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Title: A Novel Phosphatidylinositol 4,5-Bisphosphate Binding Domain Mediates Plasma Membrane Localization of ExoU and Other Patatin-like Phospholipases

Journal Article · · Journal of Biological Chemistry
 [1];  [1];  [2];  [1];  [1];  [1];  [2];  [1];  [1]
  1. Northwestern Univ., Evanston, IL (United States)
  2. Univ. of Illinois, Chicago, IL (United States)
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Bacterial toxins require localization to specific intracellular compartments following injection into host cells. In this study, we examined the membrane targeting of a broad family of bacterial proteins, the patatin-like phospholipases. The best characterized member of this family is ExoU, an effector of the Pseudomonas aeruginosa type III secretion system. Upon injection into host cells, ExoU localizes to the plasma membrane, where it uses its phospholipase A2 activity to lyse infected cells. The targeting mechanism of ExoU is poorly characterized, but it was recently found to bind to the phospholipid phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2), a marker for the plasma membrane of eukaryotic cells. We confirmed that the membrane localization domain (MLD) of ExoU had a direct affinity for PI(4,5)P2, and we determined that this binding was required for ExoU localization. Previously uncharacterized ExoU homologs from Pseudomonas fluorescens and Photorhabdus asymbiotica also localized to the plasma membrane and required PI(4,5)P2 for this localization. A conserved arginine within the MLD was critical for interaction of each protein with PI(4,5)P2 and for localization. Furthermore, we determined the crystal structure of the full-length P. fluorescens ExoU and found that it was similar to that of P. aeruginosa ExoU. Each MLD contains a four-helical bundle, with the conserved arginine exposed at its cap to allow for interaction with the negatively charged PI(4,5)P2. Overall, these findings provide a structural explanation for the targeting of patatin-like phospholipases to the plasma membrane and define the MLD of ExoU as a member of a new class of PI(4,5)P2 binding domains.

Research Organization:
Argonne National Laboratory (ANL), Argonne, IL (United States)
Sponsoring Organization:
USDOE
Grant/Contract Number:
CCSG P30 CA060553; HHSN272200700058C; HHSN272201200026C
OSTI ID:
1352274
Journal Information:
Journal of Biological Chemistry, Vol. 290, Issue 5; ISSN 0021-9258
Publisher:
American Society for Biochemistry and Molecular BiologyCopyright Statement
Country of Publication:
United States
Language:
ENGLISH
Citation Metrics:
Cited by: 29 works
Citation information provided by
Web of Science

References (76)

