Abstract
Edema Factor (EF), the toxic sub-unit of the Bacillus anthracis Edema Toxin (ET) is a calmodulin-dependent adenylate cyclase whose detrimental activity in the infected host results in severe edema. EF is therefore a major virulence factor of B. anthracis. We describe a simple, rapid and reliable functional adenylate-cyclase assay based on inhibition of a luciferase-mediated luminescence reaction. The assay exploits the efficient adenylate cyclase-mediated depletion of adenosine tri-phosphate (ATP), and the strict dependence on ATP of the light-emitting luciferase-catalyzed luciferin-conversion to oxyluciferin, which can be easily visualized. The assay exhibits a robust EF-dose response decrease in luminescence, which may be specifically reverted by anti-EF antibodies. The application of the assay is exemplified in: (a) determining the presence of EF in B. anthracis cultures, or its absence in cultures of EF-defective strains; (b) evaluating the anti-EF humoral response in experimental animals infected/vaccinated with B. anthracis; and (c) rapid discrimination between EF producing and non-producing bacterial colonies. Furthermore, the assay may be amenable with high-throughput screening for EF inhibitory molecules.
Highlights
The gram-positive spore-forming bacterium Bacillus anthracis is the causative agent of anthrax, a rare fatal disease that is initiated, in its most severe form, by inhalation of infectious spores
We document a novel simple, rapid and sensitive functional assay for Edema Factor (EF) activity based on the ability of this enzyme to promote efficient depletion of adenosine tri-phosphate (ATP) which may be quantitatively evaluated by inhibition of a luciferase-mediated light-emitting reaction
We show that this simple and cost-effective method may be implemented for evaluation of EF activity in crude B. anthracis culture supernatants, may be used for detection of anti-EF antibodies or other EF-inhibitory molecules
Summary
The gram-positive spore-forming bacterium Bacillus anthracis is the causative agent of anthrax, a rare fatal disease that is initiated, in its most severe form, by inhalation of infectious spores. We document a novel simple, rapid and sensitive functional assay for EF activity based on the ability of this enzyme to promote efficient depletion of ATP which may be quantitatively evaluated by inhibition of a luciferase-mediated light-emitting reaction. We show that this simple and cost-effective method may be implemented for evaluation of EF activity in crude B. anthracis culture supernatants, may be used for detection of anti-EF antibodies or other EF-inhibitory molecules.
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