Figure 1.
CyaA entrains CD11b/CD18 into lipid rafts.
J774A.1 cells were mock-treated with buffer, or incubated with 1 nM CyaA at 37°C for 10 min (CyaA-treated). Cells were placed on ice and extracted at 4°C for 60 minutes in buffer containing 1% Triton X-100. Cell lyzates were fractionated by flotation in buoyant sucrose density gradients at 150,000×g in a Beckman SW60Ti rotor at 4°C for 16 h. Gradient fractions were analyzed by Western blotting. (A) Unless otherwise stated, CyaA was detected using the 9D4 monoclonal antibody (MAb) recognizing the C-terminal RTX repeats. The 3D1 MAb was used to specifically detect the distal part (aa 373–400) of the AC domain of CyaA [37]. The full-length CyaA (∼200 kDa) and the processed form of CyaA (∼160 kDa) are indicated by a crossline (×) and a square symbol (□), respectively. (B) CD11b was detected with the OKM-1 antibody on native immunoblots of Blue-native PAGE gels, while conventional Western blots were used for detection of CyaA and CD11a by 9D4 and MEM-25 antibodies, respectively. (C) Chinese hamster ovary (CHO) cells were mock-treated or incubated with 113 nM CyaA at 37°C for 10 min. (D) CHO cells stably transfected with genes encoding the human CD11b/CD18 integrin subunits (CHO-CD11b/CD18) were incubated with 1 nM CyaA at 37°C for 10 min. CyaA and CD18 were detected with 9D4 and MEM-48, respectively. Lanes 1–8 correspond to gradient fractions. Representative immunoblots from at least 3 independent experiments are shown. (E) Distribution of CyaA (filled bars) and CD11b (open bars) across sucrose density gradient. The values for relative amounts of CyaA and CD11b, detected in a given fraction of the gradient and expressed as % of total detected protein, were derived from densitometric analysis of immunoblots. (F) Relative abundance of the processed form of CyaA (160 kDa) was expressed as % of total CyaA detected within a given fraction of the gradient. Values represent the mean ± S.D. of three independent experiments. N.D., not determined.
Figure 2.
Mobilization of CyaA into lipid rafts depends on CyaA-mediated Ca2+ influx.
(A) J774A.1 cells were incubated with 1 nM CyaA-derived proteins at 37°C for 10 min, cell lyzates were fractionated on sucrose gradients and proteins were detected by immunoblots as described for Fig. 1. (B) J774A.1 cells were incubated at 37°C for 10 min with 6 nM CyaA proteins labeled with Alexa Fluor 488 before 5 µg/ml of Alexa Fluor 594-labeled recombinant cholera toxin subunit B (CtxB) was added for additional 5 min. The cell-bound proteins were visualized by fluorescence microscopy and co-localization of CyaA (green) with CtxB (red) was assessed in the merged images (yellow). Representative images from two independent experiments are shown. Note that intact CyaA induced the reported cAMP-dependent ruffling of J774A.1 cells [2]. (C and D) CyaA induces increase of cytosolic calcium concentration ([Ca2+]i). J774A.1 cells were loaded with the Ca2+ probe Fura-2/AM (3 µM) at 25°C for 30 min and exposed to 17 nM (C) or 113 nM (D) CyaA proteins. Time course of Ca2+ entry was recorded as the ratio of fluorescence intensities (excitation at 340/380 nm, emmision 505 nm), as previously described [18]. The shown curves are representative of at least three independent experiments.
Table 1.
Relative toxin activities of CyaA-derived constructs.
Figure 3.
Binding of 3D1 antibody uncouples AC translocation from membrane insertion and CyaA-mediated Ca2+ influx.
