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The mechanism of action of the Pseudomonas aeruginosa-encoded type III cytotoxin, ExoU. EMBO J 2003 Jun 16;22(12):2959-69

Date

06/14/2003

Pubmed ID

12805211

Pubmed Central ID

PMC162142

DOI

10.1093/emboj/cdg290

Scopus ID

2-s2.0-0038376087   267 Citations

Abstract

Pseudomonas aeruginosa delivers the toxin ExoU to eukaryotic cells via a type III secretion system. Intoxication with ExoU is associated with lung injury, bacterial dissemination and sepsis in animal model and human infections. To search for ExoU targets in a genetically tractable system, we used controlled expression of the toxin in Saccharomyces cerevisiae. ExoU was cytotoxic for yeast and caused a vacuolar fragmentation phenotype. Inhibitors of human calcium-independent (iPLA(2)) and cytosolic phospholipase A(2) (cPLA(2)) lipase activity reduce the cytotoxicity of ExoU. The catalytic domains of patatin, iPLA(2) and cPLA(2) align or are similar to ExoU sequences. Site-specific mutagenesis of predicted catalytic residues (ExoUS142A or ExoUD344A) eliminated toxicity. ExoU expression in yeast resulted in an accumulation of free palmitic acid, changes in the phospholipid profiles and reduction of radiolabeled neutral lipids. ExoUS142A and ExoUD344A expressed in yeast failed to release palmitic acid. Recombinant ExoU demonstrated lipase activity in vitro, but only in the presence of a yeast extract. From these data we conclude that ExoU is a lipase that requires activation or modification by eukaryotic factors.

Author List

Sato H, Frank DW, Hillard CJ, Feix JB, Pankhaniya RR, Moriyama K, Finck-Barban??on V, Buchaklian A, Lei M, Long RM, Wiener-Kronish J, Sawa T

Authors

Jimmy B. Feix PhD Professor in the Biophysics department at Medical College of Wisconsin
Dara W. Frank PhD Professor in the Microbiology and Immunology department at Medical College of Wisconsin
Cecilia J. Hillard PhD Associate Dean, Center Director, Professor in the Pharmacology and Toxicology department at Medical College of Wisconsin
Roy M. Long PhD Assistant Dean, Associate Professor in the Medical School Regional Campuses department at Medical College of Wisconsin




MESH terms used to index this publication - Major topics in bold

Amino Acid Sequence
Animals
Bacterial Proteins
Carboxylic Ester Hydrolases
Cell Line
Genes, Reporter
Humans
Lipase
Molecular Sequence Data
Phenotype
Phospholipases A
Plant Proteins
Pseudomonas aeruginosa
Recombinant Fusion Proteins
Saccharomyces cerevisiae
Sequence Alignment
Solvents