replicative vacuole and binds to the bacterial effector LpnE , 2009, Cellular microbiology.

[33]  H. Hilbi,et al.  Rab1 Guanine Nucleotide Exchange Factor SidM Is a Major Phosphatidylinositol 4-Phosphate-binding Effector Protein of Legionella pneumophila , 2009, Journal of Biological Chemistry.

[34]  M. Heidtman,et al.  Large‐scale identification of Legionella pneumophila Dot/Icm substrates that modulate host cell vesicle trafficking pathways , 2009, Cellular microbiology.

[35]  S. Emr,et al.  Assembly of the PtdIns 4-kinase Stt4 complex at the plasma membrane requires Ypp1 and Efr3 , 2008, The Journal of cell biology.

[36]  H. Hilbi,et al.  The Legionella pneumophila phosphatidylinositol‐4 phosphate‐binding type IV substrate SidC recruits endoplasmic reticulum vesicles to a replication‐permissive vacuole , 2008, Cellular microbiology.

[37]  G. D’Angelo,et al.  Function and dysfunction of the PI system in membrane trafficking , 2008, The EMBO journal.

[38]  G. D’Angelo,et al.  Phosphoinositides as regulators of membrane trafficking in health and disease. , 2008, Cellular and molecular life sciences : CMLS.

[39]  G. D’Angelo,et al.  Membrane traffic in the secretory pathway , 2008, Cellular and Molecular Life Sciences.

[40]  Giovanni D'Angelo,et al.  The multiple roles of PtdIns(4)P – not just the precursor of PtdIns(4,5)P2 , 2008, Journal of Cell Science.

[41]  K. Shokat,et al.  Maintenance of hormone-sensitive phosphoinositide pools in the plasma membrane requires phosphatidylinositol 4-kinase IIIalpha. , 2007, Molecular biology of the cell.

[42]  D. Lambright,et al.  Legionella pneumophila proteins that regulate Rab1 membrane cycling , 2007, Nature.

[43]  R. Isberg,et al.  A Bifunctional Bacterial Protein Links GDI Displacement to Rab1 Activation , 2007, Science.

[44]  W. Dietrich,et al.  Restriction of Legionella pneumophila growth in macrophages requires the concerted action of cytokine and Naip5/Ipaf signalling pathways , 2007, Cellular microbiology.

[45]  P. De Camilli,et al.  A role of the Lowe syndrome protein OCRL in early steps of the endocytic pathway. , 2007, Developmental cell.

[46]  Pietro De Camilli,et al.  Phosphoinositides in cell regulation and membrane dynamics , 2006, Nature.

[47]  D. Toomre,et al.  The Legionella pneumophila effector protein DrrA is a Rab1 guanine nucleotide-exchange factor , 2006, Nature Cell Biology.

[48]  Christopher J. R. Loewen,et al.  Inter-organelle membrane contact sites: through a glass, darkly. , 2006, Current opinion in cell biology.

[49]  R. Isberg,et al.  Targeting of host Rab GTPase function by the intravacuolar pathogen Legionella pneumophila. , 2006, Developmental cell.

[50]  T. Balla,et al.  Phosphatidylinositol 4-kinases: old enzymes with emerging functions. , 2006, Trends in cell biology.

[51]  C. G. Robinson,et al.  Attachment and fusion of endoplasmic reticulum with vacuoles containing Legionella pneumophila , 2006, Cellular microbiology.

[52]  H. Hilbi,et al.  Legionella pneumophila Exploits PI(4)P to Anchor Secreted Effector Proteins to the Replicative Vacuole , 2006, PLoS pathogens.

[53]  W. Dietrich,et al.  Flagellin-Deficient Legionella Mutants Evade Caspase-1- and Naip5-Mediated Macrophage Immunity , 2006, PLoS pathogens.

[54]  W. Dietrich,et al.  The Birc1e cytosolic pattern-recognition receptor contributes to the detection and control of Legionella pneumophila infection , 2006, Nature Immunology.

[55]  V. Niggli.  Regulation of protein activities by phosphoinositide phosphates. , 2005, Annual review of cell and developmental biology.

[56]  E. Eisenberg,et al.  Lowe syndrome protein OCRL1 interacts with clathrin and regulates protein trafficking between endosomes and the trans-Golgi network. , 2005, Molecular biology of the cell.