(A) 17 nM CyaA was preincubated for 20 min at 37°C with 20 µg/ml of 3D1 MAb (CyaA+3D1) or the Tu-01 IgG1 isotype control MAb (CyaA+isotype control), before 6 nM toxin was added to J774A.1 cells. CyaA binding was determined as the amount of total cell-associated AC enzyme activity upon cells incubation with 6 nM CyaA for 10 min at 37°C in the presence or absence of the indicated antibody. AC domain translocation was assessed by determining the intracellular concentration of cAMP generated in cells in the presence or absence of the indicated antibodies, following incubation of cells with four different toxin concentrations from within the linear range of the dose-response curve (0.05, 0.1, 0.25, and 0.5 nM). The % of cAMP accumulation in cells at each toxin concentration was calculated, taking cAMP values for CyaA preincubated in buffer alone as 100%. The average of such determined % activity values is given. An asterisk indicates a statistically significant difference (*, p<0.01; Student's t test). (B) J774A.1 cells were exposed to 1 nM CyaA alone, or to 1 nM CyaA preincubated with 3D1 or IgG1 isotype MAb as above. Cell lyzates were separated on sucrose density gradients and analyzed as in Fig. 1. The 3D1 and isotype IgG1 MAbs were detected with anti-mouse IgG antibody. The relative amounts of processed CyaA (∼160 kDa) within individual gradient fractions were determined as in Fig. 1F. Values represent the mean ± S.D. of three independent experiments. An asterisk indicates a statistically significant difference (*, p<0.05; Student's t test). N.D., not determined. (C) J774A.1 cells were loaded with Fura-2/AM as above and exposed to 17 nM CyaA alone, or to CyaA preincubated with 3D1 or an IgG1 isotype control MAb. Ca2+ influx was recorded as above and the shown curves are representative of three independent experiments.
Figure 4.
Mobilization of CyaA into lipid rafts depends on influx of extracellular Ca2+ ions into cells.
(A) J774A.1 cells were incubated at 37°C for 10 min with 1 nM CyaA and CyaA-E570K+E581P, in the presence or absence of 500 nM ionomycin , or of 1 µM thapsigargin, respectively. Cell lyzates were analyzed on sucrose density gradients as above. The blots are representative of four independent experiments. (B) Distribution of CyaA proteins in gradient fractions was quantified as in Fig. 1 and the values represent the mean ± S.D. of three independent experiments. An asterisk indicates a statistically significant difference (*, p<0.05; Student's t test).
Figure 5.
Mobilization of CyaA into lipid rafts depends on talin cleavage by calpain.
(A) J774A.1 cells were incubated with 30 nM CyaA proteins at 37°C for 10 min (left panel). Talin, CyaA and α-tubulin were detected in cell lyzates by Western blotting using 8d4, 9D4 and Tu-01 MAbs, respectively. Calpain activation was inhibited by preincubation of cells with 100 µM calpeptin at 37°C for 30 min (right panel). Talin processing was determined from Western blots as relative increase of the amount of the 220-kDa talin fragment, taking the value for untreated cells as 1. Data represent the mean ± S.D. from three experiments. (B) Cells were kept in buffer or pretreated with 100 µM calpeptin at 37°C for 30 min before incubation with 1 nM CyaA at 37°C for 10 min. Talin, talin head and CyaA were detected in cell lyzates separated on sucrose density gradients using 8d4, TA-205 and 9D4 MAbs, respectively. The shown blots are representative of four independent experiments. (C) CyaA-mediated talin cleavage promotes CD11b/CD18 release from cytoskeletal constraints. J774A.1 cells were incubated with 30 nM CyaA for 30 min at 37°C. Cell lyzates were prepared and incubated with anti-CD11b monoclonal antibody (MEM-174) covalently coupled to CNBr-activated Sepharose beads. The beads were washed, bound proteins were eluted with SDS-PAGE loading buffer, and talin and talin head were detected by Western blotting with 8d4 and TA-205 antibody, respectively. (D) DRM association of CyaA in J774A.1 cells kept in buffer or preincubated with 100 µM calpeptin was analyzed as above. (E) AC translocation into control and calpeptin-pretreated J774A.1 cells were determined as described in the legend to Fig. 3. (F) CyaA-mediated influx of Ca2+ into untreated and calpeptin-pretreated J774A.1 cells was measured as in Fig. 2. Values represent the mean ± S.D. of three independent experiments. An asterisk indicates a statistically significant difference (*, p<0.05; Student's t test). (G) J774A.1 cells grown on coverslips were kept in buffer or pretreated with 100 µM calpeptin at 37°C for 30 min before membrane distribution of fluorescently labeled CyaA-AC− (6 nM, green) and CtxB (5 µg/ml, red) was visualized as described in the legend to Fig. 2.
Figure 6.
Cholesterol depletion inhibits translocation of AC domain across target cytoplasmic membrane.
