Translocation ofClostridium difficileToxin B across Polarized Caco-2 Cell Monolayers Is Enhanced by Toxin A

Tim Du,Michelle J Alfa

doi:10.1155/2004/292580

Abstract

Clostridium difficile is 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 of C difficile toxins 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 purified C difficile toxins A and B. C difficile toxins were (125)I 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 which C difficile toxin A facilitates the translocation of toxin B from the gut into submucosal areas where it may play a role in inflammatory damage.

Highlights

Clostridium difficile is the etiological agent of antibiotic-associated diarrhea; the most common form of nosocomial infectious diarrhea
Human foreskin fibroblasts (HFF), a nontransformed cell line derived from pooled foreskins from children, was maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum, 1 mM sodium pyruvate and 2 mM L-glutamine
The electrical resistance measurements indicate the level of tight junction integrity

Summary

Introduction

Clostridium difficile is the etiological agent of antibiotic-associated diarrhea; the most common form of nosocomial infectious diarrhea. 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. La translocation de la toxine B du Clostridium difficile dans les monocouches de cellules Caco-2 polarisées est renforcée par la toxine A. Un modèle de culture polarisée de tissus faisant appel à des cellules Caco-2 cultivées sur des inserts transwell a été créé pour étudier la translocation des toxines A et B purifiées de C difficile. Les toxines du C difficile étaient étiquetées 125I et inoculées sur des monocouches de cellules Caco-2 polarisées confluentes, afin d’étudier la dynamique de translocation. Some animal studies have shown that systemic injection of minute amounts of toxin B are fatal [12,13,14]

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