Characterization of the Membrane-targeting C1 Domain in Pasteurella multocida Toxin journal June 2010
PepA, a secreted protein of Pseudomonas aeruginosa , is necessary for cytotoxicity and virulence journal February 1998
Live cell imaging of phosphoinositides with expressed inositide binding protein domains journal November 2008
Characterization of Phospholipase Activity of the Pseudomonas aeruginosa Type III Cytotoxin, ExoU journal February 2005
In situ quantitative imaging of cellular lipids using molecular sensors journal October 2011
Structural basis of the membrane-targeting and unmasking mechanisms of the radixin FERM domain journal September 2000
Bacterial protein toxins that modify host regulatory GTPases journal June 2011
Plasma membrane association of three classes of bacterial toxins is mediated by a basic-hydrophobic motif: Mechanism of 4HBM-family membrane association journal November 2011
A C-Terminal Domain Targets the Pseudomonas aeruginosa Cytotoxin ExoU to the Plasma Membrane of Host Cells journal May 2006
Type III protein secretion is associated with poor clinical outcomes in patients with ventilator-associated pneumonia caused by Pseudomonas aeruginosa journal January 2002
Protein secretion systems in bacterial-host associations, and their description in the Gene Ontology journal January 2009
Salmonella typhimurium SifA Effector Protein Requires Its Membrane-anchoring C-terminal Hexapeptide for Its Biological Function journal February 2003
Basic Local Alignment Search Tool journal October 1990
Functional Regions of the Pseudomonas aeruginosa Cytotoxin ExoU journal January 2005
Identification of a phosphoinositide binding motif that mediates activation of mammalian and yeast phospholipase D isoenzymes journal November 1999
Structural Basis for the Function of Clostridium difficile Toxin B journal September 2005
PROCHECK: a program to check the stereochemical quality of protein structures journal April 1993
Role of Phosphatidylinositol 4-Phosphate (PI4P) and Its Binding Protein GOLPH3 in Hepatitis C Virus Secretion journal June 2012
The phosphoinositide phosphatase Sac1p controls trafficking of the yeast Chs3p chitin synthase journal September 2001
PIP 2 and Proteins: Interactions, Organization, and Information Flow journal June 2002
Localization of phosphatidylinositol 3-phosphate in yeast and mammalian cells journal September 2000
MolProbity: all-atom contacts and structure validation for proteins and nucleic acids journal May 2007
Plasma membrane localization affects the RhoGAP specificity of Pseudomonas ExoS journal September 2007
Secreted bacterial phospholipase A2 enzymes: better living through phospholipolysis journal February 2007
Structural Basis for PI(4)P-Specific Membrane Recruitment of the Legionella pneumophila Effector DrrA/SidM journal March 2014
Differential Roles of Phosphatidylserine, PtdIns(4,5)P 2 , and PtdIns(3,4,5)P 3 in Plasma Membrane Targeting of C2 Domains: MOLECULAR DYNAMICS SIMULATION, MEMBRANE BINDING, AND CELL TRANSLOCATION STUDIES OF THE PKCα C2 Domain journal July 2008
Characterisation of Legionella pneumophila phospholipases and their impact on host cells journal November 2011
Structure of the Type III Secretion Effector Protein ExoU in Complex with Its Chaperone SpcU journal November 2012
ExoU expression by Pseudomonas aeruginosa correlates with acute cytotoxicity and epithelial injury journal August 1997
Inference of Macromolecular Assemblies from Crystalline State journal September 2007
Identification of Pseudomonas aeruginosa genes required for epithelial cell injury journal June 1997
A Phosphatidylserine-binding Site in the Cytosolic Fragment of Clostridium sordellii Lethal Toxin Facilitates Glucosylation of Membrane-bound Rac and Is Required for Cytotoxicity journal September 2004
DaliLite workbench for protein structure comparison journal June 2000
High-throughput fluorescence assay for membrane-protein interaction journal September 2013
Organelle identity and the signposts for membrane traffic journal November 2005
Actin Crosslinking Toxins of Gram-Negative Bacteria journal December 2009
Live cell imaging with protein domains capable of recognizing phosphatidylinositol 4,5-bisphosphate; a comparative study journal January 2009
Structural Basis of Cytotoxicity Mediated by the Type III Secretion Toxin ExoU from Pseudomonas aeruginosa journal April 2012
Role of the Membrane Localization Domain of the Pseudomonas aeruginosa Effector Protein ExoU in Cytotoxicity journal May 2010
Cloning, expression, purification and characterization of patatin, a novel phospholipase A: Active site characterization of patatin journal October 2001
Relative Contributions of Pseudomonas aeruginosa ExoU, ExoS, and ExoT to Virulence in the Lung journal December 2004
