JOURNAL ARTICLE

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

Marc A Benson, Katherine M Schmalzer, Dara W Frank
Clinica Chimica Acta; International Journal of Clinical Chemistry 2010, 411 (3-4): 190-7
19900431

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.

DISCUSSION: 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.

Full Text Links

Find Full Text Links for this Article

Discussion

You are not logged in. Sign Up or Log In to join the discussion.

Related Papers

Remove bar
Read by QxMD icon Read
19900431
×

Save your favorite articles in one place with a free QxMD account.

×

Search Tips

Use Boolean operators: AND/OR

diabetic AND foot
diabetes OR diabetic

Exclude a word using the 'minus' sign

Virchow -triad

Use Parentheses

water AND (cup OR glass)

Add an asterisk (*) at end of a word to include word stems

Neuro* will search for Neurology, Neuroscientist, Neurological, and so on

Use quotes to search for an exact phrase

"primary prevention of cancer"
(heart or cardiac or cardio*) AND arrest -"American Heart Association"