A sensitive fluorescence-based assay for the detection of ExoU-mediated PLA(2) activity.
BACKGROUND
Pseudomonas aeruginosa is an opportunistic pathogen that causes disease in immunocompromised individuals, burn victims, and cystic fibrosis patients. Strains that secrete ExoU induce host cell lysis and damage epithelial tissue, which can lead to severe outcomes including sepsis and mortality. ExoU is classified as an A2 phospholipase (PLA(2)) and activity is dependent on the eukaryotic protein, superoxide dismutase 1 (SOD1).


METHODS
A sensitive and low background in vitro fluorescence-based assay was developed to detect ExoU activity using the fluorogenic substrate, PED6.


RESULTS
The optimized assay enabled us to perform the first kinetic evaluation of the activation of ExoU (apparent K(m) of 13.2+/-1.5mumol/l PED6 and an apparent V(max) of 42nmol/min/mg). An inhibitor study using the inhibitor, methyl arachidonyl fluorophosphonate (MAFP), yielded an IC(50) of 13.8+/-1.1nmol/l and validated the use of high-throughput inhibitor screens using the assay. Most notably, the in vitro fluorescence-based activity assay was sensitive enough to detect catalytically active ExoU injected into eukaryotic cells.


CONCLUSIONS
The use of the fluorescence-based activity assay to study the mechanism of ExoU activation may lead to the development of potential therapeutics to reduce P. aeruginosa-associated mortality.
Benson
Marc A
MA
Center for Biopreparedness and Infectious Diseases, Department of Microbiology and Molecular Genetics, Medical College of Wisconsin, 8701 Watertown Plank Rd., Milwaukee, WI 53226, United States.
Schmalzer
Katherine M
KM
Frank
Dara W
DW
D001095:Arachidonic Acids / Q000494:pharmacology; D001426:Bacterial Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D004789:Enzyme Activation; D057075:Enzyme Assays / Q000379:methods*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006367:HeLa Cells; D057166:High-Throughput Screening Assays; D006801:Humans; D042541:Intracellular Space / Q000201:enzymology; D063065:Organophosphonates / Q000494:pharmacology; D064801:Phospholipase A2 Inhibitors; D054467:Phospholipases A2 / Q000378:metabolism*; D021381:Protein Transport; D011550:Pseudomonas aeruginosa / Q000201:enzymology* / Q000502:physiology; D015203:Reproducibility of Results; D012970:Sodium Glutamate / Q000494:pharmacology; D013050:Spectrometry, Fluorescence
D001095:Arachidonic Acids; D001426:Bacterial Proteins; D004791:Enzyme Inhibitors; D063065:Organophosphonates; D064801:Phospholipase A2 Inhibitors; C104810:methyl arachidonylfluorophosphonate; C111749:pseudomonas exoprotein A protein, Pseudomonas aeruginosa; D054467:Phospholipases A2; D012970:Sodium Glutamate