Ubiquitin and ubiquitin-modified proteins activate the Pseudomonas aeruginosa T3SS cytotoxin, ExoU: Ubiquitin activation of ExoU journal November 2011
Identification of a conserved membrane localization domain within numerous large bacterial protein toxins journal March 2010
Colonization strategies of Pseudomonas fluorescens Pf0-1: activation of soil-specific genes important for diverse and specific environments journal January 2013
Members of a Legionella pneumophila Family of Proteins with ExoU (Phospholipase A) Active Sites Are Translocated to Target Cells journal June 2006
Pseudomonas aeruginosa ExoU, a Toxin Transported by the Type III Secretion System, Kills Saccharomyces cerevisiae journal July 2003
Analysis Tool Web Services from the EMBL-EBI journal May 2013
bdhA-patD Operon as a Virulence Determinant, Revealed by a Novel Large-Scale Approach for Identification of Legionella pneumophila Mutants Defective for Amoeba Infection journal May 2009
Genome-wide Functional Annotation of Dual-Specificity Protein- and Lipid-Binding Modules that Regulate Protein Interactions journal April 2012
REFMAC 5 for the refinement of macromolecular crystal structures journal March 2011
Effectors of animal and plant pathogens use a common domain to bind host phosphoinositides journal December 2013
Intoxication of Host Cells by the T3SS Phospholipase ExoU: PI(4,5)P2-Associated, Cytoskeletal Collapse and Late Phase Membrane Blebbing journal July 2014
PI4P and PI(4,5)P2 Are Essential But Independent Lipid Determinants of Membrane Identity journal June 2012
Patatin-like proteins: a new family of lipolytic enzymes present in bacteria? journal March 2004
Comparative Genomics of Plant-Associated Pseudomonas spp.: Insights into Diversity and Inheritance of Traits Involved in Multitrophic Interactions journal July 2012
Signal-dependent membrane targeting by pleckstrin homology (PH) domains journal August 2000
The mechanism of action of the Pseudomonas aeruginosa-encoded type III cytotoxin, ExoU journal June 2003
Ionic protein–lipid interaction at the plasma membrane: what can the charge do? journal March 2014
The Emerging Human Pathogen Photorhabdus asymbiotica Is a Facultative Intracellular Bacterium and Induces Apoptosis of Macrophage-Like Cells journal December 2008
Inactivation of the Phosphoinositide Phosphatases Sac1p and Inp54p Leads to Accumulation of Phosphatidylinositol 4,5-Bisphosphate on Vacuole Membranes and Vacuolar Fusion Defects journal March 2007
PI(4,5)P2 regulation of surface membrane traffic journal August 2001
Role of phosphatidylinositol 4,5-bisphosphate in the activation of cytosolic phospholipase A2-α journal December 2006
PhosphoMARCKS drives motility of mouse melanoma cells journal July 2010
NMRPipe: A multidimensional spectral processing system based on UNIX pipes journal November 1995
Pseudomonas aeruginosa uses type III secretion system to kill biofilm-associated amoebae journal May 2008
Role of the ENTH Domain in Phosphatidylinositol-4,5-Bisphosphate Binding and Endocytosis journal February 2001
A new bioinformatics analysis tools framework at EMBL-EBI journal May 2010
Use of the Galleria mellonella Caterpillar as a Model Host To Study the Role of the Type III Secretion System in Pseudomonas aeruginosa Pathogenesis journal May 2003
Bacterial Toxin Effector-Membrane Targeting: Outside in, then Back Again journal January 2012
Secretion of the Toxin ExoU Is a Marker for Highly Virulent Pseudomonas aeruginosa Isolates Obtained from Patients with Hospital‐Acquired Pneumonia journal December 2003
Association of Botulinum Neurotoxin Serotype A Light Chain with Plasma Membrane-bound SNAP-25 journal March 2011
Phaser crystallographic software journal July 2007
Phosphatidylinositol 4,5-Bisphosphate Is a Novel Coactivator of the Pseudomonas aeruginosa Cytotoxin ExoU journal May 2013
Signal-dependent membrane targeting by pleckstrin homology (PH) domains journal August 2000
Basic local alignment search tool journal October 1990
[20] Processing of X-ray diffraction data collected in oscillation mode book January 1997

Cited By (4)

Bacterial Sphingomyelinases and Phospholipases as Virulence Factors journal June 2016
SopF, a phosphoinositide binding effector, promotes the stability of the nascent Salmonella-containing vacuole journal July 2019
Pseudomonas aeruginosa Toxin ExoU as a Therapeutic Target in the Treatment of Bacterial Infections journal December 2019
Pseudomonas aeruginosa ExoU augments neutrophil transepithelial migration journal August 2017

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Related Subjects

59 BASIC BIOLOGICAL SCIENCES
36 MATERIALS SCIENCE
Bacterial Pathogenesis
Bacterial Toxin
Crystal Structure
Phosphatidylinositol Signaling
Phospholipase
Pseudomonas aeruginosa (P. aeruginosa)