Figure 1.
dCyaA-KP toxoid unable to promote calcium influx and relocate into lipid rafts is rapidly taken up by J774A.1 cells.
(A) J774A.1 cells were loaded with the calcium probe Fura-2/AM at a 3 µM final concentration at 25°C for 30 min. After washing in HBSS medium, toxoid variants (3 µg/ml) or buffer were added at time zero (indicated by arrow) and time course of calcium entry into cytosol of cells (elevation of [Ca2+]i) was followed by spectrofluorimetry [14]. (B) J774A.1 cells were incubated for 10 minutes with 500 ng/ml of dCyaA or dCyaA-KP and detergent-resistant membrane microdomains (DRMs) were extracted with cold Triton X-100, separated by flotation in sucrose density gradient and probed in Western blots with the 9D4 antibody. The DRM fractions were defined as fractions enriched in the lipid raft marker NTAL. The transferrin receptor CD71 was used as a non-raft marker that remained in the bottom fractions of the gradient. (C) J774A.1 cells grown on glass bottom microwell dishes were incubated for 20 minutes at 37°C with 5 µg/ml of Dyomics 647-labeled dCyaA or Alexa Fluor 488-labeled dCyaA-KP proteins in the presence or absence of the anti-CD11b MAb M1/70 (20 µg/ml). Endocytic uptake of toxoids was analyzed at indicated time points by live cell imaging using an Olympus CellR IX 81 microscope with a 60×/1.35 oil objective (UPlanSApo). (D) The numbers of endosomes localized in cytoplasm of cells and loaded with dCyaA or dCyaA-KP were counted as described in detail in the legend to Supplementary figure S2, using a script based on WCIF ImageJ software (http://rsb.info.nih.gov/ij, http://www.uhnresearch.ca/facilities/wcif/imagej). The plot shows mean values plus standard deviations, as calculated on 20 to 40 cells per time point for one representative experiment out of three performed (n = 3). (E) J774A.1 cells (3×105) were incubated for 30 min on ice at three different toxoid concentrations within the linear range of the dose-response curve (0.5, 1 and 5 µg/ml) and in the presence or absence of the anti-CD11b monoclonal antibody M1/70 (20 µg/ml, 15 min of preincubation of cells). Binding to cells was assessed as cell-associated toxoid fluorescence by FACS analysis. The % of toxoid binding at each concentration was calculated, taking the value for dCyaA toxoid in the absence of M1/70 as 100%. The average ± standard deviations are shown.
Figure 2.
Intact dCyaA toxoid is endocytosed by a clathrin-dependent mechanism.
(A) J774A.1 cells were incubated for 20, 40 and 60 minutes with 1 µg/ml of Alexa Fluor 488-labeled dCyaA or dCyaA-KP. For simultaneous visualization of early and recycling endocytic compartments, cells were co-incubated at 37°C for the last 20 minutes with Dyomics 547-labeled transferrin (10 µg/ml). Cells were washed in cold PBS, fixed with 4% PFA, and observed using a Leica confocal microscope TCS SP2 with a 63×/1.40 oil objective (HCX PL APO). (B) Co-localization of toxoids with transferrin was quantified using ImageJ software as described in details in Materials and Methods. (C) Murine RAW 264.7 macrophages transfected by dominant-negative mutant (DIII) of Eps-15 protein fused to GFP (blue) were incubated at 37°C for 30 min with the dCyaA or dCyaA-KP toxoids (1 µg/ml) labeled with Dyomics-647 (green). Dyomics 547-labeled transferrin (10 µg/ml) was added for the last 10 minutes of incubation (red), cells were washed with 0.1 M sodium acetate, 150 mM NaCl, pH 3.5 to remove surface-associated transferrin, fixed and observed with Olympus CellR microscope using a 100× oil immersion objective (N.A. 1.3). The calculated values of Pearson's correlation coefficients for transferrin (Dyomics 547) and toxoid (Dyomics 647) in non-transfected cells were 0.319 and 0.067 for dCyaA and dCyaA-KP, respectively.
Figure 3.
Inactive dCyaA-KP toxoid is endocytosed by a rapid macropinocytic pathway.
