Calcium Influx Rescues Adenylate Cyclase-Hemolysin from Rapid Cell Membrane Removal and Enables Phagocyte Permeabilization by Toxin Pores

Bordetella adenylate cyclase toxin-hemolysin (CyaA) penetrates the cytoplasmic membrane of phagocytes and employs two distinct conformers to exert its multiple activities. One conformer forms cation-selective pores that permeabilize phagocyte membrane for efflux of cytosolic potassium. The other conformer conducts extracellular calcium ions across cytoplasmic membrane of cells, relocates into lipid rafts, translocates the adenylate cyclase enzyme (AC) domain into cells and converts cytosolic ATP to cAMP. We show that the calcium-conducting activity of CyaA controls the path and kinetics of endocytic removal of toxin pores from phagocyte membrane. The enzymatically inactive but calcium-conducting CyaA-AC− toxoid was endocytosed via a clathrin-dependent pathway. In contrast, a doubly mutated (E570K+E581P) toxoid, unable to conduct Ca2+ into cells, was rapidly internalized by membrane macropinocytosis, unless rescued by Ca2+ influx promoted in trans by ionomycin or intact toxoid. Moreover, a fully pore-forming CyaA-ΔAC hemolysin failed to permeabilize phagocytes, unless endocytic removal of its pores from cell membrane was decelerated through Ca2+ influx promoted by molecules locked in a Ca2+-conducting conformation by the 3D1 antibody. Inhibition of endocytosis also enabled the native B. pertussis-produced CyaA to induce lysis of J774A.1 macrophages at concentrations starting from 100 ng/ml. Hence, by mediating calcium influx into cells, the translocating conformer of CyaA controls the removal of bystander toxin pores from phagocyte membrane. This triggers a positive feedback loop of exacerbated cell permeabilization, where the efflux of cellular potassium yields further decreased toxin pore removal from cell membrane and this further enhances cell permeabilization and potassium efflux

Membrane Restructuring by Bordetella pertussis Adenylate Cyclase Toxin, a Member of the RTX Toxin Family

Adenylate cyclase toxin (ACT) is secreted by Bordetella pertussis, the bacterium causing whooping cough. ACT is a member of the RTX (repeats in toxin) family of toxins, and like other members in the family, it may bind cell membranes and cause disruption of the permeability barrier, leading to efflux of cell contents. The present paper summarizes studies performed on cell and model membranes with the aim of understanding the mechanism of toxin insertion and membrane restructuring leading to release of contents. ACT does not necessarily require a protein receptor to bind the membrane bilayer, and this may explain its broad range of host cell types. In fact, red blood cells and liposomes (large unilamellar vesicles) display similar sensitivities to ACT. A varying liposomal bilayer composition leads to significant changes in ACT-induced membrane lysis, measured as efflux of fluorescent vesicle contents. Phosphatidylethanolamine (PE), a lipid that favors formation of nonlamellar (inverted hexagonal) phases, stimulated ACT-promoted efflux. Conversely, lysophosphatidylcholine, a micelle-forming lipid that opposes the formation of inverted nonlamellar phases, inhibited ACT-induced efflux in a dose-dependent manner and neutralized the stimulatory effect of PE. These results strongly suggest that ACT-induced efflux is mediated by transient inverted nonlamellar lipid structures. Cholesterol, a lipid that favors inverted nonlamellar phase formation and also increases the static order of phospholipid hydrocarbon chains, among other effects, also enhanced ACT-induced liposomal efflux. Moreover, the use of a recently developed fluorescence assay technique allowed the detection of trans-bilayer (flip-flop) lipid motion simultaneous with efflux. Lipid flip-flop further confirms the formation of transient nonlamellar lipid structures as a result of ACT insertion in bilayers

dCyaA-KP toxoid unable to promote calcium influx and relocate into lipid rafts is rapidly taken up by J774A.1 cells.

<p>(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 <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002580#ppat.1002580-Fiser1" target="_blank">[14]</a>. (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 Cell^R 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 <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002580#ppat.1002580.s002" target="_blank">figure S2</a>, using a script based on WCIF ImageJ software (<a href="http://rsb.info.nih.gov/ij" target="_blank">http://rsb.info.nih.gov/ij</a>, <a href="http://www.uhnresearch.ca/facilities/wcif/imagej" target="_blank">http://www.uhnresearch.ca/facilities/wcif/imagej</a>). 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.</p

Both toxoids end up in MHC II-positive organelles.

<p>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 Cell^R 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.</p

Endocytosis of CyaA is not affected by cAMP.

<p>(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.</p

Endocytic removal from cell membrane controls the cytolytic potency of CyaA toxin.

<p>(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 <i>Bp</i>-CyaA purified from <i>B. pertussis</i> 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 <i>Bp</i>-CyaA and the shown curves are representative of three independent experiments.</p

Influx of Ca²⁺ decelerates macropinocytic uptake of CyaA-ΔAC and enables cell permeabilization by hemolysin pores.

<p>(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 <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002580#ppat-1002580-g001" target="_blank">Figure 1B</a>. 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.</p

Ca²⁺ influx and K⁺ efflux promoted <i>in trans</i> decelerate the endocytic uptake of enzymatically active CyaA.

<p>J774A.1 macrophages (3×10⁶/ml) in D-MEM medium were incubated for 5 minutes at 37°C with 200 ng/ml of r<i>Ec</i>-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 r<i>Ec</i>-CyaA-biotin <i>per</i> 3×10⁵ cells was detected with PE-conjugated Streptavidine (0.5 mg/ml) by FACS analysis (n = 3).</p

Calcium influx into J774A.1 macrophages decelerates endocytic uptake of the dCyaA-KP toxoid.

<p>(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 <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002580#ppat-1002580-g001" target="_blank">Figure 1B</a>. 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 <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002580#ppat-1002580-g004" target="_blank">Figure 4</a>.</p