doi: 10.1128/IAI.70.9.4997-5007.2002.
Anthrax edema toxin requires influx of calcium for inducing cyclic AMP toxicity in target cells
Affiliations
- PMID:12183546
- PMCID: PMC128280
- DOI: 10.1128/IAI.70.9.4997-5007.2002
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Anthrax edema toxin requires influx of calcium for inducing cyclic AMP toxicity in target cells
Praveen Kumar et al. Infect Immun.2002 Sep.
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Abstract
The anthrax edema toxin comprises two proteins: protective antigen and edema factor. Anthrax protective antigen binds to the receptors on the surface of target cells and facilitates the entry of edema factor into these target cells. Edema factor (EF) is an adenylate cyclase that catalyzes the synthesis of cyclic AMP (cAMP) in the cytosol of the host cells. In this study, we examined the requirement of extracellular calcium for anthrax edema toxin-induced toxicity in host cells. The cAMP response generated by edema toxin was analyzed in a variety of cells, including CHO, macrophage-like RAW264.7, human neutrophils, and human lymphocytes. Our investigations reveal that after EF reaches the cell cytosol, a rapid influx of calcium is triggered in the host cell that has a pivotal role in determining the cAMP response of the affected cells. Although the cAMP response generated by edema toxin in different cell types varied in intensity and in the time of initiation, the influx of calcium invariably preceded cAMP accumulation. Agents that blocked the uptake of calcium also inhibited edema toxin-induced accumulation of cAMP in the host cells. This is the first report that demonstrates that edema toxin induces accumulation of cAMP in lymphocytes. By accumulating cAMP, a potent inhibitor of immune cell function, edema toxin may actually be poisoning the immune system and thus facilitating the survival of the bacteria in the host.
Figures
Requirement of extracellular calcium for anthrax edema toxin-induced toxicity in CHO cells. CHO cells plated in 96-well plates were washed with calcium-free medium and then incubated with toxin (12 nM PA, along with the indicated concentration of EF) in calcium-free medium (•) or in medium containing 2 mM calcium (○). After 2 h of incubation, the intracellular cAMP concentration was measured. The basal level of cAMP in the untreated cells was 40 pmol/mg of cell protein (in presence of calcium) and 38 pmol/mg of cell protein (in absence of calcium). Cells treated with PA or EF alone maintained basal levels of cAMP, both in the presence or in the absence of calcium. The total protein content of the cells was 7 μg per well. All of the values are mean ± the standard deviation (SD) of three experiments done in triplicate.
Dependence of anthrax edema toxin-induced cAMP response of CHO cells on extracellular calcium concentration. CHO cells were grown to confluence. They were washed with calcium-free media. The cells were then treated with edema toxin (1 nM EF, along with 12 nM PA [•]) in medium containing known concentration of calcium. Control cells were not treated with toxin (○) but were incubated with medium containing known concentration of calcium. Intracellular cAMP concentrations were determined after 2 h of toxin treatment. The total protein content of the cells was 7 μg per well. All values are mean ± the SD of three different experiments done in triplicate.
45Ca2+ uptake and cAMP accumulation in toxin-treated CHO cells in the absence or the presence of antagonists. (A) The CHO cells were treated with 11 nM wild-type EF plus 12 nM wild-type PA (solid symbols) or with 11 nM wild-type EF plus 12 nM translocation-defective PA (open symbols) in calcium-containing medium. At the indicated times, the cAMP accumulation (solid line) and45Ca2+ influx (dotted line) were determined, in parallel experiments.45Ca2+ uptake data are presented as the percent uptake of45Ca2+ in the treated cells relative to that observed in the control cells (that were not treated with the toxin).45Ca2+ uptake in the control cells was 4,250 ± 45 cpm per 10 min per well of CHO cells. cAMP levels in the control cells did not vary with time and was equal to 40 ± 3 pmol/mg of CHO cell protein. The total protein content of the cells was 7 μg per well. The data are mean ± the SD of three different experiments done in triplicates. (B) The CHO cells were treated with a 100 μM concentration of antagonist before additiona of the wild-type edema toxin to the cells.45Ca2+ influx was determined over a period of 40 min for cells treated with toxin in absence of any inhibitor (•) or in the presence of EGTA (○), LaCl3 (▾), nifedipine (▿), verapamil (▪), flunarizine (□), dantrolene (♦), or diltiazem (⋄). In a parallel experiment, cAMP accumulation was determined after 40 min in these cells (data are presented as a bar diagram). The values presented here are the means ± the SD of data collected from three different experiments performed in triplicate.
