Abstract
Enterotoxigenic Escherichia coli produce various heat-labile and heat-stable enterotoxins. STb is a low molecular weight heat-resistant toxin responsible for diarrhea in farm animals, mainly young pigs. A previous study demonstrated that cells having internalized STb toxin induce epithelial barrier dysfunction through changes in tight junction (TJ) proteins. These modifications contribute probably to the diarrhea observed. To gain insight into the mechanism of increased intestinal permeability following STb exposure we treated human colon cells (T84) with purified STb toxin after which cells were harvested and proteins extracted. Using a 1% Nonidet P-40-containing solution we investigated the distribution of claudin-1, a major structural and functional TJ protein responsible for the epithelium impermeability, between membrane (NP40-insoluble) and the cytoplasmic (NP-40 soluble) location. Using immunoblot and confocal microscopy, we observed that treatment of T84 cell monolayers with STb induced redistribution of claudin-1. After 24 h, cells grown in Ca++-free medium treated with STb showed about 40% more claudin-1 in the cytoplasm compare to the control. Switching from Ca++-free to Ca++-enriched medium (1.8 mM) increased the dislodgement rate of claudin-1 as comparable quantitative delocalization was observed after only 6 h. Medium supplemented with the same concentration of Mg++ or Zn++ did not affect the dislodgement rate compared to the Ca++-free medium. Using anti-phosphoserine and anti-phosphothreonine antibodies, we observed that the loss of membrane claudin-1 was accompanied by dephosphorylation of this TJ protein. Overall, our findings showed an important redistribution of claudin-1 in cells treated with STb toxin. The loss of phosphorylated TJ membrane claudin-1 is likely to be involved in the increased permeability observed. The mechanisms by which these changes are brought about remain to be elucidated.
Highlights
Enterotoxigenic Escherichia coli (ETEC) represent an important cause of severe diarrhea in newborn animals [1] and diarrhea in humans following the ingestion of contaminated food and water [2]
stable enterotoxin b (STb) affects the distribution of claudin-1 Since STb toxin was previously shown to increase paracellular permeability [31], we wanted to address whether translocation of tight junction (TJ) proteins was involved in this process
Enteric pathogens have developed strategies that induce the production of diarrhea in infected hosts, through disruption of intercellular TJs [45,46]
Summary
Enterotoxigenic Escherichia coli (ETEC) represent an important cause of severe diarrhea in newborn animals [1] and diarrhea in humans following the ingestion of contaminated food and water [2] Expression of both colonization factors and toxins are required for disruption of intestinal fluid homeostasis, leading to diarrhea [3]. The calcium increase activates phospholipases A2 responsible for the release of arachidonic acid from membrane phospholipids leading to production of prostaglandin E2 (PGE2) and 5hydroxytryptamine (5-HT) [11,12] These molecules mediate transport of water and HCO3- from enterocytes into the intestinal lumen and prevent Na+ absorption resulting in watery diarrhea [7]
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