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.

Keywords: ATP-depletion; Bacillus anthracis; Edema Factor; adenylate cyclase; luciferase; luminescent assay.

Figure 1

A luminescent adenosine tri-phosphate (ATP)-depletion assay for identification of edema factor (EF), based on its adenylate cyclase activity. (A) Schematic description of the assay. Step I: EF or “unknown” samples are incubated together with Calmodulin (CaM), ATP and luciferin in microtiter plate wells for the indicated period of time at room temperature. Step II: Luciferase is added to the wells. Step III: The microtiter plate is scanned in a luminescence scanner. The amount of luminescence is directly proportional to the amount of ATP in the reaction which decreases due to the AC activity of EF. The light emitting reaction is described as an equation under the scheme. (B) Dose-response curve of luminescence as a function of the amount of EF in the reaction. (C) The dose-dependent decrease in luminescence caused by EF is paralleled by an increase in the amount of cyclic adenosine mono-phosphate (cAMP) resulting from the conversion of ATP. (D) Dose-response of the luminescence as a function of the amount of luciferase added at step II of the assay. Routinely, an amount of luciferase affording maximal light emission (100 ng) was used in the assay. (E) Time course of the luminescence decrease in the presence of 10 ng EF (black histograms) compared to luminescence in the absence of EF (gray histograms). In panels B–E, luminescence is expressed in photon counts per second (cps); this value depends on the sensitivity and the internal calibration of the scanner and may vary in different scanners. Reactions in the experiments described in B–D were carried out for 3 h. The data in panels B–E represent the geometrical mean (±SD) obtained in at least three independent experiments. See Experimental Section for details.

Figure 2

The luminescent ATP-depletion assay is implemented for determining the presence of active EF in B. anthracis culture supernatants and the neutralizing activity of anti-EF antibodies. (A) Presence of EF in B. anthracis culture-supernatants. Histogram 1: Luminescence in the absence of ATP. Histogram 2 (control): Luminescence measured in the luciferase/luciferin reaction. Histogram 3: Luminescence obtained following addition of 10 ng pure EF. Histograms 4–5: Luminescence following addition of 10 μL of B. anthracis cultures (8 h growth in DMEM [Dulbecco’s modified Eagle’s medium] in an 8% CO₂-enriched atmosphere at 37 °C) of WT (wild-type) Vollum (pXO1⁺, histogram 4) or ∆Vollum (pXO1⁻, histogram 5) strains. (B) Inhibitory effect of EF is reversed by anti-EF antibodies. Histogram 1 (control): Luminescence measured in the luciferase/luciferin reaction. Histogram 2: Luminescence inhibition promoted by EF. Histogram 3–5: Effect of inclusion of sera. Reversion of EF inhibitory effect is promoted by pre-incubation of EF with sera obtained from guinea-pigs immunized with an EF producing B. anthracis strain (histogram 3) but not with sera from animals immunized with a B. anthracis ∆cya strain (histogram 4) or by sera from naïve animals (histogram 5). Histogram 6: EF-dependent luminescence inhibition is reversed by pre-incubation of EF with commercial anti-EF antibodies (10 μL) in the reaction. Luminescence is expressed in photon counts per second (cps). The experiment was independently performed at least three times in triplicate. The data represent the average luminescence level (+SD) obtained in a representative experiment. * Statistical analysis of the inhibitory effect of EF on luminescence was performed using the student t-test (p < 0.001). The presence (+) or absence (-) of a particular element included in the assay is indicated in the table below the histogram chart.

Figure 2

The luminescent ATP-depletion assay is implemented for determining the presence of active EF in B. anthracis culture supernatants and the neutralizing activity of anti-EF antibodies. (A) Presence of EF in B. anthracis culture-supernatants. Histogram 1: Luminescence in the absence of ATP. Histogram 2 (control): Luminescence measured in the luciferase/luciferin reaction. Histogram 3: Luminescence obtained following addition of 10 ng pure EF. Histograms 4–5: Luminescence following addition of 10 μL of B. anthracis cultures (8 h growth in DMEM [Dulbecco’s modified Eagle’s medium] in an 8% CO₂-enriched atmosphere at 37 °C) of WT (wild-type) Vollum (pXO1⁺, histogram 4) or ∆Vollum (pXO1⁻, histogram 5) strains. (B) Inhibitory effect of EF is reversed by anti-EF antibodies. Histogram 1 (control): Luminescence measured in the luciferase/luciferin reaction. Histogram 2: Luminescence inhibition promoted by EF. Histogram 3–5: Effect of inclusion of sera. Reversion of EF inhibitory effect is promoted by pre-incubation of EF with sera obtained from guinea-pigs immunized with an EF producing B. anthracis strain (histogram 3) but not with sera from animals immunized with a B. anthracis ∆cya strain (histogram 4) or by sera from naïve animals (histogram 5). Histogram 6: EF-dependent luminescence inhibition is reversed by pre-incubation of EF with commercial anti-EF antibodies (10 μL) in the reaction. Luminescence is expressed in photon counts per second (cps). The experiment was independently performed at least three times in triplicate. The data represent the average luminescence level (+SD) obtained in a representative experiment. * Statistical analysis of the inhibitory effect of EF on luminescence was performed using the student t-test (p < 0.001). The presence (+) or absence (-) of a particular element included in the assay is indicated in the table below the histogram chart.

Figure 3

The luminescent ATP-depletion assay is implemented to distinguish between wild-type (WT, parental) and cya-gene disrupted colonies of B. anthracis. (A) 24 discrete colonies obtained after gene disruption of the cya-gene by a homologous recombination gene-targeting protocol [29,30,31] were used to inoculate 24 wells of a micro-titer plate (left panel) containing 200 μL DMEM and grown for 2 h. Ten microliters of the supernatant of each well were transferred to an ATP-depletion luminescence-inhibition assay microtiter plate (as described in Figure 1A). The assay was carried-out for 45 min. Following Luciferase addition, the plate was analyzed for 10 s with a luminescent-image analyzer (right panel). Luminescent wells (visualized as black dots) indicate that the bacteria did not secrete functional EF: (B) quantitative analysis of the plate by the VICTOR³TM luminometer; and (C) confirmation of abrogation of EF synthesis by Western-blot analysis of colony 3 (which did not exhibit luminescence inhibition in the assay, marked ∆cya) compared to colony 5 (which inhibited the luminescence in the ATP-depletion assay, marked WT-wild type); all EF-devoid colonies selected by the luminescent assay demonstrated the desired phenotype by Western-blot analysis.