*Article* **A Robust and Sensitive Spectrophotometric Assay for the Enzymatic Activity of Bacterial Adenylate Cyclase Toxins**

**Marilyne Davi 1, Mirko Sadi 1,2, Irene Pitard 3,4, Alexandre Chenal <sup>1</sup> and Daniel Ladant 1,\***


**Abstract:** Various bacterial pathogens are producing toxins that target the cyclic Nucleotide Monophosphate (cNMPs) signaling pathways in order to facilitate host colonization. Among them, several are exhibiting potent nucleotidyl cyclase activities that are activated by eukaryotic factors, such as the adenylate cyclase (AC) toxin, CyaA, from *Bordetella pertussis* or the edema factor, EF, from *Bacillus anthracis*. The characterization of these toxins frequently requires accurate measurements of their enzymatic activity in vitro, in particular for deciphering their structure-to-function relationships by protein engineering and site-directed mutagenesis. Here we describe a simple and robust in vitro assay for AC activity based on the spectrophotometric detection of cyclic AMP (cAMP) after chromatographic separation on aluminum oxide. This assay can accurately detect down to fmol amounts of *B. pertussis* CyaA and can even be used in complex media, such as cell extracts. The relative advantages and disadvantages of this assay in comparison with other currently available methods are briefly discussed.

**Keywords:** adenylate cyclase toxin; *Bordetella pertussis*; cyclic nucleotide; cAMP; spectrophotometric enzymatic assay

**Key Contribution:** A simple and robust in vitro spectrophotometric assay for adenylate cyclase activity is described and shown to be capable of detecting fmol amounts of *Bordetella pertussis* CyaA toxin.
