title: Role of the membrane localization domain of the Pseudomonas aeruginosa effector protein ExoU in cytotoxicity.
abstract: ExoU is a potent effector protein that causes rapid host cell death upon injection by the type III secretion system of Pseudomonas aeruginosa. The N-terminal half of ExoU contains a patatin-like phospholipase A(2) (PLA(2)) domain that requires the host cell cofactor superoxide dismutase 1 (SOD1) for activation, while the C-terminal 137 amino acids constitute a membrane localization domain (MLD). Previous studies had utilized insertion and deletion mutations to show that portions of the MLD are required for membrane localization and catalytic activity. Here we further characterize this domain by identifying six residues that are essential for ExoU activity. Substitutions at each of these positions resulted in abrogation of membrane targeting, decreased ExoU-mediated cytotoxicity, and reductions in PLA(2) activity. Likewise, each of the six MLD residues was necessary for full virulence in cell culture and murine models of acute pneumonia. Purified recombinant ExoU proteins with substitutions at five of the six residues were not activated by SOD1, suggesting that these five residues are critical for activation by this cofactor. Interestingly, these same five ExoU proteins were partially activated by HeLa cell extracts, suggesting that a host cell cofactor other than SOD1 is capable of modulating the activity of ExoU. These findings add to our understanding of the role of the MLD in ExoU-mediated virulence.
authors.affiliation: Department of Microbiology/Immunology, Northwestern University, Chicago, Illinois, USA.
authors.forename: Jeff L
authors.initials: JL
authors.lastname: Veesenmeyer
authors.forename: Heather
authors.initials: H
authors.lastname: Howell
authors.forename: Andrei S
authors.initials: AS
authors.lastname: Halavaty
authors.forename: Sebastian
authors.initials: S
authors.lastname: Ahrens
authors.forename: Wayne F
authors.initials: WF
authors.lastname: Anderson
authors.forename: Alan R
authors.initials: AR
authors.lastname: Hauser
mesh_terms: D019943:Amino Acid Substitution / Q000235:genetics; D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics / Q000633:toxicity*; D002462:Cell Membrane / Q000737:chemistry; D002470:Cell Survival; D004847:Epithelial Cells / Q000382:microbiology; D005260:Female; D006367:HeLa Cells; D006801:Humans; D051379:Mice; D008807:Mice, Inbred BALB C; D020125:Mutation, Missense; D018410:Pneumonia, Bacterial / Q000382:microbiology; D021382:Protein Sorting Signals; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D011552:Pseudomonas Infections / Q000382:microbiology; D011550:Pseudomonas aeruginosa / Q000472:pathogenicity*; D037521:Virulence Factors / Q000235:genetics / Q000633:toxicity*
chemical_list: D001426:Bacterial Proteins; D021382:Protein Sorting Signals; D037521:Virulence Factors; C111749:pseudomonas exoprotein A protein, Pseudomonas aeruginosa