Cholesterol-rich membrane microdomains mediate cell cycle arrest induced by Actinobacillus actinomycetemcomitans cytolethal-distending toxin

Abstract

We have previously shown that Actinobacillus actinomycetemcomitans cytolethal-distending toxin (Cdt) is a potent immunosuppressive agent that induces G2/M arrest in human lymphocytes. In this study, we explored the possibility that Cdt-mediated immunotoxicity involves lipid membrane microdomains. We first determined that following treatment of Jurkat cells with Cdt holotoxin all three Cdt subunits localize to these microdomains. Laser confocal microscopy was employed to colocalize the subunits with GM1-enriched membrane regions which are characteristic of membrane rafts. Western blot analysis of isolated lipid rafts also demonstrated the presence of Cdt peptides. Cholesterol depletion, using methyl beta-cyclodextrin, protected cells from the ability of the Cdt holotoxin to induce G2 arrest. Moreover, cholesterol depletion reduced the ability of the toxin to associate with Jurkat cells. Thus, lipid raft integrity is vital to the action of Cdt on host cells. The implications of our observations with respect to Cdt mode of action are discussed.

Fig. 1

Detection of Cdt subunits associated with the Jurkat cell surface following treatment with Cdt holotoxin. Jurkat cells were exposed to Cdt holotoxin (2 μg ml⁻¹) for 2 h and then treated with control murine IgG (dotted lines), anti-CdtA mAb [solid line in (A); mAb Cdt162]; anti-CdtB mAb [solid line in (B); mAb Cdt171] or anti-CdtC mAb [solid line in (C); mAb Cdt112]. Cells were then sequentially stained with goat anti-mouse Ig conjugated to biotin and streptavidin conjugated to FITC and analysed by flow cytometry. FITC fluorescence is plotted versus relative cell number. Numbers represent the mean channel fluorescence (MCF); at least 10 000 cells were analysed per sample. Results are representative of three experiments.

Fig. 2

Colocalization of CdtA with GM1. Jurkat cells were treated with CTB conjugated to AlexaFluor 647 and patched with anti-CTB sera. The cells were then treated with Cdt holotoxin and stained with control IgG (A–C) or anti-CdtA (mAb162; D–I) as described in Experimental procedures and assessed using laser confocal microscopy. Images of FITC fluorescence (A, D and G) and AlexaFluor 647 fluorescence (B, E and H) are shown as well as merged images (C, F and I) showing both FITC (green) and AlexaFluor 647 (red) fluorescence along with DAPI-stained nuclei; colocalization is shown in yellow. Analysis of the images indicate that >96% of CdtA fluorescence colocalizes with CTB fluorescence. Results are representative of three experiments.

Fig. 3

Colocalization of CdtB with GM1. Jurkat cells were treated with CTB conjugated to AlexaFluor 647 and patched with anti-CTB sera. The cells were then treated with Cdt holotoxin and stained with control IgG (A–C) or anti-CdtB (mAb171; D–I) as described in Experimental procedures and assessed using laser confocal microscopy. Images of FITC fluorescence (A, D and G) and AlexaFluor 647 fluorescence (B, E and H) are shown as well as merged images (C, F and I) showing both FITC (green) and AlexaFluor 647 (red) fluorescence along with DAPI-stained nuclei; colocalization is shown in yellow. Analysis of the images indicates that 70% of CdtB fluorescence colocalizes with CTB fluorescence. Results are representative of three experiments.

Fig. 4

Colocalization of CdtC with GM1. Jurkat cells were treated with CTB conjugated to AlexaFluor 647 and patched with anti-CTB sera. The cells were then treated with Cdt holotoxin and stained with control IgG (A–C) or anti-CdtC (mAb112; D–I) as described in Experimental procedures and assessed using laser confocal microscopy. Images of FITC fluorescence (A, D and G) and AlexaFluor 647 fluorescence (B, E and H) are shown as well as merged images (C, F and I) showing both FITC (green) and AlexaFluor 647 (red) fluorescence along with DAPI-stained nuclei; colocalization is shown in yellow. Analysis of the images indicates that >88% of CdtC fluorescence colocalizes with CTB fluorescence. Results are representative of three experiments.

