THE BACTEROIDES-FRAGILIS TOXIN FRAGILYSIN DISRUPTS THE PARACELLULAR BARRIER OF EPITHELIAL-CELLS

Bacteroides fragilis is a member of the normal colonic microflora of m ost mammals and is the most commonly isolated anaerobe from human clin ical specimens. Some strains produce a toxin (fragilysin, a zinc-metal loproteinase) implicated as a cause of diarrheal disease in farm anima ls and humans. Studies in our laboratory confirm that the proteolytic activity of this toxin is responsible for the fluid secretion and tiss ue damage observed in vivo. In this study, we investigated the effects of fragilysin on the paracellular barrier of epithelial cells. Resear chers suggest that, since the toxin rapidly intoxicates HT-29 cells, i t may be internalized. However, we could not prevent cell rounding by using inhibitors of receptor-mediated endocytosis, which indicates tha t the toxin may act outside the cell. Based on these observations, we studied the effects of the highly purified B. fragilis fragilysin on t he barrier function of cultured epithelial cells. Fragilysin rapidly i ncreased the permeability of the paracellular barrier of epithelial ce lls to ions (decrease in electrical resistance across monolayers) and to larger molecules (increase in mannitol flux across monolayers). We tested a human colon cell line and cell lines from the lung and the ki dney; the human colon cell line was most sensitive, but all three were affected in the same manner. Our studies show that B. fragilis fragil ysin alters the barrier function of the epithelial lining, possibly by degrading the tight junction proteins, such as ZO-1. The proteolytic activity is required to cause this effect. The toxin's action has been assumed to be limited to the intestine; however, our studies show tha t fragilysin could also contribute to the pathogenesis of B. fragilis in extraintestinal infections.