A simple luminescent adenylate-cyclase functional assay for evaluation of Bacillus anthracis edema factor activity

Máayan Israeli, Shahar Rotem, Uri Elia, Erez Bar-Haim, Ofer Cohen, Theodor Chitlaru

Research output: Contribution to journalArticlepeer-review

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.

Original languageEnglish
Article number243
JournalToxins
Volume8
Issue number8
DOIs
StatePublished - 18 Aug 2016
Externally publishedYes

Keywords

  • ATP-depletion
  • Adenylate cyclase
  • Bacillus anthracis
  • Edema factor
  • Luciferase
  • Luminescent assay

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