Cell entry and cAMP imaging of anthrax edema toxin

Abstract

The entry and enzymatic activity of the anthrax edema factor (EF) in different cell types was studied by monitoring EF-induced changes in intracellular cAMP with biochemical and microscopic methods. cAMP was imaged in live cells, transfected with a fluorescence resonance energy transfer biosensor based on the protein kinase A regulatory and catalytic subunits fused to CFP and YFP, respectively. The cAMP biosensor was located either in the cytosol or was membrane-bound owing to the addition of a tag determining its myristoylation/palmitoylation. Real-time imaging of cells expressing the cAMP biosensors provided the time course of EF catalytic activity and an indication of its subcellular localization. Bafilomycin A1, an inhibitor of the vacuolar ATPase proton pump, completely prevented EF activity, even when added long after the toxin. The time course of appearance of the adenylate cyclase activity and of bafilomycin A1 action suggests that EF enters the cytosol from late endosomes. EF remains associated to these compartments and its activity shows a perinuclear localization generating intracellular cAMP concentration gradients from the cell centre to the periphery.

Figure 1

EF generates a prolonged cAMP increase in different cell lines. Cells were treated with EF 10 nM and PA 20 nM and incubated for the indicated time periods at 37°C. After centrifugation, the supernatant was removed and cells were lysed to measure intracellular cAMP with the Amersham immuno assay kit. (A) Jurkat T cells, (B) HeLa cells (grey bars) and Raw264.7 macrophages (dark grey bars). Data are the average of two or more different experiments run in triplicates and bars represent±s.d.

Figure 2

The v-ATPase proton pump bafilomycin A1 prevents EF activity long after toxin addition to cells. (A) Jurkat cells were treated with EF 10 nM plus PA 20 nM at time 0 and incubated at 37°C for 2 h; bafilomycin A1 (0.5 μM) was added at the indicated time intervals after the addition of the toxin. Cells were centrifuged, and lysed, after removal of the supernatant, to measure intracellular cAMP with the Amersham immunoassay kit. (B) The same experiment was performed with HeLa cells (grey bars) and with the Raw264.7 macrophages (dark grey bars). cAMP determined at any given time is reported as percentage of the maximum value. (C) cAMP content of HeLa cells treated with 10 μM nocodazole (Sigma) added at the indicated time intervals before or after toxin addition. The partial initial inhibitory effect of nocodazole is likely to be due to the fact that some time is required for microtubule depolymerization to take place. Data are the average of two or more different experiments run in triplicates and bars represent±s.d.

Figure 3

Different subcellular localizations of anthrax LF and EF. HeLa cells were treated either with LF+PA (10 and 20 nM, respectively) or with EF+PA (10 and 20 nM, respectively) for 1 h at 37°C and were then lysed and separated into a supernatant (PNS), which includes the cell cytosol, and a membrane fraction that was fractionated on a sucrose density gradient. Three fractions were obtained: HM, heavy membranes, EE, early endosomes and LE, late endosomes. Ten microgram proteins of these fractions were subjected to SDS–PAGE, immunoblotted and developed as described in the Materials and methods. (A) Toxin and markers distribution among the different fractions in a representative experiment, while (B) show the corresponding quantification of the bands, which includes a normalization to the total amount of proteins of each of the four fractions examined. Rab7 is a marker of late endosomes and EEA1 is a marker of early endosomes. Note the largely different distribution of LF and EF, with LF being present mainly in the cell cytosol fraction and EF on endosomes. The significant amount of LF and EF still present in the early endosomal fraction is due to the fact that the experiment was performed without low-temperature synchronization of the binding step, under the same conditions required for imaging (see text).

Figure 4

Imaging of the EF-induced rise of cAMP with PKA fluorescent probes in Jurkat cells. Jurkat cells expressing the catalytic PKA subunit coupled to YFP and the regulatory PKA subunit coupled to CFP in the cytosol or in the plasma membrane depending on the presence of a membrane localization sequence were imaged after treatment with EF 10 nM+PA 20 nM (time zero). During microscopic observations, cells were maintained in 2 ml of a balanced salt solution inside a microscope-adapted micro-incubator at 37°C and constant 5% CO₂ pressure. Images were acquired every 10 s and the ratio between CFP and YFP emissions was calculated. An increasing ratio corresponds to increasing cAMP concentrations. Similar traces were recorded in other cells and they do not depend on cell size. (A) Change of cAMP with time in a cell expressing the cytosolic probe; the inset shows the fluorescence of CFP at time O indicating a cytosolic distribution of the probe. (B) cAMP remains low in cells treated with PA only or EF only. This is revealed by both the cytosolic PKA fluorescent probe (orange trace corresponding to the cell of inset 1 which shows the CFP fluorescence at time 0) and by the membrane localized PKA probe (inset 2, blue trace, and inset 3, magenta trace, show the CFP fluorescence taken at time 0 of cells treated with PA or Ef, respectively). (C) The change of cAMP with time in a Jurkat cell expressing the membrane localized PKA probe; the inset shows the fluorescence of membrane-bound CFP at time O. (D) The Jurkat cell of (C) as pseudo-colours, which reflect the increasing cAMP concentration from green (low cAMP) to red (high cAMP) at the indicated time points of incubation with PA+EF.

Figure 5

Anthrax edema toxin creates c-AMP microdomains in HeLa cells. (A) HeLa cells expressing the cytosolic PKA-based probe cAMP fluorescence biosensor were treated with EF 10+PA 20 nM (time zero) and maintained in 2 ml of balanced salt solution at 37°C during microscopic observations. CFP/YFP ratios were measured in the indicated areas, identified with different colour contours: perinuclear regions (1, red trace; 2, orange trace) and cell periphery (3, yellow trace; 4, green trace). Notice the lower cAMP rising in the peripheral areas. (B) HeLa cell expressing the cAMP cytosolic probe treated with the B. pertussis CyaA adenylate cyclase toxin, which enters from the plasma membrane. Notice the faster rise of the ratiometic signal in the sub-plasma membrane areas identified by different colours, which are the same of those of the corresponding traces. (C, D) Pseudo-colour images, generated by CFP/YFP ratio imaging, of the intracellular cAMP at the given time points of the cell of (A) treated with PA+EF and of the cell of (B) treated with CyaA.

Figure 6

Different routes of entry of EF and of the CyaA adenylate cyclase of B. pertussis. HeLa cells expressing the membrane-bound PKA-based biosensor for cAMP were treated with 1 nM CyaA (A) or with EF 10 nM+PA 20 nM (B) and the CFP/YFP ratio was measured in plasma membrane areas of the cells (see contours in the cells of the insets) as a function of time.