Identification of superoxide dismutase as a cofactor for the pseudomonas type III toxin, ExoU. Biochemistry 2006 Aug 29;45(34):10368-75
Date
08/23/2006Pubmed ID
16922513DOI
10.1021/bi060788jScopus ID
2-s2.0-33748672064 (requires institutional sign-in at Scopus site) 63 CitationsAbstract
Pseudomonas aeruginosa is an opportunistic pathogen that uses a type III secretion system and four effector proteins to avoid innate immune responses. ExoS, ExoT, ExoY, and ExoU all possess enzymatic activities that disrupt host cellular physiology and prevent bacterial clearance by host defense mechanisms. The specificity of these toxins for eukaryotic cells depends on the presence of substrate targets and eukaryotic cofactors responsible for effector activation. We used a combined biochemical and proteomic approach to identify Cu(2+), Zn(2+)-superoxide dismutase (SOD1) as a cofactor that activates the phospholipase activity of ExoU. Recombinant ExoU (rExoU) was activated in a dose-dependent manner by either bovine liver SOD1 or the yeast ortholog, Sod1p, but not by either Fe or Mn-containing SODs from E. coli or small molecule SOD mimetics. Inhibitor studies indicated that SOD enzymatic activity was not required for the activation of rExoU. The physical interaction between rExoU and SOD was demonstrated by capture techniques using either of the two proteins immobilized onto the solid phase. Identification of SOD as a cofactor allowed us to develop a new assay using a fluorescent substrate to measure the phospholipase activity of rExoU. The ability of SOD to act as a cytoplasmic cofactor stimulating ExoU phospholipase activity has significant implications for the biological activity of the toxin. Further elucidation of the structural mechanism of ExoU activation by this eukaryotic cofactor may provide a rational approach to the design of inhibitors that can diminish tissue damage during infection by ExoU-producing strains of P. aeruginosa.
Author List
Sato H, Feix JB, Frank DWAuthors
Jimmy B. Feix PhD Professor in the Biophysics department at Medical College of WisconsinDara W. Frank PhD Professor in the Microbiology and Immunology department at Medical College of Wisconsin
MESH terms used to index this publication - Major topics in bold
ADP Ribose TransferasesAnimals
Bacterial Proteins
Bacterial Toxins
Cattle
Coenzymes
Copper
Cytoplasm
Drug Design
Enzyme Inhibitors
GTPase-Activating Proteins
Humans
Immunity, Innate
Opportunistic Infections
Phospholipases
Protein Binding
Pseudomonas Infections
Pseudomonas aeruginosa
Recombinant Proteins
Saccharomyces cerevisiae Proteins
Superoxide Dismutase
Superoxide Dismutase-1
Zinc