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A Simple Luminescent Adenylate-Cyclase Functional Assay for Evaluation of Bacillus anthracis Edema Factor Activity
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 fun...
Autores principales: | , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
MDPI
2016
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4999859/ https://www.ncbi.nlm.nih.gov/pubmed/27548219 http://dx.doi.org/10.3390/toxins8080243 |
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author | Israeli, Ma’ayan Rotem, Shahar Elia, Uri Bar-Haim, Erez Cohen, Ofer Chitlaru, Theodor |
author_facet | Israeli, Ma’ayan Rotem, Shahar Elia, Uri Bar-Haim, Erez Cohen, Ofer Chitlaru, Theodor |
author_sort | Israeli, Ma’ayan |
collection | PubMed |
description | 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. |
format | Online Article Text |
id | pubmed-4999859 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2016 |
publisher | MDPI |
record_format | MEDLINE/PubMed |
spelling | pubmed-49998592016-09-01 A Simple Luminescent Adenylate-Cyclase Functional Assay for Evaluation of Bacillus anthracis Edema Factor Activity Israeli, Ma’ayan Rotem, Shahar Elia, Uri Bar-Haim, Erez Cohen, Ofer Chitlaru, Theodor Toxins (Basel) Communication 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. MDPI 2016-08-18 /pmc/articles/PMC4999859/ /pubmed/27548219 http://dx.doi.org/10.3390/toxins8080243 Text en © 2016 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC-BY) license (http://creativecommons.org/licenses/by/4.0/). |
spellingShingle | Communication Israeli, Ma’ayan Rotem, Shahar Elia, Uri Bar-Haim, Erez Cohen, Ofer Chitlaru, Theodor A Simple Luminescent Adenylate-Cyclase Functional Assay for Evaluation of Bacillus anthracis Edema Factor Activity |
title | A Simple Luminescent Adenylate-Cyclase Functional Assay for Evaluation of Bacillus anthracis Edema Factor Activity |
title_full | A Simple Luminescent Adenylate-Cyclase Functional Assay for Evaluation of Bacillus anthracis Edema Factor Activity |
title_fullStr | A Simple Luminescent Adenylate-Cyclase Functional Assay for Evaluation of Bacillus anthracis Edema Factor Activity |
title_full_unstemmed | A Simple Luminescent Adenylate-Cyclase Functional Assay for Evaluation of Bacillus anthracis Edema Factor Activity |
title_short | A Simple Luminescent Adenylate-Cyclase Functional Assay for Evaluation of Bacillus anthracis Edema Factor Activity |
title_sort | simple luminescent adenylate-cyclase functional assay for evaluation of bacillus anthracis edema factor activity |
topic | Communication |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4999859/ https://www.ncbi.nlm.nih.gov/pubmed/27548219 http://dx.doi.org/10.3390/toxins8080243 |
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