Fig. 5

Isolation of Jurkat cell DRMs. Jurkat cells were treated with Cdt holotoxin for 2 h and DRMs were isolated as described in Experimental procedures. Two distinct low-buoyant-density bands, designated DRM1 and DRM2, were obtained. The composition of these bands was analysed to ascertain that they were indeed lipid rafts. DRM1, DRM2 and the soluble fraction were assessed for cholesterol (bottom) and GM1 (dot blot). The protein profile of the fractions was assessed by Western blot analysis for the presence of the raft-associated protein, Lck, which was enriched in these fractions. In contrast, the transferrin receptor (CD71), a non-raft-associated protein, was found in the soluble fraction. Results are representative of three experiments.

Fig. 6

Western blot analysis of Cdt peptides associated with DRMs. Jurkat cells were treated with Cdt holotoxin, CdtB peptide or both CdtA and CdtC for 2 h; DRMs were isolated as described in Experimental procedures. DRM1, DRM2 and the soluble fraction were analysed for the presence of each Cdt peptide by Western blot analysis using mAb specific for CdtA, CdtB and CdtC. The immunoblots were analysed by digitized scanning densitometry; the numbers represent the relative distribution (%) of each subunit within DRM1, DRM2 and the soluble (s) fractions respectively. Results are representative of three experiments.

Fig. 7

Effect of MβCD on Cdt-induced cell cycle arrest. Jurkat cells were pre-treated with medium (A), 2.5 mM (B), 5.0 mM (C) or 10 mM (D) MβCD for 30 min. The cells were washed, exposed to Cdt holotoxin (40 pg ml⁻¹), incubated for 18 h, stained with propidium iodide and analysed for cell cycle distribution by flow cytometry. Cell cycle distribution is based on DNA content (propidium iodide fluorescence) which is plotted versus relative cell number. G0/G1 cells are found in the first peak (MCF of 75) and G2/M cells in the second peak (MCF of 150); the percentage of cells in S phase was determined based on computer modelling as noted in Experimental procedures. Numbers represent the percentage of cells in the G0/G1, S and G2/M phases of the cell cycle. Results are representative of four experiments; 15 000 cells were analysed for each sample. Parallel experiments verified that MβCD did not adversely affect Jurkat cell viability as determined by propidium iodide exclusion.

Fig. 8

Effect of MβCD and cholesterol-saturated MβCD on Cdt-induced cell cycle arrest. Jurkat cells were pre-treated with medium (A and B), 10 mM MβCD (C) or cholesterol-saturated MβCD (D) for 30 min, washed, exposed to Cdt holotoxin (40 pg ml⁻¹; B–D), incubated for 18 h, stained with propidium iodide and analysed for cell cycle distribution by flow cytometry as described. Numbers represent the percentage of cells in the G0/G1, S and G2/M phases of the cell cycle. Results are representative of four experiments; 15 000 cells were analysed for each sample.

Fig. 9

Cholesterol repletion in MβCD-treated cells restores susceptibility to Cdt. Jurkat cells were first exposed to medium (A and B) or 5 mM MβCD (C–F) for 30 min. The cells were washed and incubated for 30 min in the presence of medium (A–D) or cholesterol-saturated MβCD (E and F; 0.5 mM). Cells were then incubated for 18 h in the presence of medium (A, C and E) or Cdt holotoxin (40 pg ml⁻¹; B, D and F), stained with propidium iodide and analysed for cell cycle distribution by flow cytometry. Numbers represent the percentage of cells in the G0/G1, S and G2/M phases of the cell cycle. Results are representative of three experiments; 15 000 cells were analysed for each sample.

Fig. 10

Effect of cholesterol depletion on Cdt binding to Jurkat cells. Jurkat cells were pretreated with medium (A, C, E and G) or 10 mM MβCD (B, D, F and H) for 30 min. The cells were washed and treated with Cdt for 1 h. Jurkat cells were then washed and stained with control IgG (A and B), anti-CdtA mAb (mAb162; C and D), anti-CdtB mAb (mAb171; E and F) or anti-CdtC mAb (mAb112; G and H); cells were then exposed to goat anti-mouse IgG conjugated to FITC as described in Experimental procedures. Cells were analysed by flow cytometry for FITC fluorescence; numbers in each panel represent the MCF. Results are representative of three experiments; at least 15 000 cells were analysed per sample.