title: A Simple Luminescent Adenylate-Cyclase Functional Assay for Evaluation of Bacillus anthracis Edema Factor Activity.
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.
authors.affiliation: Department of Biochemistry and Molecular Genetics, Israel Institute for Biological Research, Ness-Ziona 74100, Israel. maayani@iibr.gov.il.
authors.forename: Ma'ayan
authors.initials: M
authors.lastname: Israeli
authors.affiliation: Department of Biochemistry and Molecular Genetics, Israel Institute for Biological Research, Ness-Ziona 74100, Israel. shaharr@iibr.gov.il.
authors.forename: Shahar
authors.initials: S
authors.lastname: Rotem
authors.affiliation: Department of Biochemistry and Molecular Genetics, Israel Institute for Biological Research, Ness-Ziona 74100, Israel. urie@iibr.gov.il.
authors.forename: Uri
authors.initials: U
authors.lastname: Elia
authors.affiliation: Department of Biochemistry and Molecular Genetics, Israel Institute for Biological Research, Ness-Ziona 74100, Israel. erezb@iibr.gov.il.
authors.forename: Erez
authors.initials: E
authors.lastname: Bar-Haim
authors.affiliation: Department of Biochemistry and Molecular Genetics, Israel Institute for Biological Research, Ness-Ziona 74100, Israel. oferc@iibr.gov.il.
authors.forename: Ofer
authors.initials: O
authors.lastname: Cohen
authors.affiliation: Department of Biochemistry and Molecular Genetics, Israel Institute for Biological Research, Ness-Ziona 74100, Israel. theodorc@iibr.gov.il.
authors.forename: Theodor
authors.initials: T
authors.lastname: Chitlaru
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D000262:Adenylyl Cyclases / Q000378:metabolism*; D000942:Antigens, Bacterial / Q000378:metabolism*; D001408:Bacillus anthracis / Q000201:enzymology* / Q000235:genetics; D001427:Bacterial Toxins / Q000378:metabolism*; D057166:High-Throughput Screening Assays / Q000379:methods*; D007211:Indoles / Q000378:metabolism; D007700:Kinetics; D008156:Luciferases / Q000378:metabolism; D008163:Luminescent Measurements; D011719:Pyrazines / Q000378:metabolism; D015203:Reproducibility of Results
chemical_list: D000942:Antigens, Bacterial; D001427:Bacterial Toxins; D007211:Indoles; D011719:Pyrazines; C030325:anthrax toxin; C014730:oxyluciferin; D000255:Adenosine Triphosphate; D008156:Luciferases; D000262:Adenylyl Cyclases
keywords: ATP-depletion; Bacillus anthracis; Edema Factor; adenylate cyclase; luciferase; luminescent assay