Translocation ofClostridium difficileToxin B across Polarized Caco-2 Cell Monolayers Is Enhanced by Toxin A
Clostridium difficileis the etiological agent of antibiotic-associated diarrhea; the most common form of nosocomial infectious diarrhea. The basis for the shock-like systemic symptoms observed in severe cases of this infection are not known. It is hypothesized that the invasion ofC difficiletoxins A and/or B from the gut mucosa may contribute to these symptoms.A polarized tissue culture model employing Caco-2 cells grown on transwell inserts was established to study the translocation of purifiedC difficiletoxins A and B.C difficiletoxins were125I labelled and inoculated onto confluent polarized Caco-2 cell monolayers to study translocation dynamics. Electrical resistance measurements were used to monitor monolayer confluence and tight junction integrity. Samples were taken from the apical and basal sides of the insert, as well as the insert itself, and tested using the human foreskin fibroblasts cell cytotoxicity assay to monitor partitioning of the radiolabelled toxins.Toxin A produced a 50% reduction in electrical resistance in 3 h whereas the same concentration of toxin B required at least 7 h to achieve the same effect. Both toxins A and B were able to translocate across confluent monolayers of Caco-2 cells. The combination of toxin A and B together was synergistic with respect to promoting the translocation of toxin B. Although the addition of toxin A resulted in a 100% increase in the amount of toxin B able to translocate, no increases in toxin A translocation were observed. These findings suggest a model of pathogenesis in whichC difficiletoxin A facilitates the translocation of toxin B from the gut into submucosal areas where it may play a role in inflammatory damage.