Cloned Shiga toxin 2 B subunit induces apoptosis in Ramos Burkitt's lymphoma B cells

Abstract

The Shiga toxins (Stx1 and Stx2), produced by Shigella dysenteriae type 1 and enterohemorrhagic Escherichia coli, consist of one A subunit and five B subunits. The Stx1 and Stx2 B subunits form a pentameric structure that binds to globotriaosylceramide (Gb3-Cer) receptors on eukaryotic cells and promotes endocytosis. The A subunit then inhibits protein biosynthesis, which triggers apoptosis in the affected cell. In addition to its Gb3-Cer binding activity, the data in the following report demonstrate that the Stx2 B pentamer induces apoptosis in Ramos Burkitt's lymphoma B cells independently of A subunit activity. Apoptosis was not observed in A subunit-free preparations of the Stx1 B pentamer which competitively inhibited Stx2 B pentamer-mediated apoptosis. The pancaspase inhibitor, Z-VAD-fmk, prevented apoptosis in Ramos cells exposed to the Stx2 B subunit, Stx1 or Stx2. Brefeldin A, an inhibitor of the Golgi transport system, also prevented Stx2 B subunit-mediated apoptosis. These observations suggest that the Stx2 B subunit must be internalized, via Gb3-Cer receptors, to induce Ramos cell apoptosis. Moreover, unlike the two holotoxins, Stx2 B subunit-mediated apoptosis does not involve inhibition of protein biosynthesis. This study provides further insight into the pathogenic potential of this family of potent bacterial exotoxins.

FIG. 1.

Effect of Stx1 or Stx2 holotoxins or their cloned B subunits on Daudi (A) and Ramos (B) Burkitt's lymphoma B cells. B cells were incubated at 37°C for 18 h with the indicated concentrations of Stx1 or Stx2 holotoxins or their cloned B subunits. The B cells were then labeled with Annexin V-FITC and propidium iodide, and the proportion of early and late apoptotic cells in each sample was determined by flow cytometry analysis. The error bars (n = 5) represent the standard deviations.

FIG. 2.

DNA fragmentation patterns in Ramos Burkitt's lymphoma B cells exposed to Stx1 or Stx2 holotoxins or their cloned B subunits for 18 h at 37°C. Fragmented DNA was isolated and precipitated from cell lysates and analyzed by agarose gel electrophoresis by using a 2% gel. Lane 1, 1-kb DNA ladder; lane 2, no treatment; lane 3, 5 μM camptothecin; lane 4, 1 ng of Stx1/ml; lane 5, 1 ng of Stx2/ml; lane 6, 10 μg of Stx1 B subunit/ml; lane 7, 10 μg of Stx2 B subunit/ml.

FIG. 3.

Immunoblot analysis of Bcl-2 expression in Ramos and Daudi Burkitt's lymphoma B cells. Ramos and Daudi B cells were subjected to SDS-PAGE and immunoblot analysis. The immunoblots were then reacted sequentially with a murine Bcl-2 monoclonal antibody and goat polyclonal horseradish peroxidase-conjugated anti-mouse IgG. Lane 1, Ramos whole-cell lysate; lane 2, Daudi whole-cell lysate.

FIG. 4.

CD77 (Gb3-Cer) expression on Ramos, Daudi, and Namalwa Burkitt's lymphoma cell surfaces. Ramos, Daudi, and Namalwa B cells were incubated with a monoclonal anti-CD77 antibody conjugated to FITC and analyzed by flow cytometry. The error bars represent the standard deviations (n = 3).

FIG. 5.

Inhibition of apoptosis of Ramos Burkitt's lymphoma B cells. B cells were preincubated with (shaded bars) or without (open bars) Z-VAD-fmk (A) or with (shaded bars) or without (open bars) cloned Stx1 B subunit (B) or were preincubated with (shaded bars) or without (open bars) brefeldin A (BFA) (C). The Ramos cells were next incubated at 37°C for 18 h with camptothecin, Stx holotoxins, or their cloned B subunits. These Ramos cells were then labeled with Annexin V-FITC and propidium iodide, and the proportion of early and late apoptotic cells in each sample was determined by flow cytometry analysis. The error bars represent the standard deviations (n = 5).

FIG. 6.

Protein biosynthetic capacity of Ramos cells treated with Stx1 or Stx2 holotoxins or their cloned B subunits. The protein biosynthetic capacity of Ramos cells treated with Stx1 or Stx2 holotoxins or their cloned B subunits, proportional to that of untreated cells, was assessed by measuring the incorporation of [³H]leucine into the TCA-precipitable fraction. Duplicate samples were pretreated with Z-VAD-fmk. Each datum point represents the average of triplicate evaluations.

FIG. 7.

Protein biosynthetic capacity of rabbit reticulocyte lysates treated with Stx2 A subunit or the cloned Stx1 or Stx2 B subunits. The protein biosynthetic capacity of rabbit reticulocyte lysates incubated with the Stx2 A subunit or the cloned Stx1 or Stx2 B subunits, proportional to that of an untreated lysate, was assessed by measuring the incorporation of [³H]leucine into the TCA-precipitable fraction. Each datum point represents the average of triplicate determinations ± the standard deviation.