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Toxins 2016, 8(8), 243; doi:10.3390/toxins8080243

A Simple Luminescent Adenylate-Cyclase Functional Assay for Evaluation of Bacillus anthracis Edema Factor Activity

Department of Biochemistry and Molecular Genetics, Israel Institute for Biological Research, Ness-Ziona 74100, Israel
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Academic Editor: Shihui Liu
Received: 24 July 2016 / Revised: 9 August 2016 / Accepted: 15 August 2016 / Published: 18 August 2016
(This article belongs to the Collection Anthrax Toxins)
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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. View Full-Text
Keywords: Bacillus anthracis; Edema Factor; adenylate cyclase; luminescent assay; luciferase; ATP-depletion Bacillus anthracis; Edema Factor; adenylate cyclase; luminescent assay; luciferase; ATP-depletion
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This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. (CC BY 4.0).

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Israeli, M.; Rotem, S.; Elia, U.; Bar-Haim, E.; Cohen, O.; Chitlaru, T. A Simple Luminescent Adenylate-Cyclase Functional Assay for Evaluation of Bacillus anthracis Edema Factor Activity. Toxins 2016, 8, 243.

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