Open AccessRole of the Membrane Localization Domain of the Pseudomonas aeruginosa Effector Protein ExoU in Cytotoxicity
Author(s) -
Jeff L. Veesenmeyer,
Heather A. Howell,
A.S. Halavaty,
Sebastian Ahrens,
W.F. Anderson,
Alan R. Hauser
Publication year - 2010
Publication title -
infection and immunity
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.508
H-Index - 220
eISSN - 1070-6313
pISSN - 0019-9567
DOI - 10.1128/iai.00223-10
Subject(s) - effector , biology , virulence , secretion , biochemistry , phospholipase , cytotoxicity , microbiology and biotechnology , pseudomonas aeruginosa , enzyme , gene , in vitro , bacteria , genetics
ExoU is a potent effector protein that causes rapid host cell death upon injection by the type III secretion system ofPseudomonas aeruginosa . The N-terminal half of ExoU contains a patatin-like phospholipase A2 (PLA2 ) 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 PLA2 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.