Figure 1.
Effects of DTT upon iota cytotoxicity.
(A) DTT (5 and 10 mM) effects upon iota (460 pM Ia +500 pM Ib) cytotoxicity with Vero cells (n = 3 wells per calculation). “*” and “***” represent significant differences at p<0.05 and p<0.0005, respectively. ns = not significant. (B) Inhibition of iota cytotoxicity with anti-CD44 antibody. Vero cells were pre-treated with serial dilutions of an anti-CD44 monoclonal antibody for 30 min before adding iota toxin. A non-specific isotype antibody was included as a control. After 4 h of intoxication, the actin cytoskeleton was stained with Alexa-488 phalloidin. Images were acquired and integrated fluorescence intensity of Alexa-488 phalloidin staining determined with MetaXpress software. Values represent the mean of nine fields +/− standard deviation. “*” designates statistically different (ANOVA p<0.05) than isotype control antibody plus iota toxin, or toxin only. (C) Typical visual results showing Vero cells following various treatments and subsequent staining with Hoechst (nucleus), Alexa-488 phalloidin (F-actin), and CellMask Deep Red (cytoplasm and nucleus).
Figure 2.
CD44− cells are resistant to iota and iota-like toxins versus CD44+ cells.
(A) Dose-response of iota toxin on cells with controls consisting of cells in media only. The Y-axis represents the “% control” of F-actin content (Alexa-488 phalloidin stained after 5 h) in intoxicated cells versus controls in media only. (B) Like iota toxin, CD44+ RPM cells are also susceptible to C. difficile (CDT) and C. spiroforme (CST) binary toxins. Each assay was done in duplicate and values represent mean +/− standard deviation from three separate experiments.
Figure 3.
Comparative binding of Ib to CD44+, versus CD44−, cells.
Confocal microscopy was done with RPM (CD44− and CD44+) cells incubated with Cy3-labeled Ib (10−7 M in DMEM +0.1% BSA) for 3 min at 37°C, washed with PBS, and mounted in mowiol. Blue represents Dapi-stained nuclei and red indicates cell-bound Ib. A representative field of cells is shown.
Figure 4.
Binding of iota-family B components to purified CD44 in solution.
(A and B) The B component (10 µg) of each toxin was added to CD44-IgG (10 µg in 50 µl) at room temperature for 60 min. Protein A - agarose beads were then added for 5 min at room temperature, gently centrifuged, and washed with buffer. SDS-PAGE sample buffer containing reducing agent was added to the beads, the mixture heated, and protein separated from the beads by centrifugation. Supernatant proteins were then resolved on a 10% gel, transferred onto nitrocellulose, and clostridial B component detected with either 1∶1000 dilutions of rabbit anti-Ib (Panel A) or anti-C2II sera (Panel B). Protein A - peroxidase conjugate was used to detect bound antibody, and following washes, specific bands were visualized with chemiluminescent substrate. (C) Like the CD44-IgG construct, Ib also binds specifically to a CD44-GST construct. A GST version of C. botulinum C3 exoenzyme, used as a negative control, does not bind to Ib in pull-down experiments done similarly for panels A and B, with an exception being the use of glutathione-sepharose (instead of Protein A-agarose) beads.