Role of CD11b/CD18 in the process of intoxication by the adenylate cyclase toxin of Bordetella pertussis

Abstract

The adenylate cyclase toxin (ACT) of Bordetella pertussis does not require a receptor to generate intracellular cyclic AMP (cAMP) in a broad range of cell types. To intoxicate cells, ACT binds to the cell surface, translocates its catalytic domain across the cell membrane, and converts intracellular ATP to cAMP. In cells that express the integrin CD11b/CD18 (CR3), ACT is more potent than in CR3-negative cells. We find, however, that the maximum levels of cAMP accumulation inside CR3-positive and -negative cells are comparable. To better understand how CR3 affects the generation of cAMP, we used Chinese hamster ovary and K562 cells transfected to express CR3 and examined the steps in intoxication in the presence and absence of the integrin. The binding of ACT to cells is greater in CR3-expressing cells at all concentrations of ACT, and translocation of the catalytic domain is enhanced by CR3 expression, with ∼80% of ACT molecules translocating their catalytic domain in CR3-positive cells but only 25% in CR3-negative cells. Once in the cytosol, the unregulated catalytic domain converts ATP to cAMP, and at ACT concentrations >1,000 ng/ml, the intracellular ATP concentration is <5% of that in untreated cells, regardless of CR3 expression. This depletion of ATP prevents further production of cAMP, despite the CR3-mediated enhancement of binding and translocation. In addition to characterizing the effects of CR3 on the actions of ACT, these data show that ATP consumption is yet another concentration-dependent activity of ACT that must be considered when studying how ACT affects target cells.

Fig 1

ACT-induced intracellular cAMP accumulation in cells with and without CR3. Each figure shows pmoles cAMP/mg of target cell protein as a function of toxin concentration. ACT was added to cells for 30 min at 37°C. (A) Accumulation of intracellular cAMP in J774 macrophage-like cells which express CR3 and in BEAS-2B epithelial cells which do not express CR3. Data presented are the means ± standard errors of duplicate samples in a single experiment which is representative of 3 individual experiments. (B) cAMP accumulation in CHO cells. Data presented are the means ± standard errors of 5 experiments each performed in triplicate. (C) cAMP accumulation in K562 cells. Data presented are the means ± standard errors of 6 experiments each performed in duplicate.

Fig 2

Competition with inactive ACT (iACT) decreases WT ACT-elicited cAMP accumulation in CHO CR3⁺ and K562 CR3⁺ cells but not in the respective control cells. (A) CHO CR3⁺ cells were incubated with WT ACT at the specified concentrations in the continuous presence of 5,000 ng/ml (30 nM) iACT. (B) Same as described for panel A but with CHO control cells. (C) K562 CR3⁺ (squares) and K562 control (circles) cells were incubated with WT ACT at the specified concentrations either without (closed) or with (open) 5,000 ng/ml iACT. (D) K562 CR3⁺ (gray bars) and K562 (black bars) cells were incubated with 100 ng/ml WT ACT in the continuous presence of 5,000 ng/ml iACT. To examine blocking of cAMP generation by a MAb to CD11b, 100 ng/ml of ACT was added to cells after 1 h incubation at 4°C with 20 μg/ml MAb M1/70 to CD11b or 20 μg/ml isotype control antibody. Data presented are the means ± standard errors of duplicate samples in a single experiment which is representative of 3 (CHO) or 2 (K562) individual experiments.

Fig 3

Association of ACT with cells is greater at all concentrations in the presence of CR3. (A) ACT was added to cells, unbound ACT was washed away, and biotin-conjugated polyclonal antibody to ACT, followed by APC-conjugated streptavidin, was added to detect surface-associated ACT. Results of flow cytometry measurements are expressed as difference in mean fluorescence intensity between cells not treated with toxin and those treated with toxin (ΔMFI). Data presented are the means ± standard errors of 3 separate experiments. (B, C) Total cell-associated ACT was measured by AC enzyme activity. Background native adenylyl cyclase activity, which is minimal, was subtracted from experimental conditions. Data presented are the means ± standard errors of 3 (CHO) or 2 (K562) experiments each performed in duplicate. (D) Surface expression of CD11b on CHO cells. CHO mock-transfected control cells (gray) and CHO CR3⁺ cells (black) were stained with phycoerythrin-conjugated MAb to CD11b, clone ICRF44. Ten thousand cells were counted for each cell type. (E) Surface expression of CD11b on K562 cells. Conditions were the same as described for panel A but with K562 control cells (gray) and K562 CR3⁺ cells (black). Ten thousand cells were counted for each cell type.

Fig 4

CR3 expression enhances translocation of the catalytic domain of ACT. (A) Percent translocation of the catalytic domain was calculated by adding ACT to CHO cells at 37°C, washing away unbound ACT, and treating half of the cells with trypsin. AC enzymatic activity was used to measure the amount of AC catalytic domain protected from trypsin (translocated to the cytosol) or the total amount of AC catalytic domain bound to nontrypsinized cells. Percent translocation was calculated as [AC enzyme in trypsinized sample/AC enzyme in nontrypsinized samples] × 100. (B) Percent translocation in K562 cells with or without CR3 was calculated. Data presented are the means ± standard errors of 4 (CHO) or 2 (K562) experiments each performed in duplicate. Translocation percentages for CR3⁺ and CR3⁻ cells were compared at each concentration, and an asterisk indicates a P value of <0.05 by Student's two-tailed t test. For concentrations not marked by an asterisk, the P value is >0.05.

Fig 5

Cellular ATP depletion by ACT with and without CR3. ACT was added to cells at the indicated concentrations, and intracellular cAMP and ATP were measured on the same samples simultaneously after 30 min at 37°C. (A) Percentage of intracellular ATP was calculated for each sample with the formula (ATP in cells treated with ACT/baseline ATP in cells not treated with ACT) × 100. (B) Accumulation of intracellular cAMP in CHO cells measured in parallel with ATP. (C) ATP depletion as a function of cAMP generation, each measured in parallel. Data presented are the means ± standard errors of 3 experiments each performed in triplicate.

Fig 6

Comparison of ATP depletion, cAMP accumulation, and ACT-cell association in J774 cells. ACT was added to cells at the indicated concentrations, and intracellular cAMP, intracellular ATP, and cell-associated AC enzymatic activity were measured on the same samples simultaneously after 30 min at 37°C. (A) Intracellular ATP as a function of toxin concentration in J774 cells. Percentage of intracellular ATP was calculated for each sample with the formula (ATP in cells treated with ACT/baseline ATP in cells not treated with ACT) × 100. (B) Prior to addition of ACT, J774 cells were either untreated or treated with DNP/DOG which depleted ATP to 15% ± 0.4% of that in untreated cells. Subsequently, 30 ng/ml of ACT was added to cells for 30 min at 37°C and intracellular cAMP was measured. (C) Intracellular cAMP in J774 cells treated with the indicated concentrations of ACT. (D) Cell-associated ACT measured by AC enzymatic assay. (E) cAMP accumulation as a function of cell-associated ACT representing the amount of cAMP generated per unit of cell-associated ACT. Data presented are the means ± standard errors of 3 experiments each performed in triplicate.