45Ca2+ uptake and cAMP accumulation in edema toxin-treated neutrophils. (A) Neutrophils were isolated from the blood of healthy donors and were treated with edema toxin (12 nM PA, along with 1 nM EF) in calcium-containing medium.45Ca2+ uptake was measured in cells treated with edema toxin (•) and in cells treated with PA alone (○). In parallel, cAMP measurements were done. Cells that were not treated with toxin or cells treated with PA alone maintained 4 pmol of cAMP per 107 cells. The intracellular cAMP concentration in edema toxin-treated CHO cells is shown (▾). (B) Neutrophils were treated with a 100 μM concentration of the antagonist for 15 min before the addition of anthrax edema toxin.45Ca2+ uptake was measured in cells that were treated with toxin in absence of any inhibitor (•) or in the presence of nifedipine (○), diltiazem (▾), LaCl3 (▿), flunarizine (▪), or EGTA (□). cAMP measurements were done 90 min after toxin treatment (the results are presented as a bar diagram). None of the antagonist had any affect on cAMP levels of cells that were not treated with toxin. The values presented here are the means ± the SD of data collected from three different experiments done in triplicate.
45Ca2+ uptake and cAMP accumulation in edema toxin-treated RAW264.7 cells. (A) RAW264.7 cells were treated with edema toxin (12 nM PA, along with 1 nM EF) in calcium-containing medium. At the indicated times,45Ca2+ uptake was measured in toxin-treated cells (•) and also in the cells treated with PA alone (○). Cells that were not treated with toxin or those that were treated with PA alone maintained basal concentration of intracellular cAMP that did not change with time (95 ± 4 pmol/105 cells). The intracellular cAMP concentration in edema toxin-treated cells is shown (▾). (B) The cells were treated with a 100 μM concentration of antagonist for 15 min before addition of the toxin. The45Ca2+ influx was determined in cells treated with toxin in the absence of any inhibitor (•) or in the presence of EGTA (○), flunarizine (▾), nifedipine (▿), verapamil (▪), diltiazem (□), or LaCl3 (♦). After 2 h of incubation with the toxin, the intracellular cAMP level was determined in the RAW264.7 cells (the results are presented as a bar diagram). None of the antagonist had any affect on the cAMP levels of the control cells that were not treated with the toxin. The data presented here is mean ± the SD of three experiments.
45Ca2+ uptake and cAMP accumulation in toxin-treated lymphocytes. (A) Lymphocytes were isolated from the blood of healthy donors and were treated with toxin (12 nM of PA, along with 1 nM EF) in calcium-containing medium.45Ca2+ uptake was measured in edema toxin-treated cells (•) and in cells treated with PA alone (○). In parallel, cAMP measurements were done. Cells that were not treated with toxin or those that were treated with PA alone maintained 8 ±2 pmol of cAMP per 106 cells. Intracellular cAMP concentration in edema toxin-treated cells is shown (▾). (B) Lymphocytes were treated with a 100 μM concentration of antagonist for 15 min before addition of the toxin.45Ca2+ influx was measured in the toxin-treated cells and is presented as the percent increase45Ca2+ uptake over control for cells that were treated with toxin in absence of any inhibitor (•) or in the presence of EGTA (○), flunarizine (▾), nifedipine (▿), verapamil (▪), or dantrolene (□). cAMP measurements were made 90 min after toxin treatment (the results are presented as a bar diagram). None of the antagonist had any affect on cAMP levels of cells that were not treated with toxin. The values represent the mean ± the SD of data from three different experiments.
Rise in cytosolic calcium observed upon treatment of lymphocytes with edema toxin. Lymphocytes were isolated from the blood of healthy individuals and were loaded with Fura-2AM as described in Materials and Methods. We added 12 nM PA to these cells in calcium-containing medium. Changes in cytosolic calcium were assessed by measuring the variation in the fluorescence of Fura-2AM-loaded cells. We then added 1 nM EF to these cells, and the variation in fluorescence was again recorded. Tracings are representative of three individual experiments with similar results.
References
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