Ormed a series of experiments with cultured cells, as well as animals, to investigate whether CD44 is involved in the mode of action of clostridial binary toxins. Results implicate a role for CD44 during PS 1145 site intoxication by the iota-family toxins.Results Reducing Agent or Antibody Against CD44 Inhibits Iota CytotoxicityDisulfide-driven clustering of CD44 on the cell surface promotes binding of a natural ligand (hyaluronan) to cells and is inhibited by a reducing agent like dithiothreitol (DTT) [22]. As iota toxin also forms oligomers on Vero and MDCK cells [14,16,17,23], and CD44 was our top proteomics-based candidate involved in intoxication of Vero cells, we first examined if DTT had any overt effect upon iota intoxication. Figure 1A shows that either 5 or 10 mM DTT significantly delayed overt rounding due to iota activity, versus cells incubated with toxin alone. However, by 12 h the DTT-treated Vero cells eventually rounded due to iota toxin. In contrast, Vero cells incubated with high picomolar concentrations of C2 toxin were not protected by 10 mM DTT (data not shown). Control cells incubated with either 5 or 10 mM DTT alone showed no change in morphology. Regarding the effects of DTT upon each component of iota toxin, we first excluded that DTT (10 and 50 mM) interfered with A component (Ia)-catalyzed ADP-ribosylation of actin from Vero cell lysate in vitro (data not shown) [7]. Furthermore, Ib binding to cells was not inhibited by DTT (10 mM) as determined by Western blot analysis (data not shown). In conjunction with the earlier proteomic-based findings [19], sensitivity to DTT suggested that CD44 might play a role in iota cytotoxicity; however, the nonspecific nature of this reducing agent upon any disulfidecontaining protein found on a cell surface required more targeted experimentation. Experiments were next done to neutralize the cytotoxic effects of iota toxin 1531364 via a specific monoclonal antibody against CD44 (Figs. 1B and 1C). Pretreatment of Vero cells with this antibody afforded protection against iota intoxication, as evidenced by inhibited rounding and disruption of the actin cytoskeleton. The latter was quantitatively measured by binding of fluor-labeled phalloidin to filamentous (F) actin. Figure 1B summarizes the dosedependent, protective effect of anti-CD44 upon iota intoxication with representative images in Figure 1C. With nine fields per sample, the mean buy Lecirelin readings of cells incubated with iota toxin and varying antibody concentrations (15.6?25 mg/ml) were significantly different versus controls consisting of: 1) isotype control antibody plus iota toxin; or 2) iota toxin only. These results strongly suggested a crucial role, whether direct or indirect, for CD44 during intoxication of cells by iota toxin.cells to iota toxin was very similar to Vero cells commonly used for cytotoxicity assays (Fig. 2A). When higher concentrations (1027 M) of iota toxin were incubated with non-transfected RPM cells, there was some cytotoxicity. Further confirmation of these findings was evident by confocal microscopy showing that Cy3-labeled Ib more readily bound to the CD44+, versus CD442, RPM cells (Fig. 3). These results demonstrated that reconstitution of CD44 expression in RPM cells increased binding, and subsequent cytotoxic effects, of iota toxin. Prompted by these observations with iota toxin, it was important to examine if other closely related binary toxins produced by C. difficile and C. spiroforme similarly affected the CD44+/C.Ormed a series of experiments with cultured cells, as well as animals, to investigate whether CD44 is involved in the mode of action of clostridial binary toxins. Results implicate a role for CD44 during intoxication by the iota-family toxins.Results Reducing Agent or Antibody Against CD44 Inhibits Iota CytotoxicityDisulfide-driven clustering of CD44 on the cell surface promotes binding of a natural ligand (hyaluronan) to cells and is inhibited by a reducing agent like dithiothreitol (DTT) [22]. As iota toxin also forms oligomers on Vero and MDCK cells [14,16,17,23], and CD44 was our top proteomics-based candidate involved in intoxication of Vero cells, we first examined if DTT had any overt effect upon iota intoxication. Figure 1A shows that either 5 or 10 mM DTT significantly delayed overt rounding due to iota activity, versus cells incubated with toxin alone. However, by 12 h the DTT-treated Vero cells eventually rounded due to iota toxin. In contrast, Vero cells incubated with high picomolar concentrations of C2 toxin were not protected by 10 mM DTT (data not shown). Control cells incubated with either 5 or 10 mM DTT alone showed no change in morphology. Regarding the effects of DTT upon each component of iota toxin, we first excluded that DTT (10 and 50 mM) interfered with A component (Ia)-catalyzed ADP-ribosylation of actin from Vero cell lysate in vitro (data not shown) [7]. Furthermore, Ib binding to cells was not inhibited by DTT (10 mM) as determined by Western blot analysis (data not shown). In conjunction with the earlier proteomic-based findings [19], sensitivity to DTT suggested that CD44 might play a role in iota cytotoxicity; however, the nonspecific nature of this reducing agent upon any disulfidecontaining protein found on a cell surface required more targeted experimentation. Experiments were next done to neutralize the cytotoxic effects of iota toxin 1531364 via a specific monoclonal antibody against CD44 (Figs. 1B and 1C). Pretreatment of Vero cells with this antibody afforded protection against iota intoxication, as evidenced by inhibited rounding and disruption of the actin cytoskeleton. The latter was quantitatively measured by binding of fluor-labeled phalloidin to filamentous (F) actin. Figure 1B summarizes the dosedependent, protective effect of anti-CD44 upon iota intoxication with representative images in Figure 1C. With nine fields per sample, the mean readings of cells incubated with iota toxin and varying antibody concentrations (15.6?25 mg/ml) were significantly different versus controls consisting of: 1) isotype control antibody plus iota toxin; or 2) iota toxin only. These results strongly suggested a crucial role, whether direct or indirect, for CD44 during intoxication of cells by iota toxin.cells to iota toxin was very similar to Vero cells commonly used for cytotoxicity assays (Fig. 2A). When higher concentrations (1027 M) of iota toxin were incubated with non-transfected RPM cells, there was some cytotoxicity. Further confirmation of these findings was evident by confocal microscopy showing that Cy3-labeled Ib more readily bound to the CD44+, versus CD442, RPM cells (Fig. 3). These results demonstrated that reconstitution of CD44 expression in RPM cells increased binding, and subsequent cytotoxic effects, of iota toxin. Prompted by these observations with iota toxin, it was important to examine if other closely related binary toxins produced by C. difficile and C. spiroforme similarly affected the CD44+/C.