[57]  Simon C Watkins,et al.  Distinct Golgi Populations of Phosphatidylinositol 4-Phosphate Regulated by Phosphatidylinositol 4-Kinases* , 2005, Journal of Biological Chemistry.

[58]  P. Várnai,et al.  A plasma membrane pool of phosphatidylinositol 4-phosphate is generated by phosphatidylinositol 4-kinase type-III alpha: studies with the PH domains of the oxysterol binding protein and FAPP1. , 2005, Molecular biology of the cell.

[59]  P. Cossart,et al.  Subversion of phosphoinositide metabolism by intracellular bacterial pathogens , 2004, Nature Cell Biology.

[60]  T. Levine,et al.  Multiple Pools of Phosphatidylinositol 4-Phosphate Detected Using the Pleckstrin Homology Domain of Osh2p* , 2004, Journal of Biological Chemistry.

[61]  P. Majerus,et al.  The inositol polyphosphate 5-phosphatase Ocrl associates with endosomes that are partially coated with clathrin. , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[62]  M. Pypaert,et al.  Legionella Subvert the Functions of Rab1 and Sec22b to Create a Replicative Organelle , 2004, The Journal of experimental medicine.

[63]  R. Isberg,et al.  Legionella pneumophila Replication Vacuole Formation Involves Rapid Recruitment of Proteins of the Early Secretory System , 2004, Infection and Immunity.

[64]  D. Alessi,et al.  FAPPs control Golgi-to-cell-surface membrane traffic by binding to ARF and PtdIns(4)P , 2004, Nature Cell Biology.

[65]  Diana Murray,et al.  Genome-wide analysis of membrane targeting by S. cerevisiae pleckstrin homology domains. , 2004, Molecular cell.

[66]  D. Lambright,et al.  Membrane Recognition and Targeting by Lipid-Binding Domains , 2003, Science's STKE.

[67]  C. Roy,et al.  Legionella phagosomes intercept vesicular traffic from endoplasmic reticulum exit sites , 2002, Nature Cell Biology.

[68]  S. Munro,et al.  Targeting of Golgi-Specific Pleckstrin Homology Domains Involves Both PtdIns 4-Kinase-Dependent and -Independent Components , 2002, Current Biology.

[69]  C. G. Robinson,et al.  How the parasitic bacterium Legionella pneumophila modifies its phagosome and transforms it into rough ER: implications for conversion of plasma membrane to the ER membrane. , 2001, Journal of cell science.

[70]  S. Dowler,et al.  Identification of pleckstrin-homology-domain-containing proteins with novel phosphoinositide-binding specificities. , 2000, The Biochemical journal.

[71]  P. Novick,et al.  The yeast phosphatidylinositol-4-OH kinase Pik1 regulates secretion at the Golgi , 1999, Nature Cell Biology.

[72]  Péter Várnai,et al.  Visualization of Phosphoinositides That Bind Pleckstrin Homology Domains: Calcium- and Agonist-induced Dynamic Changes and Relationship to Myo-[3H]inositol-labeled Phosphoinositide Pools , 1998, The Journal of cell biology.

[73]  R. Isberg,et al.  Analysis of the Legionella pneumophila fliI gene: intracellular growth of a defined mutant defective for flagellum biosynthesis , 1997, Infection and immunity.

[74]  L. Cantley,et al.  Subcellular Locations of Phosphatidylinositol 4-Kinase Isoforms* , 1997, The Journal of Biological Chemistry.

[75]  R. Isberg,et al.  Topology of Legionella pneumophila DotA: an inner membrane protein required for replication in macrophages , 1997, Infection and immunity.

[76]  K. Catt,et al.  Characterization of a soluble adrenal phosphatidylinositol 4-kinase reveals wortmannin sensitivity of type III phosphatidylinositol kinases. , 1996, Biochemistry.

[77]  C J Marshall,et al.  A CAAX or a CAAL motif and a second signal are sufficient for plasma membrane targeting of ras proteins. , 1991, The EMBO journal.

[78]  T. Murata,et al.  Analysis of Rab1 recruitment to vacuoles containing Legionella pneumophila. , 2005, Methods in enzymology.