Identification of superoxide dismutase as a cofactor for the pseudomonas type III toxin, ExoU.
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.
Sato
Hiromi
H
Department of Biophysics, Medical College of Wisconsin, 8701 Watertown Plank Road, Milwaukee, Wisconsin 53226, USA.
Feix
Jimmy B
JB
Frank
Dara W
DW
D036002:ADP Ribose Transferases / Q000276:immunology / Q000378:metabolism; D000818:Animals; D001426:Bacterial Proteins / Q000037:antagonists & inhibitors / Q000276:immunology / Q000378:metabolism*; D001427:Bacterial Toxins / Q000276:immunology / Q000378:metabolism; D002417:Cattle; D003067:Coenzymes / Q000276:immunology / Q000378:metabolism*; D003300:Copper / Q000276:immunology / Q000378:metabolism; D003593:Cytoplasm / Q000201:enzymology / Q000276:immunology; D015195:Drug Design; D004791:Enzyme Inhibitors / Q000627:therapeutic use; D020690:GTPase-Activating Proteins / Q000276:immunology / Q000378:metabolism; D006801:Humans; D007113:Immunity, Innate; D009894:Opportunistic Infections / Q000188:drug therapy / Q000201:enzymology / Q000276:immunology / Q000382:microbiology; D010740:Phospholipases / Q000276:immunology / Q000378:metabolism*; D011485:Protein Binding / Q000276:immunology; D011552:Pseudomonas Infections / Q000188:drug therapy / Q000201:enzymology / Q000276:immunology; D011550:Pseudomonas aeruginosa / Q000201:enzymology* / Q000276:immunology; D011994:Recombinant Proteins / Q000276:immunology / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000276:immunology / Q000378:metabolism; D013482:Superoxide Dismutase / Q000276:immunology / Q000378:metabolism*; D000072105:Superoxide Dismutase-1; D015032:Zinc / Q000276:immunology / Q000378:metabolism
D001426:Bacterial Proteins; D001427:Bacterial Toxins; D003067:Coenzymes; D004791:Enzyme Inhibitors; C477521:ExoT protein, Pseudomonas aeruginosa; D020690:GTPase-Activating Proteins; D011994:Recombinant Proteins; C000606290:SOD1 protein, human; D029701:Saccharomyces cerevisiae Proteins; C111749:pseudomonas exoprotein A protein, Pseudomonas aeruginosa; D003300:Copper; D013482:Superoxide Dismutase; D000072105:Superoxide Dismutase-1; D036002:ADP Ribose Transferases; C444820:exoenzyme S; D010740:Phospholipases; D015032:Zinc