(A) J774A.1 macrophages were incubated with BSA-Dyomics 547 (50 µg/ml) together with Alexa Fluor 488-labeled dCyaA or dCyaA-KP (1 µg/ml), respectively. After 10 and 30 minutes, the cells were washed, fixed and observed as above. (B) Co-localization of toxoids with BSA was quantified using ImageJ software as described in details in Materials and Methods. (C) J774A.1 cells were incubated for 30 minutes in HBSS with wortmannin (1 µg/ml), dynasore (40 µM) or chlorpromazine (5 µg/ml), before 5 µg/ml of Alexa Fluor 488-labeled dCyaA-KP was added and time course of toxoid uptake was followed by live cell imaging and (D) quantified as above (n = 3).
Figure 4.
Both toxoids end up in MHC II-positive organelles.
Transfected RAW 264.7 macrophages expressing Rab-7-EGFP were incubated for 60 minutes with Dyomics 647-labeled toxoids (1 µg/ml), washed, fixed and analyzed by live cell imaging. (B) Co-localization of toxoids with Rab-7-EGFP was quantified using ImageJ software. (C) Cells from MHC Class II/EGFP knock-in mice (green) were incubated at 37°C in DMEM without serum for 2 hours with 1 µg/ml of Dyomics 647-labeled (red) dCyaA or dCyaA-KP. Cells were fixed in 4% PFA and mounted in Mowiol. Images were captured using an inverted Olympus CellR microscope equipped with a 60×/1.35 oil immersion objective (UPlanSApo). (D) Co-localization of toxoids with MHC II/EGFP was quantified using ImageJ software.
Figure 5.
Macropinocytosed dCyaA-KP toxoid is rapidly degraded and its capacity to deliver epitopes for presentation on MHC class II molecules is compromised.
(A) 106 J774A.1 cells were preincubated for 30 minutes at 37°C in 1 ml of D-MEM medium alone, or in D-MEM containing 1 mM chloroquine plus a cocktail of protease inhibitors (Complete Mini, Roche), or in D-MEM containing 10 mM 2DG and 0.01% of sodium azide, respectively. dCyaA or dCyaA-KP toxoids were added to a final concentration of 1 µg/ml and after continued incubation the cells were washed three times in ice-cold D-MEM at the indicated times and lyzed on ice during 30 minutes in TBS buffer containing 1% Triton X-100 and the protease inhibitor cocktail (Complete Mini, Roche). Cell nuclei were removed by centrifugation at 13,000 RPM for 10 min and supernatants of lyzed cells were separated by 7.5% SDS-PAGE. CyaA fragments were detected in Western blots using the 9D4 antibody recognizing the C-terminal RTX repeats of CyaA. The arrow indicates migration of full-length (undegraded) 200 kDa CyaA. (B) BMDC from C57BL/6 mice were pulsed with indicated concentrations of dCyaA or dCyaA-KP, or (C) with dCyaA-OVA258–276 or dCyaA-OVA258–276-KP and following medium disposal, the MalE-specific hybridoma CRMC3 (B) or OVA258–276-specific hybridoma MF2.2D9 cells (C) were added, respectively. Secretion of IL-2 by antigen-stimulated CRMC3 hybridoma (B) was determined as proliferation of the IL-2-dependent CTL-L cell line and the results are expressed in cpm ± SE of duplicate samples. The concentration of IL-2 produced by MF2.2D9 cells upon antigenic stimulation (C) was determined by sandwich ELISA. A representative Western blot from 3 independent experiments and the averaged values from two independent antigen presentation experiments performed in duplicates (n = 4), are shown.
Figure 6.
Calcium influx into J774A.1 macrophages decelerates endocytic uptake of the dCyaA-KP toxoid.
(A) J774A.1 cells were incubated for 20 minutes at 37°C with 5 µg/ml of Dyomics 647-labeled dCyaA-KP alone, or in the presence of ionomycin (5 µM and 10 µM), or as a 1∶1 mixture with Alexa 488-labeled dCyaA. (B) Uptake of toxoids into cells was analyzed by live cell imaging as above and (C) P.c.c. values for the mixed sample of dCyaA and dCyaA-KP were calculated. (D) J774A.1 cells were incubated for 10 minutes with 500 ng/ml of dCyaA, dCyaA-KP-biotin or with 1∶1 mixture of dCyaA and dCyaA-KP-biotin toxoids. Detergent-resistant membrane microdomains (DRMs) were extracted, separated and probed as in Figure 1B. Biotin-labeled dCyaA-KP was detected with streptavidin HRP conjugate (GE Healthcare, Chalfont St. Giles, UK). (E) Transfected RAW 264.7 macrophages expressing Rab-7-EGFP were incubated for 30 and 60 minutes with 1 µg/ml of Dyomics 547 (dCyaA) and Dyomics 647-labeled toxoids (dCyaA-KP), washed, fixed and analyzed fluorescence imaging and (F) P.c.c. values were determined as in Figure 4.
