PMC

Comparison of the “β-barrel” containing heptamer of ^AsymCDTb to the analogous heptamer from the protective antigen (PA) of the anthrax toxin. (A). Heptamers from PA of anthrax toxin are superimposed with electron density from the “β-barrel heptamer” observed in the ^AsymCDTb di-heptamer structure. The RBD in “β-barrel form” of the PA from anthrax toxin were not modeled in the corresponding cryo-EM model and are placed here using alignment with the soluble form of the toxin. (B) Structural comparison of heptameric forms A (Upper) and B (Lower) from ^AsymCDTb (green) and anthrax toxin (red). Cryo-EM densities are shown for all molecules except anthrax toxin form B for which the 2Fo–Fc map is shown and derived from the corresponding crystal structure.

Localization of cellular S1. (A) Amounts of S1 from various cell fractions determined by Western blotting by using monoclonal antibody 3CX4. The lanes contained 50 ng of purified pertussis toxin (Purified PTX), BP338 whole cells (Whole Cells), the BP338 soluble fraction (Soluble), the BP338 membrane fraction (Membranes), and BPRA (pertussis toxin mutant) whole cells (ΔPTX) as a negative control. (B) BP338 whole cells and soluble and membrane fractions probed with anti-pertactin antibody BB05. PRN, pertactin. (C) BP338 whole cells and soluble and membrane fractions probed with anti-PtlF antibody.

GST-Srl enhances the cytotoxicity of both toxin A and toxin B in epithelial cells. (A) Morphological changes of MDCK cells after treatment with toxin A (0.25 ng/ml [panels b, c, and d] versus 25 ng/ml [panels i, j, and k]) or toxin B (0.1 ng/ml [panels e, f, and g] versus 10 ng/ml [panels l, m, and n]) in the presence of either GST-Srl or GST. After 24 h of incubation, cells were fixed and labeled with TRITC-phalloidin. + and − denote positive and negative results, respectively. Bar, 10 μm. (B) MDCK cells were incubated with 250 ng/ml toxin A, 100 ng/ml toxin B, 10 μg/ml GST-Srl, or 10 μg/ml GST alone. After incubation for 24 h, cell viability was measured by an aqueous soluble tetrazolium/formazan assay and expressed as a percentage of the results from buffer-treated controls (−). All reactions were performed in triplicate, and the results are means ± SD of three independent measurements. n.s., not significant. *, P < 0.01 versus toxin A or toxin B. (C) MDCK cells were incubated for 24 h with the indicated concentrations of GST-Srl or GST in the presence of either 250 ng/ml toxin A or 100 ng/ml toxin B. The viability of MDCK cells was determined as in panel B. Results are means ± SD of three independent measurements. *, P < 0.05; **, P < 0.01. (D) MDCK cells were incubated with 250 ng/ml toxin A, 100 ng/ml toxin B, 10 μg/ml GST-Srl, or 10 μg/ml GST alone. After incubation for 24 h, lysates were prepared, and apoptotic oligosomal DNA fragmentation was assessed using a cell death ELISA kit. All reactions were performed in triplicate, and the results are expressed as optical density (OD.) at 405 nm. Mean values ± SD are indicated for three independent measurements. −, buffer-treated control. (E) MDCK cells were incubated for 24 h with the indicated concentrations of GST-Srl or GST in the presence of 250 ng/ml toxin A or 100 ng/ml toxin B. The cell death induced in MDCK cells was detected as in panel D. *, P < 0.05; **, P < 0.01.

Reconstituted high-density lipoproteins (rHDL) as toxin decoys. (A) rHDL consists of a discoidal patch of lipid membrane bounded by apolipoprotein A-I. (B) rHDL embedded with ganglioside monosialotetrahexosylganglioside (GM1) (purple) can act as decoys for soluble toxins such as cholera toxin (red) to protect live cells (gray). (C-E) Human retinal epithelial cells, as a model target cell line, incubated with cholera toxin B only (CTB) (C), toxin + vesicles (D), or toxin + rHDL (E). Cells appear in gray with NBD-labeled rHDL in green, Alexa594-CTB in red, and FITC-CTB in cyan (C only). Vesicles afford little protection from toxin while rHDL provide virtually complete protection. Reprinted with permission from [].