BACTEROIDES-FRAGILIS TOXIN EXHIBITS POLAR ACTIVITY ON MONOLAYERS OF HUMAN INTESTINAL EPITHELIAL-CELLS (T84 CELLS) IN-VITRO

Strains of Bacteroides fragilis associated with diarrhea in children ( termed enterotoxigenic B. fragilis, or ETBF) produce a heat-labile ca, 20-kDa protein toxin (BFT), The purpose of this study was to examine the activity of BFT on polarized monolayers of human intestinal epithe lial cells (T84 cells), In Ussing chambers, BFT had two effects, First , BFT applied to either the apical or basolateral surfaces of T84 mono layers diminished monolayer resistance, However, the time course, magn itude, and concentration dependency differed when BFT was applied to t he apical versus basolateral membranes, Second, only basolateral BFT s timulated a concentration-dependent and short-lived increase in short circuit current (I-sc; indicative of Cl- secretion), Time course exper iments indicated that I-sc returned to baseline as resistance continue d to decrease, indicating that these two electrophysiologic responses to BFT are distinct. Light microscopic studies of BFT-treated monolaye rs revealed only localized cellular changes after apical BFT, whereas basolateral BFT rapidly: altered the morphology of nearly every cell i n the monolayer, Transmission and scanning electron microscopy after b asolateral BFT confirmed a striking loss of cellular microvilli and co mplete dissolution of some tight junctions (zonula occludens) and zonu la adherens without loss of desmosomes, The F-actin structure of BFT-t reated monolayers (stained with rhodamine-phalloidin) revealed diminis hed and flocculated staining at the epical tight junctional ring and t hickening of F-actin microfilaments in focal contacts at the basolater al monolayer surface compared to those in similarly stained control mo nolayers, BPT did not injure T84 monolayers, as assessed by lactic deh ydrogenase release and protein synthesis assays. These studies indicat e that BFT is a nonlethal toxin which acts in a polar manner on T84 mo nolayers to stimulate Cl- secretion and to diminish monolayer resistan ce by altering the apical F-actin structure of these cells. BFT may co ntribute to diarrheal disease associated with ETBF infection by alteri ng epithelial barrier function and stimulating Cl- secretion.