Figure 7.
Rapid removal from the plasma membrane reduces toxoid-mediated K+ efflux from monocytes.
(A) PBFI/AM loaded J774A.1 monocytes in HBSS buffer were exposed to 3 µg/ml of enzymatically inactive CyaA-AC− toxoid, or to its CyaA-ΔAC variant, and K+ efflux at 25°C was monitored in time as described under Materials and Methods. (B) PBFI/AM loaded J774A.1 macrophages were preincubated for 15 minutes in HBSS buffer containing 0.01% sodium azide and 10 mM 2DG prior to addition of CyaA-AC− or CyaA-ΔAC proteins and time course of K+ efflux was recorded. The shown curves are representative of six independent experiments. (C) 106 J774A.1 cells were incubated for 15 minutes in D-MEM or in D-MEM containing the glycolysis inhibitor 2-deoxy-D-glucose (2DG, 10 mM) instead of glucose and 0,01% sodium azide. ATP content in cells was determined using the ATP Bioluminescence Assay Kit CLS II (Roche). (D) Prior to addition of the CyaA-ΔAC protein, 2×105 J774A.1 macrophages were preincubated for 15 minutes at 37°C in D-MEM alone or in D-MEM medium without glucose and containing 0.01% sodium azide plus 10 mM 2DG. The extent of cell lysis was determined using the Cytotox 96 assay kit (Promega) as the amount of lactate dehydrogenase released into culture media in 2 hours. Average values from three independent experiments performed in duplicates are shown. (E) CyaA-ΔAC, in contrast to CyaA-AC− is rapidly internalized into J774A.1 cells. J774A.1 cells were preincubated for 15 minutes in HBSS, or in glucose-free HBSS with 2DG and sodium azide, before 5 µg/ml of Fluor 488-labeled CyaA-ΔAC or CyaA-AC− were added. Toxoid endocytosis was analyzed by live cell imaging and (F) Numbers of endosomes containing CyaA-ΔAC and CyaA-AC− were determined as above. (G) J774A.1 cells were preincubated for 15 minutes in HBSS or in HBSS containing sodium azide and 2DG prior to addition of CyaA-ΔAC (500 ng/ml). Detergent-resistant membrane microdomains (DRMs) were extracted, separated and probed as in Figure 1B.
Figure 8.
Influx of Ca2+ decelerates macropinocytic uptake of CyaA-ΔAC and enables cell permeabilization by hemolysin pores.
(A) J774A.1 cells were loaded with Fura-2/AM and exposed to 3 µg/ml of CyaA-ΔAC (arrow) preincubated for 15 minutes at room temperature with 3D1, or with the isotype control antibody TU-01 (20 µg/ml) and Ca2+ influx was recorded. The shown curves are representative of three independent experiments. (B) J774A.1 cells were exposed for 10 minutes at 37°C to CyaA protein variants (500 ng/ml) preincubated with 3D1 or TU-01 (20 µg/ml) and cell lyzates were separated on sucrose density gradients as in Figure 1B. The 3D1 and isotype IgG1 mAbs were detected with anti-mouse IgG antibody. (C) 3 µg/ml of CyaA-ΔAC or of CyaA (D) were preincubated with 3D1 or TU-01 (20 µg/ml), added to PBFI/AM loaded J774A.1 macrophages and time course of K+ efflux from J774A.1 was recorded. The curves are representative of four independent experiments. (E) J774A.1 cells were exposed to 5 µg/ml of Alexa 488-labeled CyaA-ΔAC preincubated with 3D1 or TU-01 mAb (20 µg/ml). The time course of CyaA-ΔAC endocytosis was followed by live cell imaging and (F) quantified.
