A sensitive fluorescence-based assay for the detection of ExoU-mediated PLA(2) activity.

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