(A) J774A.1 cells were kept in buffer or treated with 10 mM MβCD at 37°C for 30 min, before CyaA was added for additional 10 min. CyaA binding and AC domain translocation were determined as described in the legend to Fig. 3. Values represent the mean ± S.D. from four independent experiments performed in triplicates. An asterisk indicates a statistically significant difference (*, p<0.01; Student's t test). (B) J774A.1 cells were kept in buffer or pretreated with 10 mM MβCD at 37°C for 30 min and incubated with 1 nM CyaA for 10 min. Cell lyzates were analyzed as in Fig. 1A. (C) J774A.1 cells were loaded with the Ca2+ probe Fura-2/AM prior to cholesterol extraction with 10 mM MβCD at 30°C for 30 min. CyaA-mediated Ca2+ influx was recorded as above. Standard deviations were calculated for mean values at indicated time points and the shown curves are representative of at least three independent experiments. (D) J774A.1 cells grown on coverslips were kept in buffer or pretreated with 10 mM MβCD at 37°C for 30 min. Membrane distribution of fluorescently labeled CyaA-AC− (6 nM, green) and CtxB (5 µg/ml, red) was visualized as described in the legend to Fig. 2. (E) U937 cells were grown for 48 hours in RPMI medium supplemented with 10% fetal calf serum (standard serum) or with 10% lipoprotein-deficient serum from fetal calf (delipidated serum). CyaA binding and AC domain translocation were determined as described in the legend to Fig. 3. Values represent the mean ± S.D. from four independent experiments performed in triplicates. An asterisk indicates a statistically significant difference (*, p<0.01; Student's t test). (F) U937 cells grown in standard or delipidated serum were incubated with 1 nM CyaA at 37°C for 10 min, cell lyzates were separated on sucrose density gradients and analyzed as in Fig. 1. (G) U937 cells grown in standard or delipidated serum were loaded with the Ca2+ probe Fura-2/AM (3 µM) at 25°C for 30 min, exposed to CyaA (17 nM) and the time course of Ca2+ entry was recorded as above. Standard deviations were calculated for mean values at indicated time points and the shown curves are representative of at least three independent experiments. (H) U937 cells cultured with standard or delipidated serum were mounted on polylysin-coated coverslips and membrane distribution of fluorescently labeled CyaA-AC− (6 nM, green) and CtxB (5 µg/ml, red) was visualized as described above.
Table 2.
Cholesterol content in J774 and U937 cells.
Figure 7.
AC domain of CyaA translocates across cellular membrane from lipid rafts.
(A) J774A.1 cells were incubated at 37°C for 10 min with 1 nM individual CyaA proteins (upper panel) or with their 1∶1 mixtures (lower panel). CyaA and biotinylated CyaA-E570K+E581P (CyaA-E570K+E581P-biotin) were detected in gradient fractions using 9D4 and streptavidine, respectively. (B) J774A.1 cells were incubated at 37°C for 10 min with the indicated pairs of proteins (12 nM) mixed in a 1∶1 molar ratio. Binding of CyaA-E570K+E581P-biotin was determined as the amount of total cell-associated AC enzyme activity upon exposure of cells to the protein mixtures and expressed as relative value. Enhancement of the residual capacity of CyaA-E570K+E581P to translocate AC domain into J774A.1 cell cytosol (1.8±0.5% of intact CyaA activity) was measured by determination of intracellular cAMP amounts accumulated in 106 cells upon exposure for 10 min at 37°C to the indicated 1∶1 protein mixtures (12 nM). The values represent the mean ± S.D. from four independent experiments performed in duplicate. An asterisk indicates a statistically significant difference (*, p<0.01; Student's t test).
Figure 8.
Model of CyaA translocation across target cell membrane.
In the first step, CyaA binds the CD11b/CD18 integrin receptor dispersed in the bulk of the membrane phase outside of lipid rafts, having the cytoplasmic tail of the CD18 subunit tethered to actin cytoskeleton via the linker protein talin. Upon receptor engagement, a ‘translocation intermediate’ of CyaA inserts into the lipid bilayer of cell membrane with the AC domain partially penetrating into cell membrane together with the pore-forming segments of the toxin and participating in formation of a transiently opened Ca2+-conducting path across cell membrane. Influx of external calcium ions into cells induces activation of the Ca2+-dependent protease calpain, yielding talin cleavage and liberation of the CyaA-CD11b/CD18 complex from binding to actin cytoskeleton. Consequently, CyaA is recruited with CD11b/CD18 into cholesterol-enriched lipid rafts, where the liquid-ordered packing of lipids and the specific presence of cholesterol enable completion of AC domain translocation across cytoplasmic membrane. Upon exposure at the cytosolic side of cell membrane, the AC domain is cleaved-off from the RTX cytolysin moiety of CyaA by a protease residing inside the cell. Binding of cytosolic calmodulin (CaM) then activates the AC enzyme and unregulated conversion of ATP to cAMP is catalyzed.