Figure 9.
Potassium efflux delays endocytosis of dCyaA toxoid.
A) J774A.1 cells were incubated for 20 minutes at 37°C with 5 µg/ml of Alexa 488-labeled dCyaA in HBSS containing 5 mM KCl or in HBSS containing 50 mM KCl. Uptake of dCyaA into cells was analyzed by live cell imaging and (B) numbers of endosomes in cell cytoplasm were quantified (n = 3).
Figure 10.
Endocytosis of CyaA is not affected by cAMP.
(A) The capacity to translocate the AC domain across cytoplasmic membrane was assessed as the capacity to elevate cytosolic concentrations of cAMP. J774A.1 cells were incubated with CyaA or CyaA-KP-Alexa 488 for 30 minutes at 37°C and the amounts of accumulated cAMP were determined in cell lyzates. The shown curves are representative of two independent experiments performed in duplicates. (B) J774A.1 cells were incubated for 20 minutes at 37°C with 5 µg/ml of Alexa 488-labeled dCyaA-KP or CyaA-KP constructs, the protein uptake was analyzed by live cell imaging and (C) the numbers of endosomes in cytoplasm were quantified as above.
Figure 11.
Ca2+ influx and K+ efflux promoted in trans decelerate the endocytic uptake of enzymatically active CyaA.
J774A.1 macrophages (3×106/ml) in D-MEM medium were incubated for 5 minutes at 37°C with 200 ng/ml of rEc-CyaA-biotin, washed in D-MEM and further incubated at 4°C or 37°C, respectively, in the presence or absence of 1 µg/ml of unlabeled CyaA-AC−. At indicated time points cell-surface localized rEc-CyaA-biotin per 3×105 cells was detected with PE-conjugated Streptavidine (0.5 mg/ml) by FACS analysis (n = 3).
Figure 12.
Endocytic removal from cell membrane controls the cytolytic potency of CyaA toxin.
(A) 2×105 J774A.1 macrophages were preincubated for 15 minutes in D-MEM medium alone or in D-MEM without glucose containing 0.01% sodium azide and 10 mM 2DG prior to addition of Bp-CyaA purified from B. pertussis 18323/pHSP9. The extent of cell lysis was determined as the amount of lactate dehydrogenase released into culture media in 2 hours, using the Cytotox 96 assay kit (Promega). The results represent the average of values obtained in three independent experiments performed in duplicates. (B) PBFI/AM-loaded J774A.1 macrophages were preincubated for 15 minutes in HBSS alone, or in HBSS buffer without glucose but containing 0.01% sodium azide and 10 mM 2DG. K+ efflux was monitored upon addition of 300 ng/ml of Bp-CyaA and the shown curves are representative of three independent experiments.
Figure 13.
Model of the calcium influx-triggered positive feedback mechanism of self-exacerbating potassium efflux due to protracted cell permeabilization by toxin pores.
Upon binding to the αMβ2 integrin (CD11b/CD18), the pore precursor conformers of dCyaA oligomerize within the bulk phase of target cell membrane and permeabilize cells for efflux of cytosolic potassium ions. In parallel, toxin translocation precursors insert into cellular membrane and conduct extracellular Ca2+ into cell cytosol. Elevation of [Ca2+]i in the submembrane compartment results in activation of talin cleavage by calpain and liberates the receptor with bound toxin translocation precursor within target membrane for lateral relocation into lipid rafts, where the translocation of the AC domain across plasma membrane is completed [15]. The entry of Ca2+ provokes deceleration of clathrin-dependent endocytic uptake of CyaA, thereby delaying removal of toxin pores from the cytoplasmic membrane. Protracted persistence of CyaA pores within cellular membrane then triggers a self-amplifying (positive) feedback loop mechanism that exacerbates cell permeabilization. The more potassium leaks out form cells, the more the clathrin-dependent endocytic removal of toxin pores from cellular membrane is decelerated and the more is the cell permeabilization and potassium leakage exacerbated. In contrast, the mutated dCyaA-KP and the CyaAΔAC hemolysin variants of CyaA are unable to conduct calcium ions into cells and are thus rapidly removed from the cytosolic membrane of cells by a membrane macropinocytosis mechanism that directs the mutant toxoids for rapid proteolytic degradation.