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

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

Figure 1

Photometric detection of AC. Panel (A): Spectrophotometric measurements of cAMP. Each line corresponds to an independent assay with the indicated nucleotide addition. Two 0.3 mL samples taken from the Al₂O₃ supernatant for each assay were transferred to a microplate and the absorption at 260 and 340 nm were recorded. The absorption at 340 nm provides a good estimate of the light scattering that arises from contaminating Al₂O₃ particles that are inadvertently aspirated in the 0.3 mL supernatant samples. Subtracting the absorption at 340 nm from the absorption at 260 nm yields a very reliable quantification of the cAMP content. Panel (B): Correspondence between the absorption at 260 nm minus absorption at 340 nm (A₂₆₀–A₃₄₀) of the Al₂O₃ supernatant as a function of cAMP (4 independent data points for each concentration) when it is added to the standard assay medium containing 2 mM ATP. Subtraction of the absorption at 340 nm allows for the correction of the potential light scattering arising from residual Al₂O₃ particles in the supernatant (see Material and methods for detail). Panel (C): Time and concentration dependence of B. pertussis AC activity. Reactions were carried out for the indicated times with the indicated final concentrations of AC384 in the presence of 2 mM ATP and 1 μM CaM (except in bar labeled “No CaM”). The calculated k_cat is indicated above the bar (mean of 4 independent experiments).

Figure 2

CaM-dependent AC activity of B. pertussis AC384 (blue) and B. cereus EF enzymes (green). AC activities (expressed in mol of cAMP produced per mol of enzyme per sec) at the indicated CaM concentrations were fitted to equation: A = (A_Max × [CaM])/(K_D + [CaM]). Maximal activities (A_Max) of 2165 ± 30 and 1183 ± 22 s⁻¹ were calculated for AC384 and EF, respectively, while the CaM concentration at half-maximal activation (≈K_D) were 0.11 ± 0.01 and 1.04 ± 0.13 nM for AC384 and EF, respectively.

Figure 3

Salt and urea concentration dependence of AC384 activity. NaCl (Panel (A)) and urea (Panel (B)) concentration dependence of B. pertussis AC activity. Reactions were carried out for 10 min with 0.32 nM AC384 in the presence of 2 mM ATP and 1 μM CaM (mean and STD of 4 independent experiments). Control experiments (not shown) showed that ATP and cAMP binding to the Al₂O₃ were not modified by the presence of NaCl or urea at the tested concentrations. Panel (C): B. pertussis AC384 activities were measured at the indicated CaM concentrations in standard conditions (i.e., in Tris buffer, blue square) or in the presence of 0.2 M NaCl (green circle) or 0.2 M urea (red diamond). Data were fitted as in Figure 2. Maximal activities (in mol of cAMP per mol of AC384 per sec) were 2225 ± 95, 1351 ± 50, and 1440 ± 32 s⁻¹ for assays in Tris buffer, in 0.2 M NaCl, and 0.2 M urea, respectively, while the CaM concentrations at half-maximal activation (≈K_D) were 0.20 ± 0.06, 0.32 ± 0.08, and 0.24 ± 0.04 nM, respectively.

Figure 4

Characterization of a detoxified CyaAE5-OVA vaccine. AC activity of the purified CyaAE5-OVA protein (stored at 5.8 μM in 6.6 M urea, 20 mM Hepes-Na) at the indicated concentrations was monitored at 30 °C in the presence of 2 mM ATP and 1 μM CaM for 10 min (bars 1 to 6). As a control, in bars 4–6, 0.32 nM AC384 were spiked in the reaction mixtures in addition to the CyaAE5-OVA protein, in order to check that the residual urea concentration in each assay (indicated on the bottom line) did not affect the enzymatic activity. Results are the mean and STD of four independent experiments.

Figure 5

Assay of CyaA binding to erythrocyte. Panels A and B: Erythrocytes (RBC) were incubated at 30 °C with 2 mM CaCl₂, for 30 min without CyaA (Panel (A)) or with 11.2 nM CyaA (panel (B)), and then extensively washed as described in the Methods section. After lysis, 20 μL of RBC lysates were tested in standard AC assays for the indicated times and the absorption of the Al₂O₃ supernatants were recorded at 260, 340, 405, and 595 nm. The cyclic AMP content in the Al₂O₃ supernatants was determined for sample A after 30 min of incubation and for sample B after 10 min of incubation by a specific ELISA assay and compared to the cAMP quantification deduced from the absorption at 260 nm (Abs₂₆₀). Panel (C): Erythrocytes were incubated without (no CyaA, black) or with 11.2 nM CyaA at 4 or 30 °C with 2 mM CaCl₂ or 2 mM EDTA for 30 min (as indicated). After extensive washing and cell lysis, 20 μL of RBC lysates were tested in standard AC assays as above for the indicated times. The relative fractions of “bound” CyaA activity, as compared to the total CyaA added to each cell suspension, correspond to about 2.4% for the incubation at 30 °C in the presence of CaCl₂ (blue), about 1.1% for the incubation at 4 °C in the presence of CaCl₂ (green), and less than 0.15% for the incubation at 30 °C in the presence of EDTA (red).