Clostridium difficile toxins A and B can alter epithelial permeability and promote bacterial paracellular migration through HT-29 enterocytes.

Clostridium difficile toxins A and B are the widely recognized etiologic agents of antibiotic-associated diseases ranging from diarrhea to pseudomembranous colitis. We hypothesized that C. difficile toxins may alter intestinal epithelial permeability and facilitate bacterial penetration of the intestinal epithelial barrier. Experiments were designed to clarify the effects of C. difficile toxins A and B on the flux of inert particles across HT-29 enterocyte monolayers, and to correlate these results with bacteria-enterocyte interactions. In all experiments, mature, confluent HT-29 cultures were preincubated 16 h with toxin A or B (1-100 ng/mL). To study alterations in epithelial permeability, toxin-treated enterocytes were incubated with 5 pM solutions of 10- and 40-kD inert dextran particles. Toxin A, but not toxin B, was associated with increased dextran flux through enterocyte monolayers. To study bacteria-enterocyte interactions, toxin-treated enterocytes were incubated with 10(8) Salmonella typhimurium, Proteus mirabilis, or Escherichia coli. Although numbers of internalized bacteria were generally unaffected, both toxins were associated with increased bacterial adherence, as well as increased bacterial transmigration through enterocyte monolayers. Bacterial transmigration was significantly greater using toxin A- compared to toxin B-treated enterocytes, consistent with the observation that dextran flux was significantly greater using toxin A- compared to toxin B-treated enterocytes. Thus intestinal colonization with toxigenic C. difficile may facilitate bacterial penetration of the intestinal epithelium by a mechanism involving increased permeability of the intestinal epithelial barrier.

Clostridium difficile toxins may augment bacterial penetration of intestinal epithelium.

BACKGROUND: Clostridium difficile can be recovered from many high-risk hospitalized patients receiving broad-spectrum antibiotic therapy. Clostridium difficile toxins A and B have been associated with increased intestinal permeability in vitro and there is growing evidence that increased intestinal permeability may be a common mechanism whereby enteric bacteria penetrate the intestinal epithelium. HYPOTHESIS: Clostridium difficile-induced alterations in the intestinal barrier facilitate microbial penetration of the intestinal epithelium, which in turn facilitates the translocation of intestinal bacteria. DESIGN: Mature Caco-2 enterocytes were pretreated with varying concentrations of toxin A or toxin B followed by 1 hour of incubation with pure cultures of either Salmonella typhimurium, Escherichia coli, or Proteus mirabilis. The effects of toxins A and B on enterocyte viability, cytoskeletal actin, and ultrastructural topography were assessed using vital dyes, fluorescein-labeled phalloidin, and scanning electron microscopy, respectively. The toxins' effects on bacterial adherence and bacterial internalization by cultured enterocytes were assessed using enzyme-linked immunosorbent assay and quantitative culture, respectively. Epithelial permeability was assessed by changes in transepithelial electrical resistance and by quantifying paracellular bacterial movement through Caco-2 enterocytes cultivated on permeable supports. RESULTS: Neither toxin A nor toxin B had a measurable effect on the numbers of enteric bacteria internalized by Caco-2 enterocytes; however, both toxins were associated with alterations in enterocyte actin, decreased transepithelial electrical resistance, and increased bacterial adherence and paracellular transmigration. CONCLUSION: Clostridium difficile toxins A or B may facilitate bacterial adherence and penetration of the intestinal epithelial barrier.

Subacute ethanol consumption reverses p-xylene-induced decreases in axonal transport.

Human exposure to organic solvents is often complicated by ethanol ingestion and the literature is replete with demonstrations of metabolic interactions between ethanol and organic solvents at a pharmacokinetic level. Because of the possible modulation of xylene toxicity by ethanol consumption, the present group of studies characterizes the effect of ethanol on the p-xylene-induced decrease in axonal transport in the rat optic system previously reported by our laboratory. Long-Evans, hooded, male rats were divided randomly into two groups: those receiving 10% ethanol in their drinking water and those receiving water only. These two groups were further subdivided into two groups which were either exposed by inhalation to 1600 ppm p-xylene for 6 h/day, 5 days/week for 8 exposure-days or were treated identically except that they were exposed to air while in the inhalation chambers. The ethanol-drinking rats were given ethanol 6 days prior to and on the days of the inhalation exposure. Immediately after removal from the inhalation chambers on the last exposure day, the animals were injected intraocularly with [35S]methionine and [3H]fucose to measure the synthesis and rapid axonal transport of proteins and glycoproteins, respectively, in the retinal ganglion cells. The animals were sacrificed 20 h later, and the amount of radioactivity in different areas of the retinal ganglion cells was determined by liquid scintillation counting. As in previous experiments, the xylene exposure group showed a significant reduction in axonal transport of proteins and glycoproteins, whereas the ethanol exposure alone produced no significant reductions in the transport of either proteins or glycoproteins. In the animals receiving both ethanol and xylene, however, the ethanol treatment prevented the decreased transport characteristic of the xylene only animals, i.e. in all areas of the optic projections the level of transport were similar to the level present in the control groups. These data suggest that the xylene-induced reduction in rapid axonal transport was reversed (or prevented) by subacute ethanol consumption.

Construction and expression of the complete Clostridium difficile toxin A gene in Escherichia coli.

Cloned fragments constituting the 8.1-kb toxin A gene of Clostridium difficile were used to reconstruct the intact gene. The recombinant toxin expressed in Escherichia coli was cytotoxic, enterotoxic, and lethal. In addition, toxic lysate caused hemagglutination of rabbit erythrocytes. The toxic activities were inhibited by antibody specific for toxin A. Our findings demonstrate that the biological activities exhibited by native toxin A are functions of a single protein encoded by the 8.1-kb toxin A gene, independent of any other C. difficile gene products.