Ve proteomic analysis of Vero cells [19] revealing a potential role of CD44 during intoxication by iotaCD44 and Iota-Family ToxinsCD44 and Iota-Family ToxinsFigure 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 Alexa488 phalloidin. Images were acquired and integrated fluorescence Title Loaded From File intensity of Alexa-488 phalloidin staining determined with MetaXpress software. Values represent the mean of nine fields +/2 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 Title Loaded From File CellMask Deep Red (cytoplasm and nucleus). doi:10.1371/journal.pone.0051356.gfamily toxins from clostridia. In contrast, current results revealed that the related C2 toxin of C. botulinum does not use CD44 for intoxication. Upon comparing lipid rafts of Ib-treated cells, versus controls from cells incubated in media only, CD44 was most highly enriched among ninety possible candidates. It is important to note intriguing biological correlations that exist between CD44 and iota toxin. For example, CD44 associates with lipid rafts in epithelial cells, forms cell-surface clusters, is basolaterally located in polarized cells, and use of a cholesterol extracting agent (methylb-cyclodextrin) disperses CD44 throughout the cell surface [21]. Discovery that C. perfringens iota toxin also associates with lipid rafts [14,17], rapidly forms temperature-dependent clusters on cells [16], preferentially intoxicates polarized cells through the basolateral surface [18,27], and that methyl-b-cyclodextrin reduces Ib binding, oligomer formation and delays iota cytotoxicity [17] remarkably parallels the findings for CD44. Upon internalization by a clathrin-independent mechanism, CD44 traffics through an acidified endosome and recycles back to the cell surface, like that reported for clostridial binary toxins [27?2]. Altogether, the similar biological aspects of CD44 and iota toxin, along with proteomic-based clues from lipid rafts, logically provided enticing evidence for us to pursue this current study. Furthermore, pre-treatment of Vero cells with pronase effectively prevents Ib binding and thus suggests a protein-based receptor [13]. It is known that membrane-bound CD44 is susceptible to proteolysis, as evidenced by a membrane-associated metalloprotease that promotes metastasis [33]. Cells incubated with DTT also do not bind hyaluronan through CD44 [22] after reduction of a disulfide bond within the transmembrane domain of CD44 [34]. Modification of select cysteines prevents CD44 dimerization and clustering into lipid rafts [35]. Our initial experiments also showed that pre-treatment of Vero cells with DTT transiently prevented iota intoxication, but this is not due to inhibiting either Ia modification of actin or Ib binding to the cell.Ve proteomic analysis of Vero cells [19] revealing a potential role of CD44 during intoxication by iotaCD44 and Iota-Family ToxinsCD44 and Iota-Family ToxinsFigure 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 Alexa488 phalloidin. Images were acquired and integrated fluorescence intensity of Alexa-488 phalloidin staining determined with MetaXpress software. Values represent the mean of nine fields +/2 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). doi:10.1371/journal.pone.0051356.gfamily toxins from clostridia. In contrast, current results revealed that the related C2 toxin of C. botulinum does not use CD44 for intoxication. Upon comparing lipid rafts of Ib-treated cells, versus controls from cells incubated in media only, CD44 was most highly enriched among ninety possible candidates. It is important to note intriguing biological correlations that exist between CD44 and iota toxin. For example, CD44 associates with lipid rafts in epithelial cells, forms cell-surface clusters, is basolaterally located in polarized cells, and use of a cholesterol extracting agent (methylb-cyclodextrin) disperses CD44 throughout the cell surface [21]. Discovery that C. perfringens iota toxin also associates with lipid rafts [14,17], rapidly forms temperature-dependent clusters on cells [16], preferentially intoxicates polarized cells through the basolateral surface [18,27], and that methyl-b-cyclodextrin reduces Ib binding, oligomer formation and delays iota cytotoxicity [17] remarkably parallels the findings for CD44. Upon internalization by a clathrin-independent mechanism, CD44 traffics through an acidified endosome and recycles back to the cell surface, like that reported for clostridial binary toxins [27?2]. Altogether, the similar biological aspects of CD44 and iota toxin, along with proteomic-based clues from lipid rafts, logically provided enticing evidence for us to pursue this current study. Furthermore, pre-treatment of Vero cells with pronase effectively prevents Ib binding and thus suggests a protein-based receptor [13]. It is known that membrane-bound CD44 is susceptible to proteolysis, as evidenced by a membrane-associated metalloprotease that promotes metastasis [33]. Cells incubated with DTT also do not bind hyaluronan through CD44 [22] after reduction of a disulfide bond within the transmembrane domain of CD44 [34]. Modification of select cysteines prevents CD44 dimerization and clustering into lipid rafts [35]. Our initial experiments also showed that pre-treatment of Vero cells with DTT transiently prevented iota intoxication, but this is not due to inhibiting either Ia modification of actin or Ib binding to the cell.

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