PHYSIOLOGY AND CLONING OF B FRAGILIS ENTEROTOXIN

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Enterotoxigenic Bacteroides fragilis (ETBF) have been isolated from the feces of 10 to 20% of diarrheic livestock and is a newly recognized human enteric pathogen. In both naturally and experimentally infected animals, the predominant site of altered histopathology has been the colon. To date, conventional tissue culture and animal assays used to detect the biologic activity of bacterial enterotoxins have failed with ETBF. We have developed a tissue culture assay using the cloned human colonic epithelial cell line, HT29/C1, which is 89% sensitive and 100% specific in detecting ETBF strains as defined by the lamb ligated intestinal loop assay. Subconfluent HT29/C1, cells treated with less than 1 nanogram of the purified ETBF heat-labile protein toxin rapidly develop dramatic morphologic changes with rounding and detachment from adjacent cells. These in vitro morphologic changes mimic those observed in vivo in natural and experimental infection. Furthermore, in polarized confluent cell monolayers, the transepithelial resistance of the monolayers is diminished and electrogenic active chloride secretion is stimulated without any alteration in cell viability. These data indicate that the ETBF toxin is an enterotoxin and a nonlethal cytotoxin. We hypothesize that the ETBF toxin is a key virulence factor of B. fragilis strains associated with diarrheal disease and that this toxin alters the intestinal epithelial barrier by effecting a change in cell shape resulting in disruption of tight junctions. Intestinal secretion may result from leakiness of the paracellular transport pathway and direct stimulation of transcellular chloride secretion. To address this hypothesis, the specific aims of this project are: I. To identify the cytoskeletal effects of the ETBF toxin. The morphologic changes induced by ETBF and its toxin in HT29/C1 cells and lamb intestine over time will be characterized by light and electron microscopy; nd by studies of the cytoskeletal proteins, actin, tubulin and keratin. II. To investigate the effects of the ETBF toxin on cellular function. The time course and mechanism(s) by which the ETBF toxin diminishes the transepithelial resistance and stimulates chloride secretion in polarized monolayers of HT29/C1 cells will be examined; and III. To establish the importance of the ETBF toxin as a virulence factor. The ETBF toxin gene will be cloned and the importance of this toxin in the pathogenesis of ETBF infections will be established by studies utilizing constructed isogenic strains and by use of an ETBF toxin DNA probe in human epidemiologic investigations. These studies utilizing a human intestinal epithelial cell line and lamb intestine will begin to characterize by cell biology, physiology and genetic techniques the importance in disease pathogenesis of the ETBF toxin, a newly identified virulence factor of B. fragilis.