Cytolethal distending toxin-induced cell cycle arrest of lymphocytes is dependent upon recognition and binding to cholesterol

Abstract

Induction of cell cycle arrest in lymphocytes after exposure to the Aggregatibacter actinomycetemcomitans cytolethal distending toxin (Cdt) is dependent upon the integrity of lipid membrane microdomains. In this study we further demonstrate that the association of Cdt with lymphocyte plasma membranes is dependent upon binding to cholesterol. Depletion of cholesterol resulted in reduced toxin binding, whereas repletion of cholesterol-depleted cells restored binding. We employed fluorescence resonance energy transfer and surface plasmon resonance to demonstrate that toxin association with model membranes is dependent upon the concentration of cholesterol; moreover, these interactions were cholesterol-specific as the toxin failed to interact with model membranes containing stigmasterol, ergosterol, or lanosterol. Further analysis of the toxin indicated that the CdtC subunit contains a cholesterol recognition/interaction amino acid consensus (CRAC) region. Mutation of the CRAC site resulted in decreased binding of the holotoxin to cholesterol-containing model membranes as well as to the surface of Jurkat cells. The mutant toxin also exhibited reduced capacity for intracellular transfer of the active toxin subunit, CdtB, as well as reduced toxicity. Collectively, these observations indicate that membrane cholesterol serves as an essential ligand for Cdt and that this association can be blocked by either depleting membranes of cholesterol or mutation of the CRAC site.

FIGURE 1.

Cdt holotoxin association with Jurkat cells is dependent upon the presence of cholesterol. Jurkat cells were exposed to medium (panels A and D) or 5 mm MβCD (panels B and E (Sigma)) for 30 min. Cells were washed, and some of the MβCD-treated cells were incubated with cholesterol-saturated MβCD (0.5 mm) for 30 min (panels C and F). Cells were incubated with Cdt holotoxin (2 μg/ml) for 1 h, washed, and treated with control murine IgG (data not shown) or anti-CdtC monoclonal antibody conjugated to Alexafluor 488. Jurkat cells were then analyzed by flow cytometry (panels A–C), or images of cells were taken on a Bio-Rad Radiance 2100 Confocal Microscope (Bio-Rad); representative images are shown in panels D–F. Results are representative of three experiments. The relative level of cholesterol in 10⁷ Jurkat cells was compared by semiquantitative TLC. Cholesterol was identified based on calculated Rf values and color after detection by charring with sulfuric acid/EtOH (1:1 vol:vol). The intensity of cholesterol for each sample was compared with the intensity of known standards using digital densitometry; values were 11.8, 0.37, and 20.7 μg cholesterol/10⁷ cells for control, MβCD-treated, and cholesterol-saturated MβCD-treated cells, respectively.

FIGURE 2.

Cdt holotoxin preferentially binds to LUVs containing cholesterol. The interaction of Cdt holotoxin with LUVs containing varying amounts of cholesterol was analyzed by FRET and SPR. Panel A shows FRET analysis of Cdt with LUVs containing varying amounts (10–40 mol %) cholesterol. Values are the mean ± S.D. (n = 3), expressed as relative % energy transfer. Results are statistically significant (p < 0.05; multivariant analysis of variance with post-hoc Scheffe test) for differences in energy transfer as cholesterol concentration is increased. Panel B shows the SPR results of Cdt interaction with LUVs. An overlay of sensorgrams shows the interaction of Cdt (10 μg/ml) with immobilized LUVs containing decreasing concentrations of cholesterol; data points were collected every 0.2 s. Data are plotted as response units versus time and are representative of three experiments. Panel C shows the results of SPR analysis for the interaction of two concentrations of Cdt (10 and 20 μg/ml) with immobilized LUVs containing 5, 10, and 20% cholesterol; data are the mean ± S.D. of three experiments and are plotted as the number of response units obtained from each sensorgram after 3 min post-injection. Results are statistically significant (p < 0.29; multivariant analysis of variance) for differences in response units as toxin concentration is increased. Panel D shows the results of SPR analysis of Cdt (20 μg/ml) with immobilized LUVs containing 20% of either cholesterol, lanosterol, ergosterol, or stigmasterol. The mean ± S.D. of the maximum response is plotted for three experiments. Results are statistically significant for differences between cholesterol and lanosterol (p < 0.001), cholesterol and ergosterol (p = 0.029), and cholesterol and stigmasterol (p = 0.029). To verify that liposomes contained comparable levels of sterol, aliquots of liposomes were extracted, and the amount of sterol was determined as described in Fig. 1; extraction yields were 7.9 μg (lanosterol), 8.6 μg (ergosterol), 7.6 μg (stigmasterol), and 7.8 μg (cholesterol).

FIGURE 3.

CdtABC preferentially associates with cholesterol and lipid rafts. LUVs were prepared containing either 13% (black bars) or 26% (gray bars) SM along with 20% cholesterol. The LUVs were assessed in real time for binding to immobilized Cdt. The maximum response units (mean ± S.D. of five experiments) is plotted versus Cdt concentration. The asterisk denotes a statistically significant difference between 13 and 26% sphingomyelin (p = 0.040); no significant difference was detected at the lower toxin concentrations.

FIGURE 4.

Localization of the CRAC site on CdtC. Ribbon representation (left) of A. actinomycetemcomitans Cdt holotoxin (PDB accession number 2F2F). CdtC is represented in blue with residues Leu-68—Lys-74 of the CRAC site colored red. CdtA is shown in yellow, and CdtB is shown in gray. Shown is a surface representation (right) of the holotoxin indicating the accessibility of the CRAC site.

FIGURE 5.

Cdt holotoxin containing a CRAC mutation (CdtABC^Y71P) exhibits reduced ability to associate with cholesterol. Panel A shows the results of the FRET analysis of the ability of CdtABC^WT versus that of CdtABC^Y71P to bind to LUVs containing 10–40 mol % cholesterol. Results are plotted as the percentage of energy transfer versus cholesterol concentration and represent the mean ± S.D. of three experiments. Panel B, SPR analysis of the ability of immobilized CdtABC^WT (broken line) and CdtABC^Y71P (solid line) to bind LUVs containing 20% cholesterol; results are representative of three experiments. Results are statistically significant (p < 0.035) for the differences between CdtABC^wt and CdtABC^Y71P at all concentrations of cholesterol.

FIGURE 6.

CdtABC^Y71P exhibits a reduction in association with Jurkat cells as well as in its ability to deliver CdtB. Jurkat cells were exposed to medium (panels A and D), CdtABC^WT (panels B and E), or CdtABC^Y71P (panels C and F) for 1 h and then analyzed by immunofluorescence for the presence of surface CdtC (panels A–C) and intracellular CdtB (panels D–F). Alexafluor fluorescence is plotted versus relative cell number. Numbers represent the mean channel fluorescence; at least 10,000 cells were analyzed per sample. Results are representative of three experiments.

FIGURE 7.

CdtABC^Y71P exhibits reduced ability to induce cell cycle arrest. Jurkat cells were incubated with medium alone (panel A), 50 pg/ml CdtABC^WT (panel B), or 50 pg/ml CdtABC^Y71P (panel C) for 18 h, stained with propidium iodide, and analyzed for cell cycle distribution by flow cytometry as described under “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 three experiments; 15,000 cells were analyzed for each sample. PI, propidium iodide.